Sommaire du brevet 2949171 - Base de données sur les brevets canadiens (2024)

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IMPROVED CULTURE METHOD FOR ORGANOIDS
Background to the invention
Recently, in the small intestine, the gene Lgr5 was identified which is
specifically
expressed in cycling Crypt Base Columnar (CBC) cells, which are small cells
that are
interspersed between the Paneth cells (Barker et al., 2007. Nature 449: 1003-
1007).
Using a mouse in which a GFP/tamoxifen-inducible Cre recombinase cassette was
integrated into the Lgr5 locus, it was shown by lineage tracing that the Lgr5+
CBC cells
constitute multipotent stem cells which generate all cell types of the
epithelium even when
assessed 14 months after Cre induction.
The existence of Lgr5 in other tissues was described in WO 2009/022907 and the
existence of Lgr5 cells in the liver was later described in WO 2012/014076.
Methods for
culturing epithelial stem cells and obtaining organoids starting from
epithelial stem cells
were provided in WO 2010/090513, WO 2012/014076 and WO 2012/168930
(KONINKLIJKE NEDERLANDSE AKADEMIE VAN WETENSCHAPPEN). Although these
methods advantageously allow the expansion of epithelial stem cells, and
provide longer-
term expansion than the previously known methods, it would be advantageous to
increase
the length of time that the stem cells can be expanded even further,
particularly for human
cells.
WO 2013061608 describes methods for isolating and culturing colorectal
epithelial stem
cells in a medium comprising serum albumin, Wnt3a and Rspondin. The methods do
not
involve a culture medium comprising a TGF-beta inhibitor, FGF, nicotinamide or
a BMP
pathway activator and expansion times are limited.
It is, therefore, an object of the present invention to provide a method for
increasing the
expansion time of epithelial stem cells.
The basic architectural unit of the liver is the liver lobule. Each lobule
consists of plates of
hepatocytes lined by sinusoidal capillaries that radiate toward a central
efferent vein. Liver
lobules are roughly hexagonal with each of six corners demarcated by the
presence of a
portal triad (portal vein, bile duct, and hepatic artery). Although
hepatocytes are the major
parenchymal cell type of the liver they function in concert with
cholangiocytes (biliary
epithelial cells), endothelial cells, sinusoidal endothelial cells, Kupffer
cells, natural killer
cells and hepatic stellate cells. This complex architecture is important for
hepatic function.
The existence of liver stem cells remains controversial. On one hand, tissue
maintenance
in the liver and liver regeneration upon certain types of injury, are not
driven by stem cells
but rather by division of the mature cells (hepatocytes or cholangiocytes).
However, liver
injury models in which hepatocyte proliferation have been inhibited also
demonstrated the

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ability of the organ to regenerate in response to damage. This suggests that
the liver can
be considered as an organ with facultative stem cells.
Liver cultures derived from hepatocytes, or by differentiation of embryonic
stem cells (ES)
or induced pluripotent stem cells, are known but these do not expand and self-
renew for
long periods.
Here there is provided a method to culture epithelial stem cells and to obtain
organoids
that shows longer-lived maintenance, and are able to differentiate to all
major differentiated
cell lineages present in the corresponding in vivo tissue. The method has been
exemplified
with epithelial cells derived from the liver, and has also been demonstrated
to work with
epithelial cells derived from the pancreas. Thus the method is envisaged to be
relevant to
the culture of all epithelial cell types.
Summary of the invention
Accordingly, the invention provides a method for culturing epithelial stem
cells, wherein
said method comprises culturing one or more epithelial stem cells in contact
with an
extracellular matrix in the presence of a culture medium as described herein.
In particular, the invention provides a method for culturing epithelial stem
cells, wherein
said method comprises culturing one or more epithelial stem cells in contact
with an
extracellular matrix in the presence of an expansion medium as described
herein. The
method may optionally further comprise the step of culturing one or more
epithelial cells
that have been cultured in an expansion medium of the invention with a
differentiation
medium as described herein.
Accordingly, the invention provides a method for culturing epithelial stem
cells, wherein
said method comprises:
culturing one or more epithelial stem cells in contact with an extracellular
matrix in
the presence of an expansion medium, the expansion medium comprising a basal
medium
for animal or human cells to which is added:
one or more receptor tyrosine kinase ligands, one or more Wnt agonist wherein
the
Wnt agonist is an Lgr5 agonist, a TGF-beta inhibitor and a cAMP pathway
activator.
In some embodiments, the Lgr5 agonist is Rspondin.
In some embodiments, the expansion medium further comprises a BMP pathway
activator.
In some embodiments, the expansion medium further comprises one or more
components
selected from the group consisting of: a further Wnt agonist, a BMP inhibitor,
nicotinamide,
gastrin, B27, N2, and N-Acetylcysteine.

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In some embodiments, in the expansion medium:
the one or more receptor tyrosine kinase ligands are selected from the group
consisting of: FGF, HGF and EGF, wherein the FGF is preferably an FGF able to
bind to
FGFR2 or FGFR4 and is preferably FGF10;
the TGF-beta inhibitor is a small molecule inhibitor of ALK4, ALK5 or ALK7,
optionally selected from the group consisting of: A83-01, SB-431542, SB-
505124, SB-
525334, LY 364947, SD-208 and SJN 2511;
the cAMP pathway activator is an adenylyl cyclase activator, for example,
forskolin,
a forskolin analog or cholera toxin, or a cAMP analog, for example 8-bromo-
cAMP, or
NKH477;
when Rspondin is present, the Rspondin is selected from R-spondin 1, R-spondin

2, R-spondin 3 and R-spondin 4;
when a further Wnt agonist is present, the further Wnt agonist is selected
from one
or more of Wnt-3a, Wnt-5, Wnt-6a, Norrin, and a GSK-inhibitor; and/or
when a BMP pathway activator is present, it is selected from one or more of
BMP7,
BMP4 and BMP2.
In some embodiments, the method further comprises a culturing step in a
differentiation
medium comprising a basal medium for animal or human cells to which is added
one or
more receptor tyrosine kinase ligands and a Notch inhibitor.
In some embodiments, the differentiation medium further comprises one or more
of: a
TGF-beta inhibitor, gastrin, dexamethasone and a BMP pathway activator.
In some embodiments, in the differentiation medium:
the one or more receptor tyrosine kinase ligands are selected from one or more
of
the group: FGF, HGF and EGF, wherein the FGF is preferably an FGF able to bind
to
FGFR2 or FGFR4 and is preferably FGF19;
the Notch inhibitor is a gamma-secretase inhibitor, optionally DAPT or
dibenzazepine (DBZ) or benzodiazepine (BZ) or LY-411575
when a TGF-beta inhibitor is present, it is a small molecule inhibitor of
ALK4, ALK5
or ALK7 optionally selected from the group consisting of: A83-01, SB-431542,
SB-505124,
SB-525334, LY 364947, SD-208, SJN 2511; and/or
when a BMP pathway activator is present, it is selected from one or more of
BMP7,
BMP4 and BMP2.

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The invention also provides a method for differentiating epithelial stem
cells, wherein said
method comprises:
culturing the cells in a differentiation medium comprising a basal medium for
animal
or human cells to which is added EGF, gastrin, FGF19, DAPT, dexamethasone, HGF
and
A8301.
In some embodiments, the method culturing or differentiating epithelial stem
cells further
comprises isolating one or more adult stem cells or obtaining and isolating an
organoid.
In some embodiments, the epithelial stem cells are from the liver, pancreas,
intestine,
stomach, prostate, lung, breast, ovarian, salivary gland, hair follicle, skin,
oesophagus or
thyroid.
In some embodiments, the epithelial stem cells are from the liver or pancreas.
There is also provided a method for culturing epithelial stem cells, wherein
the epithelial
stem cells are from the liver and wherein said method comprises:
culturing one or more epithelial stem cells in contact with an extracellular
matrix in
the presence of an expansion medium, the expansion medium comprising a basal
medium
for animal or human cells to which is added: EGF, FGF10, HGF, Rspondin,
Nicotinamide,
a TGF-beta inhibitor, forskolin, gastrin, N-Acetylcysteine, and N2 and/or B27,
and which is
supplemented with Noggin, a further Wnt agonist, and a ROCK inhibitor;
culturing the one or more epithelial stem cells in a second expansion medium
comprising a basal medium for animal or human cells to which is added: EGF,
FGF10,
HGF, Rspondin, Nicotinamide, a TGF-beta inhibitor, forskolin, gastrin, N-
Acetylcysteine,
and N2 and/or B27, and optionally BMP7; and optionally,
culturing the one or more expanded epithelial stem cells in a differentiation
medium
comprising a basal medium for animal or human cells to which is added EGF,
gastrin,
FGF19, DAPT, dexamethasone, HGF, a TGF-beta inhibitor, and optionally a BMP7.
There is also provided a method for culturing epithelial stem cells, wherein
the epithelial
stem cells are from the pancreas and wherein said method comprises:
culturing one or more epithelial stem cells in contact with an extracellular
matrix in
the presence of an expansion medium, the expansion medium comprising a basal
medium
for animal or human cells to which is added: EGF, FGF10, Rspondin, Noggin, a
further
Wnt agonist, Nicotinamide, a TGF-beta inhibitor, forskolin, PGE2, a p38
inhibitor, gastrin,
N2, B27, and N-Acetylcysteine and optionally BMP7; and optionally
culturing the one or more expanded epithelial stem cells in a differentiation
medium
comprising a basal medium for animal or human cells to which is added EGF,
gastrin,
FGF19, DAPT, dexamethasone, HGF, a TGF-beta inhibitor, and optionally a BMP7.
Also provided is a culture medium of the invention. In particular, the
invention provides an
expansion medium, comprising a basal medium for animal or human cells to which
is

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added one or more receptor tyrosine kinase ligands, one or more Wnt agonist
wherein the
Wnt agonist is an Lgr5 agonist, a TGF-beta inhibitor, and a cAMP pathway
activator.
In some embodiments, the Lgr5 agonist is Rspondin.
In some embodiments, the expansion medium further comprises a BMP pathway
activator.
5 In some embodiments, the expansion medium further comprises one or more
components
selected from the group consisting of: a further Wnt agonist, a BMP inhibitor,
nicotinamide,
gastrin, N-Acetylcysteine, and B27 and/or N2.
In some embodiments, in the expansion medium:
the one or more receptor tyrosine kinase ligands are selected from the group
consisting of: FGF, HGF and EGF, wherein the FGF is preferably an FGF able to
bind to
FGFR2 or FGFR4 and is preferably FGF10;
the TGF-beta inhibitor is a small molecule inhibitor of ALK4, ALK5 or ALK7,
optionally selected from the group consisting of: A83-01, SB-431542, SB-
505124, SB-
525334, LY 364947, SD-208 and SJN 2511;
the cAMP pathway activator is an adenylyl cyclase activator for example
forskolin,
a forskolin analog or cholera toxin, or a cAMP analog, for example 8-bromo-
cAMP, or
NKH477;
when Rspondin is present, the Rspondin is selected from Rspondin 1, Rspondin
2,
Rspondin 3 and Rspondin 4;
when a further Wnt agonist is present, the further Wnt agonist is selected
from one
or more of Wnt, Wnt-3a, Norrin, and a GSK-inhibitor; and/or
when a BMP pathway activator is present, it is selected from one or more of
BMP7,
BMP4 and BMP2.
In some embodiments, the expansion medium comprises EGF, FGF10, Rspondin,
Nicotinamide, A8301, forskolin, Noggin, Wnt, gastrin, B27 and N-
Acetylcysteine.
In some embodiments, the expansion medium comprises EGF, FGF10, HGF, Rspondin,
Nicotinamide, A8301, forskolin, BMP7, gastrin, N-Acetylcysteine, and N2 and/or
B27.
The invention also provides a differentiation medium comprising a basal medium
for
animal or human cells to which is added EGF, gastrin, FGF19, DAPT,
dexamethasone,
HGF and A8301 and optionally a BMP pathway activator.
Organoids and populations of cells as described herein are provided. For
example, the
invention provides an organoid obtainable or obtained by a method of the
invention.

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The invention also provides an organoid which has been cultured for at least
6, 8, 10, 12,
14,16, 18 or 20 weeks.
In some embodiments, the organoid is derived from the liver, pancreas,
intestine, stomach,
prostate, lung, breast, ovarian, salivary gland, hair follicle, skin,
oesophagus or thyroid.
The invention also provides an organoid in a culture medium according to the
invention.
In some embodiments, the organoid of the invention has a doubling time of less
than 65
hours, for example, 60, 58, 56, 54, 53, 50 hours or less.
The invention provides a liver organoid, wherein the liver organoid is derived
from a mouse
and:
expresses at least one, preferably all, of the following stem cell markers:
Igr5, Igr4,
epcam, Cd44, Tnfrsf19, Sox9, Sp5, Cd24a, Prom1, Cdca7 and E1f3; and/or
does not express the following stem cell marker: Igr6; and/or
expresses at least one, preferably all, of the following hepatocyte or
cholangiocyte
markers when grown in the expansion medium as described herein: Hnf1a, Hnf1b,
Hnf4a,
Hhex, Onecut1, Onecut2, Prox1, Cdh1, Foxa2, Gata6, Foxm1, Cebpa, Cebpb, Cebpd,
Cebpg, Glul, Krt7, Krt19 and Met; and/or
does not express at least one of the following genes when grown in the
expansion
medium described herein: afp, Ins1, Ins2, Gcg, Ptf1a, Cela1, Cela2a, Cela3b,
Neurod1,
Neurod2, Neurog1, Neurog2, Neurog3, Amy2a4, Igf1r, Igf2 and Cd34; and/or
expresses at least one of the following reprogramming genes: K1f4 and Myc;
and/or
does not express one of the following reprogramming genes: Pou5f1 and Sox2.
The invention also provides a liver organoid, wherein the liver organoid is
derived from a
human and:
expresses at least one, preferably all, of the following stem cell signature
genes:
LGR4, TACSTD1/Epcam, CD44, SOX9, SP5, CD24, PROM1, CDCA7 and ELF3; and/or
expresses at least one, preferably all, of the following reprogramming genes:
KLF4,
MYC, POU5F1 and SOX2; and/or
expresses at least one, preferably all, of the following
hepatocyte/cholangiocyte
specific genes: HNF1A, HNF1B, HNF4A, HHEX, ONECUT1, ONECUT2, PROX1, CDH1,
FOXA2, GATA6, FOXM1, CEBPA, CEBPB, CEBPD, CEBPG, GLUL, KRT7, KRT19 and
MET; and/or

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does not express at least one, preferably all, of the following
hepatocyte/cholangiocyte specific genes: NEUROG2, IGF1R and CD34, AFP, GCG and

PTF1A, for example, it does not express NEUROG2, IGF1R and CD34; and/or
expresses at least one, preferably all, of the following hepatocyte specific
genes:
TTR, ALB, FAH, TAT, CYP3A7, AP0A1, HMGCS1, PPARG, CYP2B6, CYP2C18,
CYP2C9, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4F8, CYP4V2 and SCARB1.
Uses of the organoids described herein and cells derived from the organoids
are likewise
provided. For example, the invention also provides the use of an organoid of
the invention
or a cell derived from said organoid in a drug discovery screen; toxicity
assay; research of
tissue embryology, cell lineages, and differentiation pathways; gene
expression studies
including recombinant gene expression; research of mechanisms involved in
tissue injury
and repair; research of inflammatory and infectious diseases; studies of
pathogenetic
mechanisms; or studies of mechanisms of cell transformation and aetiology of
cancer.
The invention also provides an organoid of the invention, or a cell derived
from said
organoid, for use in medicine.
The invention also provides an organoid of the invention, or a cell derived
from said
organoid, for use in treating a disorder, condition or disease.
The invention also provides an organoid of the invention, or a cell derived
from said
organoid for use in regenerative medicine, for example, wherein the use
involves
transplantation of the organoid or cell into a patient.
The invention also provides a pharmaceutical formulation comprising one or
more receptor
tyrosine kinase ligands, a Wnt agonist wherein the Wnt agonist is an Lgr5
agonist, a TGF
beta inhibitor, and a cAMP pathway activator, and a pharmaceutically
acceptable diluent
and/or excipient.
Detailed description of the invention
Methods for culturing epithelial stem cells from a variety of tissues have
previously been
described in W02010/090513, W02012/014076 and W02012/168930. The present
inventors have surprisingly found that adding a cAMP pathway activator to the
culture
medium allows human epithelial stem cells to be cultured for an increased
number of
passages compared to when the cAMP pathway activator is absent from the
medium.
The ability to keep the cells and resulting organoids alive for longer and
increase the
passage number advantageously allows more cells to be obtained from a single
starting
cell or from a collection of starting cells than was possible using previous
methods. This
enables a large number of cells to be available for various applications, for
example, drug

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screening, in which a large amount of material is required to test various
different drugs.
The ability to generate the cells from a single starting source is
advantageous for such
applications where it is necessary to compare results between experiments.
Similarly, it
means that many cells are available for use in transplants and that multiple
patients may
be transplanted with cells obtained from a useful donor.
Culturing the cells in an expansion medium allows the cells to multiply whilst
retaining their
stem or progenitor cell phenotype. Organoids are formed comprising these stem
or
progenitor cells. Use of the expansion medium is therefore advantageous for
providing
increased numbers of these useful stem or progenitor cells and for obtaining
organoids
containing these cells.
Accordingly, there is provided a method for culturing epithelial stem cells,
wherein said
method comprises culturing one or more epithelial stem cells in contact with
an
extracellular matrix in the presence of an expansion medium, the expansion
medium
comprising a basal medium for animal or human cells to which is added one or
more
receptor tyrosine kinase ligands, one or more Wnt agonist preferably wherein
the Wnt
agonist is an Lgr5 agonist, a TGF-beta inhibitor and a cAMP pathway activator.
There is also provided an expansion medium comprising a basal medium for
animal or
human cells to which is added one or more receptor tyrosine kinase ligands,
one or more
Wnt agonist preferably wherein the Wnt agonist is an Lgr5 agonist, a TGF-beta
inhibitor
and a cAMP pathway activator, for example, for use in the above method.
In some embodiments, the expansion medium comprises a basal medium and one or
more of an EGF receptor activator (e.g. EGF), an FGF receptor 2 or FGF
receptor 4
activator (e.g. FGF), an HGF receptor activator (e.g. HGF) as receptor
tyrosine kinase
ligands, a Wnt agonist preferably wherein the Wnt agonist is an Lgr5 agonist
(e.g.
Rspondin), a TGF beta inhibitora cAMP pathway activator (e.g. forskolin) and
Nicotinamide.
The invention therefore provides the use of a cAMP pathway activator for
culturing
epithelial stem cells. The invention also provides a method for culturing
epithelial stem
cells which uses an expansion medium as described in WO 2012/014076 or
W02012/168930 to which a cAMP pathway activator is added.
The cAMP pathway activator may be any suitable activator which increases the
levels of
cAMP in a cell. The cAMP pathway involves activation of many types of hormone
and
neurotransmitter G-protein coupled receptors. Binding of the hormone or
neurotransmitter
to its membrane-bound receptor induces a conformational change in the receptor
that
leads to activation of the a-subunit of the G-protein. The activated G subunit
stimulates,

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while the non-activated G subunit inhibits adenylyl cyclase. Stimulation of
adenylyl cyclase
catalyzes the conversion of cytoplasmic ATP to cAMP thus increasing the levels
of cAMP
in the cell. Therefore, the cAMP pathway activator may, for example, be an
adenylyl
cyclase activator. Examples of suitable adenylyl cyclase activators include
forskolin, a
forskolin analogue and cholera toxin. In some embodiments, the cAMP pathway
activator
is forskolin. In some embodiments, the cAMP pathway activator is not cholera
toxin. In
some embodiments the cAMP pathway activator may be a cAMP analog, for example
8-
bromo-cAMP. 8-bromo-cAMP is a cell-permeable cAMP analog having greater
resistance
to hydrolysis by phosphodiesterases than cAMP. In some embodiments, the cAMP
pathway activator is NKH477 (e.g. catalogue no. Tocris 1603).
cAMP pathway activators can be identified using methods known in the art, for
example,
using a competitive immunoassay which measures cAMP levels. The CatchPoint
Cyclic-
AMP Fluorescent Assay Kit (Molecular Devices LLC) is an example of a
commercially
available kit for carrying out such an immunoassay. The cAMP in the sample or
standard
competes with horseradish peroxidase (HRP)-labeled cAMP conjugate for binding
sites on
the anti-cAMP antibodies. In the absence of cAMP, most of the HRP-cAMP
conjugate is
bound to the antibody. Increasing concentrations of cAMP competitively
decrease the
amount of bound conjugate, thus decreasing measured HRP activity. A cAMP
pathway
activator would result in increased levels of cAMP and decreased measured HRP
activity,
compared to a control.
In some embodiments, the cAMP pathway activator is used at a concentration of
between
about 10 nM to about 500 pM, about 10 nM to about 100 pM, about 1 pM to about
50 pM,
about 1 pM to about 25 pM, about 5 pM to about 1000 pM, about 5 pM to about
500 pM,
about 5 pM to about 100 pM, about 5 pM to about 50 pM, about 5 pM to about 25
pM,
about 10 pM to about 1000 pM, about 10 pM to about 500 pM, about 10 pM to
about 100
pM, about 10 pM to about 50 pM, about 10 pM to about 25 pM, or about 20 pM. In
some
embodiments the cAMP pathway activator is used at a concentration of at least
10 nM, 20
nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 pM, at least 2 pM, at least 5 pM, at
least 10 pM, at
least 20 pM, at least 30 pM, at least 50 pM, or at least 100 pM.
The concentration selected may depend upon the cAMP pathway activator used and
can
be determined by the person skilled in the art depending upon the potency of
the cAMP
pathway activator. For example, NKH477 is generally more potent than 8-BR-cAMP
and
forskolin. A more potent cAMP pathway activator can be used at lower
concentrations to
the same effect.

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For example, NKH477 can in some embodiments be used at a concentration of
between
about 100 nM and about 10 pM, or at a concentration of about 100 nM, about 1
pM or
about 10 pM. 8-BR-cAMP or forskolin can in some embodiments be used at a
concentration of between about 1 pM and about 100 pM, or at a concentration of
about 1
5 pM, about 10 pM or about 100 pM.
Cholera toxin can in some embodiments be used at a concentration of between
about 1
ng/ml and about 500 ng/ml, about 10 ng/ml and about 100 ng/ml, about 50 ng/ml
and
about 100 ng/ml, or about 10 ng/ml, about 20 ng/ml, about 30 ng/ml, about 40
ng/ml,
about 50 ng/ml, about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90
ng/ml, about
10 100 ng/ml, about 200 ng/ml, about 300 ng/ml, about 400 ng/ml or about
500 ng/ml.
It has also surprisingly been found that the presence of a BMP pathway
activator in the
expansion medium increases the number of passages possible for human cells
compared
to the methods described in W02012/168930 and W02012/014076. For example,
Figure
2 shows it allows human liver cells to be passaged 8 times as opposed to only
5 times
using the previous techniques. Although this increase is not as significant as
the increase
obtained using a cAMP pathway activator, it is still useful as it allows more
cells to be
obtained than using the previous methods.
Thus in some embodiments, the expansion medium of the invention further
comprises a
BMP pathway activator. In some embodiments, the BMP pathway activator is
selected
from BMP7, BMP4 and BMP2. BMP7 is preferred. BMP7 induces the phosphorylation
of
SMAD1 and SMAD5. Thus in some embodiments, where BMP7 is mentioned, any
compound that induces the phosphorylation of SMAD1 or SMAD5 can be used
instead of
BMP7.
The invention therefore provides the use of a BMP activator for culturing
epithelial stem
cells. The invention also provides a method for culturing epithelial stem
cells which uses
an expansion medium as described in WO 2012/014076 or W02012/168930 to which a

BMP activator is added.
Accordingly, in some embodiments, the expansion medium comprises a cAMP
pathway
activator and a BMP activator. However, in some embodiments, the expansion
medium
comprises a cAMP pathway activator in the absence of a BMP activator. It is
also
envisaged that the expansion medium may comprise a BMP activator in the
absence of a
cAMP pathway activator.
The epithelial stem cells of the invention are epithelial cells from
epithelial tissue which
express Lgr5. They are also known as Lgr5 positive cells.

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Receptor tyrosine kinase ligands
Epidermal growth factor (EGF), fibroblast growth factor (FGF) and hepatocyte
growth
factor (HGF) are preferably all present in the expansion medium. In the
context of a
culture medium of the invention, EGF is also referred to herein as "E", FGF is
also referred
to herein as "F" and HGF is also referred to herein as "H". They are also
referred to herein
as "receptor tyrosine kinase ligands". Many receptor tyrosine kinase ligands
are mitogenic
growth factors. In some embodiments, the one or more receptor tyrosine kinase
ligands in
the expansion medium are selected from the group consisting of: FGF, HGF and
EGF,
wherein the FGF is preferably an FGF able to bind to FGFR2 or FGFR4 and is
preferably
FGF10.
In some embodiments. the one or more receptor tyrosine kinase ligands in the
expansion
medium are EGF and FGF. In some embodiments. the one or more receptor tyrosine

kinase ligands in the expansion medium are EGF and HGF. In some embodiments.
the
one or more receptor tyrosine kinase ligands in the expansion medium are HGF
and FGF.
In some embodiments, only one receptor tyrosine kinase ligand is included in
the
expansion medium, which may be selected from FGF, HGF and EGF.
Any suitable EGF may be used, for example, EGF obtained from Peprotech. EGF is

preferably added to the basal culture medium at a concentration of between 5
and 500
ng/ml. A preferred concentration is at least 10, 20, 25, 30, 40, 45, or 50
ng/ml and not
higher than 500, 450, 400, 350, 300, 250, 200, 150, or 100 ng/ml. A more
preferred
concentration is at least 50 and not higher than 100 ng/ml. An even more
preferred
concentration is about 50 ng/ml. In some embodiments, EGF is substituted with
an
alternative compound that activates the EGF receptor. For example, it is
envisaged that
IGF may be substituted for EGF.
The FGF used in the expansion medium is preferably an FGF able to bind to FGF
receptor
2 (FGFR2) or FGF receptor 4 (FGFR4), and is preferably FGF4, FGF7 or FGF10
(preferably from Peprotech), most preferably FGF10. In some embodiments, no
more than
one FGF is used. In other embodiments, two or more FGF are used, e.g. 2, 3 or
more. In
some embodiments, FGF is substituted with a compound that activates the FGFR2
or
FGFR4 pathway (a "FGF-pathway activator").
FGF10 is a protein that belongs to the fibroblast growth factor (FGF) family
of proteins.
FGF family members possess broad mitogenic and cell survival activities, and
are involved
in a variety of biological processes, including embryonic development, cell
growth,
morphogenesis, tissue repair, tumor growth and invasion. FGFs stimulate cells
by
interacting with cell surface tyrosine kinase receptors (FGFR). Four closely
related

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12
receptors (FGFR1¨FGFR4) have been identified. FGFR1¨FGFR3 genes have been
shown to encode multiple isoforms, and these isoforms can be critical in
determining
ligand specificity. Most FGFs bind more than one receptor (Ornitz J Biol Chem.
1998 Feb
27;273 (9):5349-57). However, FGF10 and FGF7 are unique among FGFs in that
they
interact only with a specific isoform of FGFR2, designated FGFR2b which is
expressed
exclusively by epithelial cells (Igarashi, J Biol Chem. 1998 273(21):13230-5).
FGF10 is a
preferred FGF able to bind to FGFR2 or FGFR4. In some embodiments, FGF is
substituted with an alternative compound that activates FGFR2 or FGFR4.
Preferred concentrations for FGF10 are about 20, 50, 100, 250, 500 ng/ml, not
higher than
500ng/ml.
Hepatocyte growth factor/scatter factor (HGF/SF) is a morphogenic factor that
regulates
cell growth, cell motility, and morphogenesis by activating a tyrosine kinase
signaling
cascade after binding to the proto-oncogenic c-Met receptor. Any suitable HGF
may be
used, for example, HGF obtained from Peprotech. In some embodiments, HGF is
substituted with a compound that activates the HGF receptor.
Preferred concentrations for HGF are about 1, 10, 20, 25, 50 ng/ml, not higher
than
5Ong/ml.
Three or more, for example, 3, 4, 5 or more receptor tyrosine kinase ligands
may be used
in the expansion medium.
During culturing of stem cells, said combination of receptor tyrosine kinase
ligands (e.g.
EGF, FGF10 and HGF) is preferably added to the culture medium when required,
for
example, daily or every other day. They may be added singularly or in
combination. It is
preferable that they are added every second day.
Wnt agonist
The expansion medium of the invention comprises a Wnt agonist. In the context
of a
culture medium of the invention, the Wnt agonist is also referred to herein as
"W". The
Wnt signalling pathway is defined by a series of events that occur when the
cell-surface
Wnt receptor complex, comprising a Frizzled receptor, LRP and LGR is
activated, usually
be an extracellular signalling molecule, such as a member of the Wnt family.
This results in
the activation of Dishevelled family proteins which inhibit a complex of
proteins that
includes axin, GSK-3, and the protein APC to degrade intracellular B-catenin.
The
resulting enriched nuclear B-catenin enhances transcription by TCF/LEF family
transcription factors.

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A Wnt agonist is defined as an agent that activates TCF/LEF-mediated
transcription in a
cell. Wnt agonists are therefore selected from true Wnt agonists that bind and
activate the
Wnt receptor complex including any and all of the Wnt family proteins, an
inhibitor of
intracellular 6-catenin degradation, a GSK inhibitor (such as CHIR9901) and
activators of
TCF/LEF.
In some embodiments, a Wnt agonist is a secreted glycoprotein including Wnt-
I/Int-1, Wnt-
2/Irp (InM -related Protein), Wnt-2b/13, Wnt-3/Int-4, Wnt-3a (R&D systems),
Wnt-4, Wnt-
5a, Wnt-5b, Wnt-6 (Kirikoshi H et al 2001 Biochem Biophys Res Com 283 798-
805), Wnt-
7a (R&D systems), Wnt-7b, Wnt-8a/8d, Wnt-8b, Wnt-9a/14, Wnt- 9b/14b/15, Wnt-
10a,
Wnt-10b/12, WnM I , and Wnt-16. An overview of human Wnt proteins is provided
in "THE
WNT FAMILY OF SECRETED PROTEINS", R&D Systems Catalog, 2004. In some
embodiments, the Wnt agonist is an inhibitor of RNF43 or ZNRF3. It has been
shown that
RNF43 and ZNRF3 reside in the cell membrane and negatively regulate levels of
the Wnt
receptor complex in the membrane, probably by ubiquitination of Frizzled.
Therefore, the
inventors hypothesise that inhibition of RNF43 or ZNRF3 with antagonistic
antibodies,
RNAi or small molecule inhibitors would indirectly stimulate the Wnt pathway.
RNF43 and
ZNRF3 have a catalytic ring domain (with ubiquitination activity), which can
be targeted in
small molecule inhibitor design. Several anti-RNF43 antibodies and several
anti-ZNRF3
antibodies are available commercially. In some embodiments, such antibodies
are suitable
Wnt agonists in the context of the invention.
The Wnt agonist in the expansion medium is preferably any agonist able to
stimulate the
Wnt pathway via the Lgr5 cell surface receptor, i.e. in a preferred
embodiment, the Wnt
agonist in the expansion medium is an Lgr5 agonist. Known Lgr5 agonists
include
Rspondin, fragments and derivatives thereof, and anti-Lgr5 antibodies (e.g.
see WO
2012/140274 and De Lau, W. et al. Nature, 2011 Jul 4;476(7360):293-7). A
preferred Lgr5
agonist is Rspondin. Any suitable Rspondin may be used, for example, it may be
selected
from one or more of Rspondin 1, Rspondin 2, Rspondin 3 and Rspondin 4 or
derivatives
thereof. For example, any of Rspondin 1 (NU206, Nuvelo, San Carlos, CA),
Rspondin 2
((R&D systems), Rspondin 3, and Rspondin-4) may be used. Rspondin 1, 2, 3, and
4 are
also referred to herein as "Rspondin 1-4". In the context of a culture medium
of the
invention, Rspondin is referred to herein as "R". A preferred expansion medium
of the
invention is referred to as "ERFHNic" supplemented with a cAMP pathway
activator. An
example of an agonistic anti-Lgr5 antibody is 1D9 (available commercially from
BD
Biosciences, BDB562733, No. :562733). Therefore, in one embodiment, the
agonist is the
antibody 1D9. The VL of antibody 1D9 is represented by SEQ ID NO: 1 and the VH
is
represented by SEQ ID NO: 2. Therefore, in one embodiment, the agonist is an
antibody

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comprising or consisting of SEQ ID NO: 1 and/or SEQ ID NO: 2. Fragments of
Rspondin
may be used as the Wnt agonist. For example, in some embodiments the Wnt
agonist is a
fragment of Rspondin comprising or consisting of the furin domain. Examples of
Rspondin
fragments are represented by the sequence of amino acids recited in SEQ ID NO:
3, SEQ
ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 or of sequences with more than 70, 80,
90 or
99% identity to any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID
NO: 6.
The Wnt agonist is preferably added to the media in an amount effective to
stimulate a Wnt
activity in a cell by at least 10%, more preferred at least 20%, more
preferred at least 30%,
more preferred at least 50%, more preferred at least 70%, more preferred at
least 90%,
more preferred at least 100%, relative to a level of said Wnt activity in the
absence of said
molecule, as assessed in the same cell type. As is known to a skilled person,
Wnt activity
can be determined by measuring the transcriptional activity of Wnt, for
example by
pTOPFLASH and pFOPFLASH Tcf luciferase reporter constructs (Korinek et al.,
1997.
Science 275:1784-1787).
A soluble Wnt agonist, such as Wnt-3a, may be provided in the form of Wnt
conditioned
media. For example, about 10% to about 30%, e.g. about 10 ng/ml to about 10
pg/ml,
preferably about 1 pg/ml, Wnt conditioned media may be used.
Rspondin 1-4 may be provided in the form of Rspo conditioned media. For
example, about
10% to about 30%, e.g. about 10 ng/ml to about 10 pg/ml, preferably about 1
pg/ml, Rspo
conditioned media may be used.
Examples of Rspondin mimics suitable for use in the invention are provided in
WO
2012/140274, which is incorporated herein by reference.
One or more, for example, 2, 3, 4 or more Wnt agonists may be used in the
expansion
medium. In one embodiment, the expansion medium comprises an Lgr5 agonist, for
example Rspondin, and additionally comprises a further Wnt agonist. In this
context, the
further Wnt agonist may, for example, be selected from the group consisting of
Wnt-3a, a
GSK-inhibitor (such as CHIR99021), Wnt-5, Wnt-6a and Norrin. In one
embodiment, the
expansion medium comprises Rspondin and additionally comprises a soluble Wnt
ligand,
such as Wnt3a. Addition of a soluble Wnt ligand has been shown to be
particularly
advantageous for expansion of human epithelial stem cells (as described in
W02012/168930).
Any suitable concentration of Wnt agonist, e.g. Rspondin, may be used, for
example, at
least 200 ng/ml, more preferred at least 300 ng/ml, more preferred at least
500 ng/ml. A
still more preferred concentration of Rspondin is at least 500 ng/ml or about
1 pg/ml.

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During culturing of stem cells, said Wnt agonist may be added to the culture
medium when
required, for example, daily or every other day. The Wnt agonist is preferably
added to the
culture medium every second day.
Antibodies, such as agonistic anti-Lgr5 antibodies or antagonistic TGF-beta
inhibitors (see
5 below), used in the invention may be any antibodies, fragments, etc. A
conventional
antibody is comprised of two identical heavy chains and two identical light
chains that are
joined by disulfide bonds. Each heavy and light chain contains a constant
region and a
variable region. Each variable region contains three CDRs which are primarily
responsible
for binding an epitope of an antigen. They are referred to as CDR1, CDR2, and
CDR3,
10 numbered sequentially from the N-terminus, of which the CDR3 region
comprises the most
variable region and normally provides a substantial part of the contact
residues to a target.
The more highly conserved portions of the variable regions are called the
"framework
regions".
The term antibody is used herein in the broadest sense and specifically
covers, but is not
15 limited to, monoclonal antibodies (including full length monoclonal
antibodies) of any
isotype such as IgG, IgM, IgA, IgD and IgE, polyclonal antibodies including
recombinant
polyclonal antibodies, Oligoclonics, multispecific antibodies, chimeric
antibodies,
nanobodies, diabodies, BiTE's, Tandabs, mimetobodies, bispecific antibodies,
humanized
antibodies, human antibodies, deimmunised antibodies and antibody fragments.
In
addition, scaffolds will be covered under this term, such as Anticalins,
Ankarins, etc. An
antibody reactive with a the specific epitopes of the Lgr proteins discussed
above can be
generated by recombinant methods such as selection of libraries of recombinant

antibodies in phage or similar vectors, or by immunizing an animal with the
Lgr epitopes of
nucleic acid encoding them.
In one embodiment, an antibody according to the invention comprises a single
domain
antibody, a F(ab')2, Fab, Fab', Facb, or single chain Fv (scFv) fragment. An
Fc fragment,
which for example activates complement and may bind to Fc receptors, can be
present but
is not required for an antibody and variants or derivatives thereof. A scFv
fragment is an
epitope-binding fragment that contains at least one fragment of an antibody
heavy chain
variable region (VH) linked to at least one fragment of an antibody light
chain variable
region (VL). The linker may be a short, flexible peptide selected to assure
that the proper
three-dimensional folding of the VL and VH regions occurs once they are linked
so as to
maintain the target molecule binding-specificity of the whole antibody from
which the
single-chain antibody fragment is derived. The carboxyl terminus of the VL or
VH

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sequence may be covalently linked by a linker to the amino acid terminus of a
complementary VL or VH sequence.
The antibody may be a diabody, mimetibody, nanobody, and/or a bispecific
antibody. A
nanobody is a single domain antibody that occurs naturally in camelids. In
contrast to
standard antibodies, nanobodies are relatively simple proteins comprising only
a heavy
chain- like variable region. Bispecific antibodies are artificially engineered
monoclonal
antibodies that consist of two distinct binding sites and are capable of
binding two different
epitopes. Examples of bispecific antibodies are discussed in more detail below
in the
section on dual-targeting and multi-targeting agonists.
The antibody may be a chimeric antibody comprising a binding portion, for
example the
variable region or part thereof of the heavy and light chains, of a non-human
antibody,
while the remainder portion, for example the constant region of the heavy and
light chains,
is of a human antibody. A chimeric antibody may be produced by recombinant
processes
well known in the art, and has an animal variable region and a human constant
region.
The antibody may be a human antibody or a humanized antibody. The term "human
antibody" means an antibody in which the variable and constant domain
sequences are
derived from human sequences. In a humanized antibody, only the
complementarity
determining regions (CDRs), which are responsible for antigen binding and
specificity are
animal derived and have an amino acid sequence corresponding to the animal
antibody,
and substantially all of the remaining portions of the molecule (except, in
some cases,
small portions of the framework regions within the variable region) are human
derived and
correspond in amino acid sequence to a human antibody. Methods for humanizing
non-
human antibodies are known in the art. As is known to the skilled person,
antibodies such
as rat antibodies can be humanized by grafting their CDRs onto the variable
light (VL) and
variable heavy (VH) frameworks of human Ig molecules, while retaining those
rat
framework residues deemed essential for specificity and affinity. Overall, CDR
grafted
antibodies consist of more than 80% human amino acid sequences.
In some embodiments, the antibody is a deimmunised antibody in which the T and
B cell
epitopes have been eliminated. They have reduced immunogenicity when applied
in vivo.
TGF-beta inhibitor
The expansion medium comprises a TGF-beta inhibitor. The presence of a TGF-
beta
inhibitor in the expansion media is advantageous because it increases human
organoid
formation efficiency. TGF-beta signalling is involved in many cellular
functions, including

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cell growth, cell fate and apoptosis. Signalling typically begins with binding
of a TGF-beta
superfamily ligand to a type II receptor which recruits and phosphorylates a
type I receptor.
The type I receptor then phosphorylates SMADs, which act as transcription
factors in the
nucleus and regulate target gene expression.
The TGF-beta superfamily ligands comprise bone morphogenic proteins (BMPs),
growth
and differentiation factors (GDFs), anti-mullerian hormone (AMH), activin,
nodal and TGF-
betas. In general, Smad2 and Smad3 are phosphorylated by the ALK4, 5 and 7
receptors
in the TGF-beta/activin pathway. By contrast, Smad1, Smad5 and Smad8 are
phosphorylated as part of the bone morphogenetic protein (BMP) pathway.
Although there
is some cross-over between pathways, in the context of this invention, a "TGF-
beta
inhibitor" or an "inhibitor of TGF-beta signalling" is preferably an inhibitor
of the TGF-beta
pathway which acts via Smad2 and Smad3. Therefore, in some embodiments the TGF-

beta inhibitor is not a BMP inhibitor, i.e. the TGF-beta inhibitor is not
Noggin. In some
embodiments, a BMP inhibitor is added to the culture medium in addition to the
TGF-beta
inhibitor (see below).
Thus the TGF-beta inhibitor may be any agent that reduces the activity of the
TGF-beta
signalling pathway, preferably the signalling pathway that acts via Smad2
and/or Smad3,
more preferably the signalling pathway that acts via ALK4, ALK5 or ALK7. There
are many
ways of disrupting the TGF-beta signaling pathway that are known in the art
and that can
be used in conjunction with this invention. For example, the TGF-beta
signaling may be
disrupted by: inhibition of TGF-beta expression by a small-interfering RNA
strategy;
inhibition of furin (a TGF-beta activating protease); inhibition of the
pathway by
physiological inhibitors; neutralisation of TGF-beta with a monoclonal
antibody; inhibition
with small-molecule inhibitors of TGF-beta receptor kinase 1 (also known as
activin
receptor-like kinase, ALK5), ALK4, ALK6, ALK7 or other TGF-beta-related
receptor
kinases; inhibition of Smad 2 and Smad 3 signaling e.g. by overexpression of
their
physiological inhibitor, Smad 7, or by using thioredoxin as an Smad anchor
disabling Smad
from activation (Fuchs, 0. Inhibition of TGF- Signaling for the Treatment of
Tumor
Metastasis and Fibrotic Diseases. Current Signal Transduction Therapy, Volume
6, Number 1, January 2011, pp. 29-43(15)).
Various methods for determining if a substance is a TGF-beta inhibitor are
known and
might be used in conjunction with the invention. For example, a cellular assay
may be
used in which cells are stably transfected with a reporter construct
comprising the human
PAI-1 promoter or Smad binding sites, driving a luciferase reporter gene.
Inhibition of
luciferase activity relative to control groups can be used as a measure of
compound
activity (De Gouville et al., Br J Pharmacol. 2005 May; 145(2): 166-177).

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A TGF-beta inhibitor according to the present invention may be a protein,
peptide, small-
molecules, small-interfering RNA, antisense oligonucleotide, aptamer or
antibody. The
inhibitor may be naturally occurring or synthetic. In one embodiment, the TGF-
beta
inhibitor is an inhibitor of ALK4, ALK5 and/or ALK7. For example, the TGF-beta
inhibitor
may bind to and directly inhibit ALK4, ALK5 and/or ALK7. Examples of preferred
small-
molecule TGF-beta inhibitors that can be used in the context of this invention
include the
small molecule inhibitors listed in table 1:
Table 1: Small-molecule TGF-beta inhibitors targeting receptor kinases
IC50
Inhibitor Targets (nIkl) Mol t Name Formula
A83-01 ALK5 12 421.52 3 -(6-M ethy1-2-
C25H19N5S
(TGF-13R1) pyridiny1)-N-
phenyl-4-
ALK4 45
(4-quinoliny1)-1H-
pyrazo le-1 -
ALK7 7.5
carb othio amide
SB-431542 ALK5 94 384.39 4- [4-(1,3-benzodioxol-
C22H16N403
ALK4
5-y1)-5 -(2-pyridiny1)-
1H-imidazol-2-
ALK7
yl]benzamide
SB-505124 ALK5 47 335.4 2-(5-benzo [1,3] dioxol-
C20H21N3 02
ALK4 129
5-y1-2-tert-butyl-
3Himidazol-
4-y1)-6-methylpyridine
hydrochloride hydrate
SB-525334 ALK5 14.3 343.42 6-[2-(1,1- C21H21N5
Dimethylethyl)-5-(6-
methy1-2-pyridiny1)-
1H-imidazol-4-
yl]quinoxaline
SD-208 ALK5 49 352.75 2-(5-Chloro-2- C17H 1
OC1FN6
fluoropheny1)-4- [(4-
pyridyl) amino] pteridine

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LY-36494 TGR-13R1 59 272.31 4- [3 -(2-Pyridiny1)-1H-
C17H12N4
TGF-PRII 400 pyrazol-4-yl] -quino line
MLK-7K 1400
SJN-2511 ALK5 23 287.32 2-(3-(6-
C17H13N5
Methylpyridine-2-y1)-
1H-pyrazol-4-y1)-1,5-
naphthyridine
In some embodiments, the TGF-beta inhibitor is a small molecule inhibitor
optionally
selected from the group consisting of: A83-01, SB-431542, SB-505124, SB-
525334, LY
364947, SD-208 and SJN 2511.
In some embodiments, no more than one TGF beta inhibitor is present in the
expansion
medium. In other embodiments, more than one TGF beta inhibitor is present in
the
expansion medium, e.g. 2, 3, 4 or more. In some embodiments, an expansion
medium of
the invention comprises one or more of any of the inhibitors listed in table
1. An expansion
medium may comprise any combination of one inhibitor with another inhibitor
listed. For
example, an expansion medium may comprise SB-525334 or SD-208 or A83-01; or SD-

208 and A83-01. The skilled person will appreciate that a number of other
small-molecule
inhibitors exist that are primarily designed to target other kinases, but at
high
concentrations may also inhibit TGF-beta receptor kinases. For example, SB-
203580 is a
p38 MAP kinase inhibitor that, at high concentrations (for example,
approximate 10 pM or
more) is thought to inhibit ALK5. Any such inhibitor that inhibits the TGF-
beta signalling
pathway can also be used in the context of this invention.
In some embodiments, the TGF beta inhibitor is present at at least 5 nM, for
example, at
least 50nM, at least 100nM, at least 300nM, at least 450nM, at least 475nM,
for example
5nM-500mM, 10nM-100mM, 50nM-700uM, 50nM-10uM, 100nM-1000nM, 350-650nM or
more preferably about 500nM.
A83-01 may be added to the expansion medium at a concentration of between 10
nM and
10 uM, or between 1 uM and 8 uM, or between 4 uM and 6 uM. For example, A83-01
may
be added to the expansion medium at about 5 uM. The skilled person would know
how to
determine the concentration of other TGF beta inhibitors for use in the
invention.
Prostaglandin pathway activator
In some embodiments, the expansion medium is supplemented with an activator of
the
prostaglandin signalling pathway (also called a prostaglandin pathway
activator). For

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example, the expansion medium may be supplemented with any one or more of the
compounds selected from the list comprising: Phospholipids, Arachidonic acid
(AA),
prostaglandin E2 (PGE2), prostaglandin G2 (PGG2), prostaglandin F2 (PGF2),
prostaglandin H2 (PGH2), prostaglandin D2 (PGD2). In some embodiments, the
expansion
5 medium is supplemented with PGE2 and/or AA. In some embodiments, PGE2 is
added to
the expansion medium to a final concentration of at least 10 nM, at least
30nM, at least
40nM, at least 45 nM, at least 50nM, for example between 10 nM and 500 nM,
between 10
nM and 400 nM, between 10 nM and 300 nM, between 10 nM and 200 nM, between 10
nM and 100 nM, between 20 nM and 50 nM. In a preferred embodiment in which
PGE2 is
10 present, PGE2 is added to the expansion medium to a final concentration
of 50 nM. In
some embodiments, AA is added to the expansion medium to a final concentration
of at
least 1 ug/ml, for example at least 3 ug/ml, at least 5 ug/ml, at least 8
ug/ml, at least 9
ug/ml, at least 10 ug/ml, between 1 ug/ml and 1000 ug/ml, between 1 ug/ ml and
500
ug/ml, between 1 ug/ml and 100 ug/ml, between 1 ug/ml and 50 ug/ml, or between
5 ug/ml
15 and 10 ug/ml. In a preferred embodiment in which AA is present, AA is
added to the
medium to a final concentration of 10 ug/ml.
In some embodiments, PGE2 and/or AA are absent from the expansion medium of
the
invention. In some embodiments, a prostaglandin pathway activator is absent
from the
expansion medium of the invention. The inventors have observed that, at least
for liver
20 cells, the presence of prostaglandin pathway activators in the expansion
medium may
change the phenotype of the cells being expanded and can result in cells that
do not
differentiate and which have lost some stem cell markers and ductal markers.
BMP Inhibitors
In some embodiments, the expansion medium may comprise any suitable BMP
inhibitor.
BMPs bind as a dimeric ligand to a receptor complex consisting of two
different receptor
serine/threonine kinases, type I and type II receptors. The type ll receptor
phosphorylates
the type I receptor, resulting in the activation of this receptor kinase. The
type I receptor
subsequently phosphorylates specific receptor substrates (SMAD), resulting in
a signal
transduction pathway leading to transcriptional activity.
A BMP inhibitor is defined as an agent that binds to a BMP molecule to form a
complex
wherein the BMP activity is neutralized, for example by preventing or
inhibiting the binding
of the BMP molecule to a BMP receptor. Alternatively, said inhibitor is an
agent that acts
as an antagonist or reverse agonist. This type of inhibitor binds with a BMP
receptor and
prevents binding of a BMP to said receptor. An example of a latter agent is an
antibody
that binds a BMP receptor and prevents binding of BMP to the antibody-bound
receptor.

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A BMP inhibitor may be added to the media in an amount effective to inhibit a
BMP-
dependent activity in a cell to at most 90%, more preferred at most 80%, more
preferred at
most 70%, more preferred at most 50%, more preferred at most 30%, more
preferred at
most 10%, more preferred 0%, relative to a level of a BMP activity in the
absence of said
inhibitor, as assessed in the same cell type. As is known to a skilled person,
a BMP activity
can be determined by measuring the transcriptional activity of BMP, for
example as
exemplified in Zilberberg et al., 2007. BMC Cell Biol. 8:41.
Several classes of natural BMP-binding proteins are known, including Noggin
(Peprotech),
Chordin and chordin-like proteins (R&D systems) comprising chordin domains,
Follistatin
and follistatin-related proteins (R&D systems) comprising a follistatin
domain, DAN and
DAN-like proteins (R&D systems) comprising a DAN cysteine-knot domain,
sclerostin
/SOST (R&D systems), decorin (R&D systems), and alpha-2 macroglobulin (R&D
systems).
Examples of BMP inhibitors for use in a method of the invention are Noggin,
DAN, and
DAN-like proteins including Cerberus and Gremlin (R&D systems). These
diffusible
proteins are able to bind a BMP ligand with varying degrees of affinity and
inhibit their
access to signalling receptors. The addition of any of these BMP inhibitors to
the basal
culture medium prevents the loss of stem cells.
A preferred BMP inhibitor is Noggin. In the context of a culture medium of the
invention,
Noggin is also referred to herein as "N". Noggin is preferably added to the
basal culture
medium at a concentration of at least 10 ng/ml, for example, at least 20
ng/ml, more
preferred at least 25 ng/ml. A still more preferred concentration is about 25
ng/ml.
During culturing of stem cells, said BMP inhibitor may be added to the culture
medium
when required, for example, daily or every other day. The BMP inhibitor is
preferably
added to the culture medium every second day. The culture medium may be
refreshed
when required, for example, daily or every other day.
The BMP inhibitor and BMP pathway activator are not normally included in the
expansion
medium at the same time. In a preferred embodiment, the BMP inhibitor is only
included in
the first few days (e.g. first 2 days) of culture, preferably when Wnt, e.g.
in the form of Wnt-
conditioned medium is present. After the first few days (e.g. first 2 days) of
culture in this
"first" expansion medium (EM1), the medium is changed to a "second" expansion
medium
(EM2) which comprises a BMP pathway activator but does not comprise a BMP
inhibitor.
Thus, in some embodiments the expansion medium (e.g. EM2) does not comprise a
BMP
inhibitor. In some embodiments, the expansion medium (e.g. EM1) does not
comprise a
BMP pathway activator. In some embodiments, the expansion medium (e.g. EM2)
does

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not comprise Wnt. In some embodiments, the expansion medium comprises either a
BMP
inhibitor or a BMP pathway activator.
Additional components
The expansion medium optionally comprises Nicotinamide and is preferably
supplemented
with one or more (e.g. 1, 2, 3 or all) of the compounds selected from the
group consisting
of gastrin, B27, N-acetylcystein and N2. Thus in some embodiments the
expansion
medium described above further comprises one or more components selected from
the
group consisting of: a further Wnt agonist, a BMP inhibitor, nicotinamide,
gastric, B27, N2
and N-Acetylcysteine. In some embodiments, the expansion medium described
above
further comprises one or more components selected from the group consisting
of:
nicotinamide, gastric, B27, N2 and N-Acetylcysteine.
In some embodiments, the expansion medium comprises gastrin.
B27 (Invitrogen), N-Acetylcysteine (Sigma) and N2 (Invitrogen), Gastrin
(Sigma) and
Nicotinamide (Sigma) are believed to control proliferation of the cells and
assist with DNA
stability. In the context of the invention, Nicotinamide is also referred to
herein as "Nic".
In some embodiments, Nicotinamide is present at 7-15mM, for example about
10mM.
In some embodiments, the B27 supplement is '1327 Supplement minus Vitamin A'
(available from Invitrogen, Carlsbad, CA; www.invitrogen.com; currently
catalog no.
12587010; and from PAA Laboratories GmbH, Pasching, Austria; www.paa.com;
catalog
no. F01-002; Brewer et al., J Neurosci Res., 35(5):567-76, 1993) may be used
to formulate
a culture medium that comprises biotin, cholesterol, linoleic acid, linolenic
acid,
progesterone, putrescine, retinyl acetate, sodium selenite, tri-iodothyronine
(T3), DL-alpha
tocopherol (vitamin E), albumin, insulin and transferrin. The B27 supplement
minus
vitamin A was shown to work particularly well in the expansion medium for the
liver. The
B27 Supplement supplied by PAA Laboratories GmbH comes as a liquid 50x
concentrate,
containing amongst other ingredients biotin, cholesterol, linoleic acid,
linolenic acid,
progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-
iodothyronine (T3),
DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin. Of these
ingredients at
least linolenic acid, retinol, retinyl acetate and tri-iodothyronine (T3) are
nuclear hormone
receptor agonists. B27 Supplement may be added to a culture medium as a
concentrate or
diluted before addition to a culture medium. It may be used at a lx final
concentration or at
other final concentrations. Use of B27 Supplement is a convenient way to
incorporate
biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine,
retinol, retinyl
acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol (vitamin
E), albumin,
insulin and transferrin into a culture medium of the invention. It is also
envisaged that

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23
some or all of these components may be added separately to the expansion
medium
instead of using the B27 Supplement. Thus, the expansion medium may comprise
some
or all of these components.
In some embodiments, retinoic acid is absent from the B27 Supplement used in
the
expansion medium, and/or is absent from the expansion medium.
'N2 Supplement' is available from lnvitrogen, Carlsbad, CA;
www.invitrogen.com; catalog
no. 17502-048; and from PAA Laboratories GmbH, Pasching, Austria; www.paa.com;

catalog no. F005-004; Bottenstein & Sato, PNAS, 76(1):514-517, 1979. The N2
Supplement supplied by PAA Laboratories GmbH comes as a 100x liquid
concentrate,
containing 500 g/m1 human transferrin, 500 g/m1 bovine insulin, 0.63 g/m1
progesterone,
1611 g/m1 putrescine, and 0.52 g/m1 sodium selenite. N2 Supplement may be
added to a
culture medium as a concentrate or diluted before addition to a culture
medium. It may be
used at a lx final concentration or at other final concentrations. Use of N2
Supplement is a
convenient way to incorporate transferrin, insulin, progesterone, putrescine
and sodium
selenite into a culture medium of the invention. It is of course also
envisaged that some or
all of these components may be added separately to the expansion medium
instead of
using the N2 Supplement. Thus, the expansion medium may comprise some or all
of
these components.
In some embodiments in which the medium comprises B27, it does not also
comprise N2.
The embodiments of the present invention can therefore be adapted to exclude
N2 when
B27 is present, if desired.
In some embodiments N2 is not present in the expansion medium.
In some embodiments in which the medium comprises N2, it does not also
comprise B27.
The embodiments of the present invention can therefore be adapted to exclude
B27 when
N2 is present, if desired.
In some embodiments B27 is not present in the expansion medium.
In some embodiments the expansion medium is supplemented with B27 and/or N2.
In some embodiments the basal medium is supplemented with 15Ong/m1 to 250
ng/ml N-
Acetylcysteine; preferably, the basal medium is supplemented with about
200ng/m1 N-
Acetylcysteine.
In some embodiments, the expansion medium further comprises one or more (e.g.
1, 2, 3,
4, 5 or all 6) of the following components: jagged 1, thiazovivin, a p38
inhibitor (e.g.
5B202190), 5B431542, IGF and valproic acid.

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In some embodiments the basal medium may be supplemented with 4Ong/m1 to
6Ong/m1
EGF; preferably, the basal medium is supplemented with about or exactly
5Ong/m1 EGF.
For example, in some embodiments the basal medium may be supplemented with 0.5

pg/ml to 1.5 pg/ml Rspondin1; preferably, the basal medium is supplemented
with about 1
pg/ml Rspondin1. For example, in some embodiments the basal medium may be
supplemented with 5nM to 15nM gastrin; preferably, the basal medium is
supplemented
with about 10nM gastrin. For example, in some embodiments the basal medium may
be
supplemented with 25-200ng/m1 FGF10, for example 70 ng/ml to 130 ng/ml FGF10;
preferably, the basal medium is supplemented with about 100 ng/ml FGF10. For
example,
in some embodiments the basal medium may be supplemented with 5mM to 15mM
Nicotinamide; preferably, the basal medium is supplemented with about 10mM
Nicotinamide. For example, in some embodiments the basal medium may be
supplemented with 25ng/m1 to 100 ng/ml HGF, for example 35ng/m1 to 65ng/m1
HGF;
preferably, the basal medium is supplemented with about 5Ong/m1 HGF. For
example, in
some embodiments the basal medium may be supplemented with 35% to 65% Wnt-
conditioned media; preferably, the basal medium is supplemented with about 50%
Wnt-
conditioned media.
Expansion medium 1 (EMI), Expansion medium 2 (EM2) and differentiation medium
(DM)
In some embodiments, particularly advantageously for liver cells, the cells
may first be
expanded in an expansion medium as described above and further comprising a
BMP
inhibitor, such as Noggin. This is also referred to as the expansion medium 1
(EM1). The
cells can then be expanded in a second expansion medium of the invention, as
described
above that does not contain Wnt or a BMP inhibitor (e.g. Noggin). This second
expansion
medium is sometimes referred to herein as expansion medium 2 (EM2). In some
embodiments, the cells are cultured in EM2 for approximately 1 day, 2 days, 3
days, 4
days, 5 days, 6 days, 7 days or a longer time period, such as 3, 4, 5, 10, 20
or more
weeks. As discussed above, the presence of the cAMP pathway activator and/or
the BMP
activator in the expansion medium, particularly in EM2, advantageously allows
human cells
to be passaged for a longer time than was possible previously. Thus, the
method may
comprise culturing the cells in an EM2 of the invention for 6 or more
passages, for
example, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 15 or more
or 18 or
more passages. This was not possible prior to the present invention.
If required, the expansion medium may then be changed to a differentiation
medium as
described herein, such as an optimised differentiation medium, e.g. that
contains a TGF-
beta inhibitor and a Notch inhibitor. In some embodiments the differentiation
medium
comprises a basal medium for animal or human cells to which is added one or
more

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receptor tyrosine kinase ligands and a Notch inhibitor. In some embodiments,
the
differentiation medium further comprises one or more of: a TGF-beta inhibitor,
gastrin and
dexamethasone. Typically, the differentiation medium does not contain a Wnt
agonist,
Rspondin or Nicotinamide. In some embodiments, the differentiation medium does
not
5 contain a prostaglandin pathway activator, such as PGE2 or AA. The
differentiation
medium encourages the differentiation of the cells, e.g. for liver cells,
towards mature
hepatocytes and cholangiocytes. These differentiated cells are preferably
suitable for
transplantation into humans or animals. See further comments on
differentiation methods
and media below.
10 Throughout this disclosure, statements referring to "culture medium" may
apply to the
"expansion medium" and/or "differentiation medium".
In some embodiments, retinoic acid is absent from the expansion medium. In
some
embodiments, one or more (e.g. 2, 3, 4, or all 5) of retinoic acid, FGF2,
DAGKi, human
sRANK ligand, and Zardaverine is absent from the expansion medium. In some
15 embodiments, a Notch inhibitor is absent from the expansion medium.
The invention further provides the use of an expansion medium of the invention
for
culturing cells for at least 6 passages, for example, at least 7, at least 8,
at least 9, at least
10, at least 11, at least 12, at least 13, at least 14, at least 15, at least
16, at least 17, at
least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at
least 50, at least 60
20 passages or for between 6-40 passages, for example about 8-35 passages,
10-30
passages, or 12-25 passages. In practice, some embodiments of the invention
comprise
the use of expansion medium for around 8-50, for example, 10-50, 15-50, 20-50,
20-40
passages of the cells. For example, in some embodiments the cells may be split
at a 4-6
or 4-8 dilution every 7-10 days for 2, 3, 4, 5 or 6 or more months. Preferably
the cells will
25 expand at a rate of more than two or more than three population
doublings a week, for
example, about 4-5 fold expansion per week. Preferably the cells are human
cells. As
mentioned above, human cells could be cultured for only around 5 passages
using the
expansion medium described in W02012/168930 and W02012/014076 could be used
for
long term culture of mouse cells. In contrast, use of a culture medium
comprising a cAMP
pathway activator allows human cells to be passaged many more times, for
example, 6 or
more, e.g. 8, 10, 12 or more, more preferably 16 or more (see Figure 2).
As explained above, long-term culture of human cells was not possible prior to
the present
invention. Using the previously available methods, it was not possible to
expand human
liver cells in culture for more than 2 months. However, using the present
invention, it is
possible to culture liver cells for more than 2 months, for example at least
10 weeks, at

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26
least 3 months, etc. However, it is not always necessary to culture the cells
for a long time
and so use of expansion medium for shorter periods of time is also envisaged.
Accordingly, in some embodiments, the invention provides the use of an
expansion
medium of the invention for culturing cells for at least 2 weeks, at least 1
month, at least 2
months, more preferably at least 3, at least 4, at least 5, at least 6, at
least 7, at least 8, at
least 9, at least 10, at least 15, at least 20, at least 24, at least 25, at
least 30 or more
months, for example 3 or more years. Preferably, the cells are human cells.
Preferably the epithelial stem cells are human epithelial stem cells. However,
culturing
non-human mammalian epithelial stem cells is also envisaged.
In some embodiments, the epithelial stem cells are selected from liver,
pancreas, intestine
(e.g. small intestine or colon), stomach, prostate, lung, breast, ovarian,
salivary gland, hair
follicle, skin, oesophagus and thyroid epithelial stem cells. In a preferred
embodiment, the
epithelial stem cell is a liver cell. In a further preferred embodiment, the
epithelial stem cell
is a pancreas cell. In some embodiments, the epithelial stem cell is a liver
or a pancreas
stem cell. In some embodiments, the epithelial stem cell is not a colon cell
or is not a small
intestine cell or is not an intestine cell.
Extracellular matrix
As described above, the method for culturing epithelial stem cells comprises
culturing one
or more epithelial stem cells in contact with an extracellular matrix. Any
suitable
extracellular matrix may be used. Isolated epithelial stem cells are
preferably cultured in a
microenvironment that mimics at least in part a cellular niche in which said
stem cells
naturally reside. This cellular niche may be mimicked by culturing said stem
cells in the
presence of biomaterials, such as an extracellular matrix that provides key
regulatory
signals controlling stem cell fate.
A cellular niche is in part determined by the stem cells and surrounding
cells, and the
extracellular matrix (ECM) that is produced by the cells in said niche. In a
preferred
method of the invention, epithelial stem cells are cultured in contact with an
ECM. "In
contact" means a physical or mechanical or chemical contact, which means that
for
separating said resulting organoid or population of epithelial stem cells from
said
extracellular matrix a force needs to be used. Preferably, the epithelial stem
cells are
embedded in the ECM.
A culture medium of the invention may be diffused into an extracellular matrix
(ECM). In a
preferred method of the invention, isolated tissue fragments or isolated
epithelial stem cells
are attached to an ECM. ECM is composed of a variety of polysaccharides,
water, elastin,
and glycoproteins, wherein the glycoproteins comprise collagen, entactin
(nidogen),

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27
fibronectin, and laminin. ECM is secreted by connective tissue cells.
Different types of
ECM are known, comprising different compositions including different types of
glycoproteins and/or different combination of glycoproteins. Said ECM can be
provided by
culturing ECM-producing cells, such as for example fibroblast cells, in a
receptacle, prior to
the removal of these cells and the addition of isolated tissue fragments or
isolated
epithelial stem cells. Examples of extracellular matrix-producing cells are
chondrocytes,
producing mainly collagen and proteoglycans, fibroblast cells, producing
mainly type IV
collagen, laminin, interstitial procollagens, and fibronectin, and colonic
myofibroblasts
producing mainly collagens (type I, Ill, and V), chondroitin sulfate
proteoglycan, hyaluronic
acid, fibronectin, and tenascin-C. Alternatively, said ECM is commercially
provided.
Examples of commercially available extracellular matrices are extracellular
matrix proteins
(Invitrogen) and basem*nt membrane preparations from Engelbreth-Holm-Swarm
(EHS)
mouse sarcoma cells (e.g. MatrigelTM (BD Biosciences)). A further example is
Reduced
Growth Factor BME 2 (Basem*nt Membrane Extract, Type 2, Pathclear). A
synthetic
extracellular matrix material, such as ProNectin (Sigma Z378666) may be used.
Mixtures
of extracellular matrix materials may be used, if desired. The use of an ECM
for culturing
stem cells enhanced long-term survival of the stem cells and the continued
presence of
undifferentiated stem cells. In the absence of an ECM, stem cell cultures
could not be
cultured for longer periods and no continued presence of undifferentiated stem
cells was
observed. In addition, the presence of an ECM allowed culturing of three-
dimensional
tissue organoids, which could not be cultured in the absence of an ECM. The
extracellular
matrix material will normally be a drop on the bottom of the dish in which
cells are
suspended. Typically, when the matrix solidifies at 37 C, the medium is added
and diffuses
into the ECM. The cells in the medium stick to the ECM by interaction with its
surface
structure, for example interaction with integrins. A fibronectin solution of
about 1mg/m1
(stock solution) used at approximately 1 ,g/cm2 may be used to coat a cell
culture vessel,
or between about 1 ,g/cm2 to about 250 mg/cm2, or at about 1 ,g/cm2 to about
150 ,g/cm2.
In some embodiments, a cell culture vessel is coated with fibronectin at
between 8 ,g/cm2
and 125 ,g/cm2.
An example of an ECM for use in a method of the invention comprises at least
one
glycoprotein, such as laminin.
A preferred ECM for use in a method of the invention comprises at least two
distinct
glycoproteins, such as two different types of collagen or a collagen and
laminin. The ECM
can be a synthetic hydrogel extracellular matrix or a naturally occurring ECM.
A further
preferred ECM comprises laminin, entactin, and collagen IV. A further
preferred ECM is
provided by MatrigelTM (BD Biosciences), which comprises laminin, entactin,
and collagen

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28
IV. In some embodiments the extracellular matrix is a laminin-containing
extracellular
matrix such as MatrigelTM (BD Biosciences).
In some embodiments, the single stem cell, population of cells, or tissue
fragment is
embedded in matrigel, which is optionally growth factor reduced and/or phenol
red-free.
In some embodiments, the culture medium is placed on top of the ECM. The
culture
medium can then be removed and replenished as and when required. In some
embodiments, the culture medium is replenished every 1, 2, 3, 4, 5, 6 or 7
days. If
components are "added" or "removed" from the media, then this can in some
embodiments
mean that the media itself is removed from the ECM and then a new media
containing the
"added" component or with the "removed" component excluded is placed on the
ECM.
In some embodiments the culture medium of the invention is in contact with an
extracellular matrix or a 3D matrix that mimics the extracellular matrix by
its interaction with
the cellular membrane proteins, such as integrins.
There is further provided an expansion medium of the invention and an
extracellular
matrix, e.g. supplied as a kit.
The method may advantageously comprise passaging the organoids obtained using
the
culture method. In some embodiments, one week to 10 day old organoids are
removed
from the extracellular matrix and mechanically dissociated into small
fragments before
being transferred to fresh extracellular matrix. The skilled person would know
how to split
the organoids in order to passage them so that they can multiply without
exceeding the
concentration limit for their container. In some embodiments, passaging is
performed for
at least 2 months, for example for 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20,
24 or more
months. In some embodiments, passaging is performed in 1:4 to 1:8 split ratios
once per
week for at least 2 months, for example for 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,
15, 18, 20, 24 or
more months. In some embodiments, the method comprises passaging the organoids
more than 6 times, e.g. more than 7, 8, 9, 10, 12, 15, 18, 20, 25, 30 times.
The passaging
interval can be adapted as necessary. Suitable examples are twice per week,
once per
week, once every 10 days, once every two weeks, for example once every 7-10
days. The
split of the culture medium can be adapted as necessary. Suitable examples are
1:3-1:10
dilutions, e.g. 1:3-1:9, 1:4-1:8, 1:4-1:6 dilutions. Prior to the present
invention, it was not
possible to passage human liver organoids this many times or to keep them in
culture
long-term. In particular, prior to the present invention, it was possible to
passage human
liver cells only 5 times, when passage at a rate of 1 passage per week, when
using the
medium described in W02012/014076 which comprises a TGF-beta inhibitor.

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In some embodiments, the method comprises culturing the organoid or a
population of
epithelial stem cells for at least 6, 8, 10, 12, 14, 16, 18, 20 or 25 weeks.
The expansion medium preferably induces or promotes the survival and/or
proliferation of
cells during at least 42, 50, 75, 100, 125, 150, 175, 200, 250, 300, 365 days
of culture.
Proliferation can be assessed using techniques known in the art such as BrdU
staining,
Edu staining, Ki67 staining and the use of growth curves assay can be done.
For example,
from 10 biliary ducts, it is possible after 6 days to dilute to 6 wells with
10 organoids per
well (60 new organoids). In each passage of 6 days we can generate around 360
organoids. By performing 1 passage/week over 32 weeks we are able to generate
over
11,000 (11520) new organoid structures in only 7 months. This is important for
the
industry, since the availability of cells and organoids for transplantation
poses a significant
problem. For a mouse transplant, for example, a minimum of 105 cells are
required.
Possibly 106, or 10 x 106 might be required for a human transplant, in order
for a graft to be
successful.
Put another way, media used according to the invention are capable of
expanding a
population of stem cells to form organoids for at least 6, at least 10, at
least 20, at least 30,
at least 40, at least 50, at least 60, at least 70, at least 80, at least 90
or at least 100,
passages under appropriate conditions.
Isolation of epithelial stem cells for culture
In a preferred embodiment, the epithelial stem cells to be cultured in the
expansion method
and/or from which the organoids are derived are obtained from adult tissue,
i.e. the
epithelial stem cells are adult epithelial stem cells. In this context "adult"
means mature
tissue, i.e. includes newly-born baby or child but excludes embryonic or
foetal. In a
preferred embodiment the epithelial stem cells are not derived from embryonic
stem cells
or embryonic stem cell lines, e.g. which have been differentiated in vitro.
Cells taken directly from live tissue, i.e. freshly isolated cells, are also
referred to as
primary cells. In some embodiments the epithelial stem cells are primary
epithelial stem
cells. Primary cells represent the best experimental models for in vivo
situations. In a
preferred embodiment of the invention, the epithelial stem cells are (or are
derived from)
primary epithelial stem cells. Primary cell cultures in can be passaged to
form secondary
cell cultures. With the exception cancer cells, traditional secondary cell
cultures have
limited lifespan. After a certain number of population doublings (e.g. 50-100
generations)
cells undergo the process of senescence and stop dividing. Cells from
secondary cultures
can become immortalized to become continuous cell lines. Immortalization can
occur
spontaneously, or may be virally- or chemically- induced. Immortalized cell
lines are also

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known as transformed cells. By contrast, the methods of the present invention
allow
continuous passaging of epithelial stem cells without immortalisation or
transformation.
Thus in some embodiments, the epithelial stem cells are not immortalised or
transformed
cells or are not derived from an immortalised cell line or a transformed cell
line. An
5 advantage of the present invention is that the epithelial stem cells,
undergoing multiple
rounds of expansion and passaging, retain the characteristics of primary cells
and have
minimal or no genotypic or phenotypic changes.
The epithelial stem cells may be obtained by any suitable method. In some
embodiments,
cells are isolated by collagenase digestion, for example, as described in the
examples and
10 in DoreII et al., 2008 (Hepatology. 2008 Oct;48(4):1282-91. Surface
markers for the murine
oval cell response. Dorrell C, Erker L, Lanxon-Cookson KM, Abraham SL,
Victoroff T, Ro
S, Canaday PS, Streeter PR, Grompe M). In some embodiments, collagenase
digestion is
performed on a tissue biopsy. In some embodiments, collagenase and accutase
digestion
are used to obtain the epithelial stem cells for use in the invention.
15 In some embodiments, the method comprises culturing a fragment of tissue
which
comprises epithelium. In some embodiments, the epithelial stem cells are
isolated from a
tissue fragment. For example, in the context of liver, the tissue fragment may
comprise a
liver biliary duct or biliary duct tissue. In the context of the intestine,
the tissue fragment
may comprise a crypt or part of a crypt.
20 Stem cells are not necessarily always present in the liver but can
appear in response to
injury or stimulation of Lgr, e.g. by Rspondin, for example in the context of
the methods or
media of the present invention. Liver epithelial stem cells can be derived
from ductal cells,
and so, as mentioned above, in some embodiments, the method of the invention
comprises culturing a liver biliary duct or biliary duct tissue, or comprises
culturing cells
25 isolated from the liver biliary duct or from biliary duct tissue. In
addition, crude hepatocyte
preparations (not differentially sorted), can be cultured to form organoids.
Such
preparations comprise both hepatocytes and residual ductal cells.
Alternatively, ductal
cells can be sorted using the EpCAM+ marker, and these cells allow organoids
to be
obtained with high efficiency (see Example 14). Therefore, in some
embodiments, the
30 method of the invention comprises culturing ductal cells or crude
hepatocyte preparations,
preferably comprising ductal cells. In some embodiments, the epithelial stem
cells cultured
in the context of the invention are not hepatocytes and/or are not derived
from
hepatocytes. In some embodiments, the invention provides the use of isolated
ductal cells
for generating organoids.

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A preferred method for obtaining the epithelial stem cells for culturing is
based on the fact
that epithelial stem cells according to the invention express Lgr5 and/or Lgr6
on their
surface; these proteins belong to the large G protein-coupled receptor (GPCR)
superfamily
(see, for example, WO 2009/022907, the contents of which are incorporated
herein in their
entirety). The Lgr subfamily is unique in carrying a large leucine-rich
ectodomain important
for ligand binding. A preferred method therefore comprises preparing a cell
suspension
from said epithelial tissue as described above, contacting said cell
suspension with an
Lgr5 and/or 6 binding compound (such as an antibody, e.g. an anti-Lgr5
monoclonal
antibody, e.g. as described in WO 2009/022907), isolating the Lgr5 and/or 6
binding
compound, and isolating the stem cells from said binding compound.
An organoid is preferably obtained using a cell from an adult tissue,
preferably an epithelial
stem cell from an adult tissue, more preferably an epithelial stem cell from
an adult tissue
expressing Lgr5.
A liver organoid may also be obtained from any cell which upon damage or
culturing
expresses Lgr5 and is therefore an Lgr5-expressing stem cell. Consequently, it
is not
necessary for the liver epithelial cells to express Lgr5 before they are
contacted with the
expansion medium. Culturing the liver epithelial cells in the expansion medium
of the
invention leads to Lgr5 expression in the stem cells.
In some embodiments the epithelial stem cells are normal cells. In alternative
embodiments, the epithelial stem cells are cancer stem cells. Thus, it is
envisaged that the
stem cells may be Lgr5 positive cancer stem cells. Accordingly, the cells may
be obtained
from a tumour, if required.
In another preferred embodiment, an organoid originates from a single cell,
preferably
expressing Lgr5. In some embodiments the single cell comprises a nucleic acid
construct
comprising a nucleic acid molecule of interest.
In some embodiments, the starting cell to be cultured is a single cell. A
single cell
suspension comprising the epithelial stem cells can be mechanically generated,
e.g. for
the liver from the isolated biliary duct. Small tissue fragments generated in
this way by
mechanical disruption are preferably split at a ratio of about 1:6. If
necessary, such
fragments can be incubated for a short time (only 2 or 3 minutes) in trypsin
at a dilution of
approximately 1:2. It has been found that at this stage epithelial stem cells
treated with
trypsin yielded rather low survival rates: if the cells are split into
individual cells, then those
with stem cell properties survive. This fraction is rather small
(approximately 3-12% of the
total cell population).

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Preferred Lgr5 and/or 6 binding compounds comprise antibodies, such as
monoclonal
antibodies that specifically recognize and bind to the extracellular domain of
either Lgr5 or
Lgr6, such as monoclonal antibodies including mouse and rat monoclonal
antibodies (see,
for example, WO 2010/016766, the contents of which are incorporated herein in
their
entirety). Using such an antibody, Lgr5 and/or Lgr6-expressing stem cells can
be isolated,
for example with the aid of magnetic beads or through fluorescence-activated
cell sorting,
as is clear to a skilled person. Using a method of the invention, it is
possible to isolate one
single Lgr5 and/or Lgr6 expressing cell and to apply a method of the invention
to it. An
organoid or a population of liver epithelial stem cells may therefore be
derived from one
single cell.
Alternatively, it is also envisaged that a population of cells may be used as
the starting
point, for example, a population of cells contained in a liver fragment as
described above.
Thus, the methods of the invention are not restricted to using single cells as
the starting
point.
In a further aspect, there is provided a method for obtaining an organoid
comprising
culturing epithelial stem cells in an expansion medium using a method as
described
herein.
In some embodiments, the method comprises culturing the epithelial stem cells
or
obtaining the organoid/population of adult epithelial stem cells from a single
cell.
Advantageously, this allows a hom*ogenous population of cells to form. In some
embodiments, the method comprises culturing the stem cells in an expansion
medium of
the invention for a period of time, for example, at least 1 month, at least 6
weeks, at least 2
months, and then dissociating the cells to a single cell density, seeding one
or more cells
at a ratio of 1 cell per container (e.g. per well), and expanding the cells
using an expansion
medium of the invention.
Following culturing, the method may further comprise obtaining and/or
isolating one or
more epithelial stem cells or an organoid. For example, following culture of
the stem cells,
it may be useful to remove one or more stem cells and/or one or more organoids
cultured
in the expansion medium from the culture medium for use in subsequent
applications. For
example, it may be useful to isolate a single cell for culture using the
differentiation
medium of the invention. Alternatively, it may be useful to obtain a
population of cells for
culture using the differentiation medium of the invention.
Any one of a number of physical methods of separation known in the art may be
used to
select the cells of the invention and distinguish these from other cell types.
Such physical
methods may involve FACS and various immuno-affinity methods based upon makers

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33
specifically expressed by the cells of the invention. As described herein,
LGR5 is a cell
marker expressed at high levels in the cells of the invention. Therefore, by
way of
illustration only, the cells of the invention may be isolated by a number of
physical methods
of separation, which rely on the presence of this marker. Similarly, any of
the other
markers expressed by the cells may be used.
In one embodiment, the cells of the invention may be isolated by FACS
utilizing an
antibody, for example, against one of these markers. As will be apparent to
one skilled in
the art, this may be achieved through a fluorescent labeled antibody, or
through a
fluorescent labeled secondary antibody with binding specificity for the
primary antibody.
Examples of suitable fluorescent labels includes, but is not limited to, FITC,
Alexa Fluor
488, GFP, CFSE, CFDA-SE, DyLight 488, PE, PerCP, PE-Alexa Fluor 700, PE-Cy5
(TRI-COLOR ), PE-Cy5.5, P1, PE-Alexa Fluor 750, and PE-Cy7 . This list is
provided by
way of example only, and is not intended to be limiting.
It will be apparent to a person skilled in the art that FACS analysis using an
anti-Lgr5
antibody will provide a purified cell population. However, in some
embodiments, it may be
preferable to purify the cell population further by performing a further round
of FACS
analysis using one or more of the other identifiable markers, for example,
Hnf4A and/or
Sox9, but others may also be used.
In another embodiment, the cells of the invention may be isolated by immuno-
affinity
purification, which is a separation method well known in the art. By way of
illustration only,
the cells of the invention may be isolated by immuno-affinity purification
directed towards
c-kit. As will be apparent to one skilled in the art, this method relies upon
the
immobilisation of antibodies on a purification column. The cell sample is then
loaded onto
the column, allowing the appropriate cells to be bound by the antibodies, and
therefore
bound to the column. Following a washing step, the cells are eluted from the
column using
a competitor which binds preferentially to the immobilised anti-c-kit
antibody, and permits
the cells to be released from the column.
It will be apparent to a person skilled in the art that immuno-affinity
purification using an
immobilised antibody will provide a purified cell population. However, in some
embodiments, it may be preferable to purify the cell population further by
performing a
further round of immuno-affinity purification using one or more of the other
identifiable
markers, for example Hnf4A, and use an aliquot of the isolated clones to
ascertain the
expression of other relevant intracellular markers.
It will be apparent to a person skilled in the art that the sequential
purification steps are not
necessarily required to involve the same physical method of separation.
Therefore, it will

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be clear that, for example, the cells may be purified through a FACS step
using an anti-
Lgr5 antibody, followed by an immuno-affinity purification step using a SSEA-1
affinity
column. In certain embodiments, the cells may be cultured after isolation for
at least about
15, at least about 20 days, at least about 25 days, or at least about 30 days.
In certain
aspects, the cells are expanded in culture longer to improve the hom*ogeneity
of the cell
phenotype in the cell population.
Other features of this method are defined in the part of the description
dedicated to
definitions. Single-cell suspensions or small clusters of cells (2-50
cells/cluster) will
normally be seeded, rather than large clusters of cells, as in known in the
art. As they
divide, such cells will be seeded onto a support at a density that promotes
cell proliferation.
Typically, when single cells are isolated the plating density of at least 1-
500 cells/well is
used, the surface of the well is 0.32 cm2. When clusters are seeded the
plating density is
preferably 250-2500 cells/cm2. For replating, a density of between about 2500
cells/ cm2
and about 5,000 cells/ cm2 may be used in some embodiments. During replating,
single-
cell suspensions or small cluster of cells will normally be seeded, rather
than large clusters
of cells, as in known in the art.
Methods and culture media for cancer cells and cancer organoids
In some embodiments, the epithelial stem cell is a cancer cell or a non-
cancerous tumour
cell e.g. is derived from a tumour. Where the term "cancer" is used herein, it
is to be
understood that it applies equally non-cancerous (benign) tumours. Cancer
cells tend to
have mutations that constitutively activate or deactivate certain growth
pathways and
which mean that certain factors in the culture medium that may normally be
required for
growth, are no longer necessary. For example, many colon cancers result in
constitutive
activation of the Wnt pathway. In such cases, a culture medium would not
require a Wnt
agonist. Other mutations would allow other factors to be left out of the
culture media
described herein. Other epithelial cancers (carcinomas) or non-cancerous
tumours (e.g.
adenomas) can also be grown in culture media of the invention.
In a preferred embodiment, a cancer organoid obtained from cancer stem cells
is grown in
a culture medium that is suitable for growth of the corresponding normal
tissue organoid
obtained from normal stem cells, optionally with certain factors excluded from
the medium.
For example, a stomach cancer organoid obtained by culturing stomach cancer
stem cells
may be grown in the same culture conditions as a normal gastric organoid
obtained by
culturing gastric stem cells, optionally with certain factors excluded from
the medium. In
another example, a pancreatic cancer organoid obtained by culturing pancreatic
cancer
stem cells may be grown in the same culture conditions as a normal pancreatic
organoid

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obtained by culturing pancreatic stem cells, optionally with certain factors
excluded from
the medium. In another example, a prostate cancer organoid obtained by
culturing
prostatic cancer stem cells may be grown in the same culture conditions as a
normal
prostate organoid obtained by culturing prostatic stem cells, optionally with
certain factors
5 excluded from the medium. In another example, a liver cancer organoid
obtained by
culturing liver cancer stem cells may be grown in the same culture conditions
as a normal
liver organoid obtained by culturing liver stem cells, optionally with certain
factors excluded
from the medium. In many situations it may be preferable (or at least more
convenient) to
grow cancer organoids in the normal tissue medium (without any factors
excluded). The
10 normal tissue medium should allow cancers with all genetic backgrounds
to grow, without
excluding any particular cancer mutations.
Therefore, in some embodiments, the invention provides a culture medium for
culturing
cancer cells, for example cancer stem cells, such as adenocarcinoma or
carcinoma cells
from a tissue type of interest, wherein the culture medium comprises or
consists of the
15 components of the culture medium used for culturing the cells from the
corresponding non-
cancerous tissue type of interest, optionally wherein one or more of the
following are
excluded from the medium that is used to culture the non-cancerous cells of
the tissue
type of interest: Forskolin, Wnt-3a, EGF, Noggin, Rspondin, TGF-beta
inhibitor, p38
inhibitor, nicotinamide, gastrin, FGF10 and HGF.
20 Expansion organoids and populations of cells
The invention provides an organoid or a population of epithelial stem cells
obtainable or
obtained by a method of the invention. Thus, in some embodiments, the method
further
comprises obtaining and/or isolating an organoid.
An organoid obtained using the expansion methods of the invention is also
referred to
25 herein as an "expansion organoid". An expansion organoid comprises at
least one Lgr5+
epithelial stem cell, which can divide and produce further Lgr5+ epithelial
stem cells or can
generate differentiated progeny, e.g. progenitor cells. It is to be understood
that in a
preferred expansion organoid, the majority of cells are expanding cells (i.e.
dividing cells)
that retain an undifferentiated phenotype. Although some spontaneous
differentiation may
30 occur, the cell population is generally an expanding population. The
length of time that the
organoids can continue to be expand whilst maintaining a core presence of
Lgr5+ stem
cells and whilst maintaining genotypic and phenotypic integrity of the cells,
is an important
feature of the organoids that distinguishes them from many of the organoids in
the prior
art. The organoids also have a distinctive structure that arises from these
cellular
35 properties, as described in detail below.

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It should be noted that, at any time, the expansion organoids, or cells from
the expansion
organoids can be transferred to a differentiation medium and be allowed to or
induced to
differentiate into all major differentiated cell lineages present in the
corresponding in vivo
tissue
Image analysis may be used to assess characteristics of cells in culture such
as cell
morphology; cell structures; evidence for apoptosis or cell lysis; and
organoid composition
and structure. Many types of imaging analysis are well known in the art, such
as electron
microscopy, confocal microscopy, stereomicroscopy, fluorescence microscopy.
Histological analysis can reveal basic architecture and cell types.
An expansion organoid of the invention preferably has a three dimensional
structure, i.e.
the organoid is preferably a three-dimensional organoid. In a preferred
embodiment the
expansion organoid comprises only epithelial cells, i.e. non-epithelial cells
are absent from
the organoid. This is because the culture medium of the invention is
specifically designed
to expand Lgr5+ epithelial stem cells. Therefore, even if other cell types are
transiently
present in the culture medium, e.g. in the tissue fragment that is the
starting material of the
invention, these cells are unlikely to survive and instead will be replaced by
the longer term
expansion of the Lgr5+ stem cells which generate a pure population of
epithelial cells.
In some embodiments, the epithelial cells surround a lumen. In some
embodiments, the
epithelial cells surrounding the lumen are polarized, (meaning that proteins
are
differentially expressed on the apical or basolateral side of the epithelial
cell). In some
embodiments the organoids comprise stem cells which are able to actively
divide and
which are preferably able to differentiate to all major differentiated cell
lineages present in
the corresponding in vivo tissue, e.g. when the organoid or cell is
transferred to a
differentiation medium.
In some embodiments, the organoid is a three-dimensional organoid, comprising
crypt-like
domains surrounding a central lumen, and contain intestinal stem cells that
are polarised,
residing in the bases of the structures that can actively divide and give rise
to all major
differentiated cell lineages present in the intestine.
In some embodiments the organoids of the invention have a section which is
formed of
multiple layers. Multiple layers of cells are also referred to herein as
regions of "pseudo-
stratified" cells. By "pseudo-stratified" it is meant that there are multiple
(more than one)
layers of cells. Such cells often tend to have their nuclei more central to
the cells, i.e. not
polarized. The cells in the multilayer section may organise themselves to
include a gap, or
lumen between the cells.

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In some embodiments the organoids of the invention comprise single monolayers
that are
folded (or invagin*ted) to form two or more layers. It can sometimes be
difficult to
distinguish between folded (or invagin*ted) monolayers and regions of
stratified cells. In
some embodiments an organoid comprises both regions of stratified cells and
regions of
folded monolayers. In some embodiments the organoids of the invention have a
section
which is formed of multiple layers and a section comprising a single monolayer
of cells. In
some embodiments the organoids of the invention comprise or consist of a
single
monolayer of cells.
During expansion liver and/or pancreas epithelial cells acquire a columnar
shape typical of
ductal cells, while when differentiating, the cells acquire a more polygonal
shape typical of
hepatocyte cells (e.g. see Figure 10). Thus in some embodiments, the cells of
the organoid
are a columnar shape or a polygonal shape.
Structurally, organoids according to the invention are often elongated in
shape. They may
include one or more budding structure ¨ a single cell epithelial layer with
similarities to
ducts or islets. Thus, organoids according to the invention may possess a
layer of cells
with at least one bud and a central lumen.
Under confocal microscopy, the structures may stain positive for keratin. They
may include
cells with polarised nuclei and small cytoplasm.
The organoids in the outside of the matrigel tend to be larger than the
organoids in the
center of the matrigel, perhaps because they have better access to the
necessary growth
factors.
In some embodiments the organoids of the invention comprise or consist of
epithelial cells.
In some embodiments, the organoids comprise or consist of a single layer of
epithelial
cells. In some embodiments non-epithelial cells are absent from the organoids.
In some
embodiments, the organoids of the invention comprise all the differentiated
cell types that
exist in their corresponding in vivo tissue counterpart.
Metaphase spreads of organoids more than 3 months old consistently revealed 46

chromosomes in each of the 20 cells taken from three different donors.
Morphologically, the cells appear like their corresponding in vivo tissue
counterpart. The
organoid may be derived from liver, pancreas, intestine (e.g. small intestine
or colon),
stomach, prostate, lung, breast, ovarian, salivary gland, hair follicle, skin,
oesophagus or
thyroid epithelial stem cells, i.e. the organoids may be liver, pancreas,
intestine (e.g. small
intestine or colon), stomach, prostate, lung, breast, ovarian, salivary gland,
hair follicle,
skin, oesophagus or thyroid organoids.

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There is provided an organoid or a population of epithelial stem cells which
has been
cultured in expansion media of the invention for at least 2 months, for
example at least 10
weeks, at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 4
months, at least
months, at least 6 months, at least 9 months, at least one year. Preferably,
the cells are
5 human cells.
Advantageously, use of forskolin and other cAMP pathway activators allows the
cells and
organoids to be cultured long-term. It is not possible to culture human
epithelial stem cells
or organoids for two months or more in the culture medium described in
W02012/014076
or W02012/168930. Therefore, such organoids or populations of epithelial stem
cells that
had been cultured long-term did not exist before the present invention.
Accordingly, there
is provided an organoid or a population of stem cells that has been
cultured/passaged or
that is capable of being cultured/passaged as described herein. For example,
there is
provided an organoid or a population of stem cells that has been passaged or
which is
capable of being passaged at a split ratio of 1:4-1:6 every 7-10 days for more
than 6 or
more weeks, for example, for 2 months, 3 months, 4 months, 5 months or 6 or
more
months. In one embodiment, there is provided an organoid or a population of
stem cells
that has been passaged or which is capable of being passaged for more than 18
passages
at a split ratio of 1:4-1:6 every 10 days for more than 5 months.
In some embodiments, the organoid or population of epithelial stem cells in
the expansion
medium has a doubling time of less than 65 hours, for example, 60, 58, 56, 54,
53, 50
hours or less. In some embodiments, the doubling time is measured between
passages 1-
3 ("P1" to "P3"). In some embodiments, the doubling time is measured between
passages
10-12 ("P10" to "P12"). In some embodiments, the doubling time between P1 to
P3 is
shorter than between P10 to P12, for example a doubling time of about 52 hours
between
P1 to P3 and a doubling time of about 57 hours at P10 to P12. In some
embodiments, the
doubling time for human epithelial stem cells in the expansion medium of the
invention is
shorter than the doubling time for human epithelial stem cells in the
expansion medium
described in W02012/014076 or W02012/168930. In some embodiments, the organoid
or
population of epithelial stem cells in the expansion medium is growing
exponentially.
In some embodiments, there is provided an organoid in the expansion medium of
the
invention, wherein there are 12 or more organoids per container (e.g. per
well), for
example, 15, 20, 30, 40, 50, 60 or more organoids per container. In some
embodiments,
the number of organoids per container is measured at passage 6 or passage 7.
Advantageously, use of the expansion medium of the invention, which contains a
cAMP
pathway activator, may make it possible to form more cells and organoids per
well than if a
cAMP pathway activator is absent because of the increased passage number
possible

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using the expansion medium of the invention. Thus, in some embodiments, there
is
provided an organoid in the expansion medium of the invention which is capable
of forming
12 or more organoids per container (e.g. per well), e.g. 15, 20, 30, 40, 50 or
60 more
organoids per container. In some embodiments, the culture method of the
invention
comprises splitting the cells in each container before the maximum number of
organoids
has been formed in each container.
Use of a cAMP pathway activator in the expansion medium allows larger
organoids to be
obtained than when a cAMP pathway activator is absent. This is apparent from
Figure 2A
which shows the size of organoids obtained in the absence of the cAMP pathway
activator
(left hand panel) with the size of organoids obtained in the presence of the
cAMP pathway
activator (right hand panel).
Accordingly, in some embodiments, an expansion organoid according to the
present
invention comprises a population of at least 1x103 cells, at least 1x104
cells, at least 1x105
cells, or more. In some embodiments, each organoid comprises between
approximately
1x103 cells and 5x103 cells; generally, 10-20 organoids may be grown together
in one well,
for example of a 24 well plate. In some embodiments, a single organoid may be
grown in a
well.
In some embodiments, organoids are at least 50 pm, at least 60 pm, at least 70
pm, at
least 80 pm, at least 90 pm, at least 100 pm, at least 125 pm, at least 150
pm, at least 175
pm, at least 200 pm, at least 250 pm or more in diameter at the widest point.
In contrast to
mature hepatocytes, for example, which grow to confluence for a short period
of time,
before dying, liver epithelial stem cells cultured using the method of the
invention are self-
renewing and can be cultured long-term. It has been found that the self-
renewing
population of cells are those which are capable of expressing Lgr5 on their
surface. Lgr5
negative cells do not self-renew. The term "self-renewing" should be
understood to
represent the capacity of a cell to reproduce itself whilst maintaining the
original
proliferation and differentiation properties of cells of the invention. Such
cells proliferate by
dividing to form clones, which further divide into clones and therefore expand
the size of
the cell population without the need for external intervention, without
evolving into cells
with a more restricted differentiation potential.
Within the context of the invention, a tissue fragment is a part of an adult
tissue, preferably
a human adult tissue. Preferably an organoid as identified herein is therefore
not a tissue
fragment.
In some embodiments, the organoid or population of epithelial stem cells that
has been
cultured in an expansion medium of the invention expresses the Lgr5 stem-cell
surface

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marker. Advantageously, use of a cAMP pathway activator such as forskolin in
the
expansion medium increases the amount of Lgr5 positive cells that are present
compared
to when the cAMP pathway activator is absent. Using previously available
methods which
did not comprise cAMP pathway activators, human liver stem cells lost their
stemness after
5 around two months in culture and then died. The present methods allow the
cells to be
cultured for longer periods of time and more cells to be obtained leading to
increased
percentage of stem cells. In some embodiments, the Lgr5 cells represent at
least 1% of
the culture population, e.g. at least 1.25%, at least 1.5%, at least 1.75%, at
least 2%, at
least 2.25%, at least 2.5%, at least 2.75%, at least 3%, at least 4%, at least
5% of the
10 culture population. For example, in some embodiments, the Lgr5 cells
represent 1%-5%,
for example 1.25-4%, 1%-3%, 1.25%-3%, 1.5-3% of the culture population. In
some
embodiments, it is envisaged that the percentage of Lgr5 positive cells may be
higher, for
example, if the cells carry a mutation that affects Lgr5 expression, such as a
mutation in
the Wnt pathway or if the expansion medium comprises high amounts of Wnt. Thus
it is
15 also envisaged that Lgr5 cells may represent at least 4%, at least 8%,
at least 10%, at
least 20%, at least 30%, at least 40% of the culture population.
Accordingly, an organoid preferably comprises cells that express Lgr5. For
example, in
some embodiments, at least 1%, more preferably at least 1.25%, at least 1.5%,
at least
1.75%, at least 2%, at least 2.25%, at least 2.5%, at least 2.75%, at least 3%
of the cells in
20 the organoid express Lgr5. Similarly, the invention provides a cell or a
population of cells
which express Lgr5, wherein said cells are obtained from an organoid of the
invention.
The progeny of such cells is also encompassed by the invention.
In some embodiments, the organoid or cell derived from said organoid in an
expansion
medium of the invention comprises cells which express Lgr5, wherein Lgr5
expression is
25 upregulated compared to expression of Lgr5 in an organoid or a
population of epithelial
stem cells in an expansion medium corresponding to a expansion medium of the
invention
but without a cAMP pathway activator. Preferably, expression of Lgr5 is
upregulated by
25%, for example, by 50%, 75%, 100%, 125%, 150%, 200%, 250%, 300% or more.
In a preferred embodiment, an organoid could be cultured during at least 2, 3,
4, 5, 6, 7, 8,
30 9, 10 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 months or longer. In some
embodiments, the
organoid is expanded or maintained in culture for at least 3 months,
preferably at least 4
months, at least 5 months, at least 6 months, at least 7 months, at least 9
months, or at
least 12 months or more. Advantageously, use of the culture methods provided
by the
present invention results in organoids and/or cell populations being formed in
which the
35 number of chromosomes remains stable when the cells or organoids are
cultured long-
term. Thus, in some embodiments, the organoids or population of epithelial
stem cells of

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the invention has a stable chromosome number after 2, 4, 6, 8, 10, 12 or 14
weeks or after
4, 5, 6 or more months in culture in an expansion medium of the invention.
Preferably, at
least 65%, at least 70%, more preferably at least 75%, more preferably at
least 80%, more
preferably at least 90%, more preferably at least 95%, more preferably at
least 99% of the
cells have the correct number of chromosomes after 2, 4, 6, 8, 10, 12 or 14
weeks or after
4, 5, 6 or more months culture in an expansion medium of the invention. For
human
epithelial cells, the correct number of chromosomes is 46. Of course, it is to
be
understood that cancer cells in cancer organoids, e.g. derived from cancer
stem cells,
would not necessarily have the correct number of chromosomes as genomic
instability is a
feature of certain cancers.
The expansion organoid of the invention preferably comprises at least 50%
viable cells,
more preferred at least 60% viable cells, more preferred at least 70% viable
cells, more
preferred at least 80% viable cells, more preferred at least 90% viable cells.
Viability of
cells may be assessed using Hoechst staining or Propidium Iodide staining in
FACS. In
some embodiments, there is provided one or more frozen organoids of the
invention. Also
provided is a method for preparing organoids for freezing comprising
dissociating organoid
cultures and mixing them with a freezing medium such as Recovery cell culture
freezing
medium (Gibco) and freezing following standard procedures. A method for
thawing frozen
organoids is also provided which comprises thawing frozen organoids, embedding
the
thawed organoids in an extracellular matrix (e.g. Matrigel) and culturing the
organoids in
an expansion medium of the invention. Advantageously, initially after thawing
the culture
medium may be supplemented with Y-27632, for example, about 10uM Y-27632. In
some
embodiments, the culture medium is supplemented with Y-27632 for the first 1,
2, 3, 4, 5 or
less days after thawing, preferably for the first 3 or 4 days. In some
embodiments, Y-
27632 is not present in the culture medium after the first 3, 4, 5, 6 or more
days, preferably
after the first 3 or 4 days. This freezing method can be used for expansion
organoids of
the invention.
Culturing in differentiation medium
Optionally, the organoids or cells expanded using the expansion medium may
then be
cultured in a differentiation medium to differentiate them into cells which
express mature
differentiation markers. Thus, in some embodiments, cells can be grown in a
first
"expansion" culture medium (also referred to herein as EM), followed by
culturing the cells
in a second "differentiation" culture medium (also referred to herein as DM).
Accordingly,
the methods of the invention may optionally further comprise the step of
culturing epithelial
stem cells in a differentiation medium using a method as described herein. The
method
may optionally comprise the further step of obtaining and/or isolating one or
more of the

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resulting organoids/cells/populations of cells from the culture medium.
However, in some
embodiments, the step of culturing in DM media is not carried out, for example
in some
methods, cells are transplanted and allowed to differentiate in vivo.
In some embodiments, the epithelial stem cells are isolated from the expansion
medium
prior to culturing in the differentiation medium. In other embodiments, cells
are not isolated
prior to culturing in the differentiation medium but the culture medium
components are
simply changed so that the components of the differentiation medium are added
rather
than replenishing the components of the expansion medium.
Accordingly, in some embodiments, the method further comprises culturing the
cells in a
differentiation medium which comprises or consists of a basal medium for
animal or human
cells to which is added: one or more receptor tyrosine kinase ligands and a
Notch inhibitor.
In some embodiments, the differentiation medium further comprises one more
more of: a
TGF-beta inhibitor, gastrin and dexamethasone.
In one embodiment the method further comprises culturing the cells in a
differentiation
medium which comprises or consists of a basal medium for animal or human cells
to which
is added: EGF, a TGF-beta inhibitor, a Notch inhibitor and a prostaglandin
pathway
activator, such as PGE2 and/or AA. This medium is described in WO 2012/168930
and is
useful for differentiating the cells. Preferably, the differentiation medium
also comprises
Noggin, Gastrin, FGF and/or HGF. In some embodiments the differentiation
medium
comprises a p38 inhibitor.
For example, in one embodiment, the differentiation medium comprises EGF, a
TGF-beta
inhibitor, FGF (for example, FGF10, FGF19, or any other suitable FGF family
member)
and a Notch inhibitor. In one embodiment, the TGF-beta inhibitor is A83-01
and/or the
Notch inhibitor is a gamma-secretase inhibitor (e.g. DAPT or DBZ). FGF may
optionally be
replaced by HGF or alternatively both FGF and HGF may be present or absent in
the
differentiation medium. In some embodiments, EGF might be replaced by HGF or
another
receptor tyrosine kinase ligand. Dexamethasone may also be added, for example
at a
concentration of between 10nM to 10uM. The differentiation medium may
optionally
include a prostaglandin pathway activator, such as PGE2 or AA. However, this
component
may also be excluded from the differentiation medium. In some embodiments,
oncostatin
M may also be added, for example at a concentration range between 1 ng/ml to
1mg/ml,
e.g. in the case of liver to help differentiation to hepatocyte fate.
In one embodiment, the differentiation medium comprises or consists of a basal
medium
for animal or human cells to which is added:
Epidermal Growth Factor, FGF10 and HGF as receptor tyrosine kinase ligands;

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a Notch inhibitor;
a TGF-beta inhibitor; and
a prostaglandin pathway activator, such as PGE2 and/or AA.
This culture medium is particularly preferred for liver cells. In some
embodiments, the DM
comprises or consists of a basal medium to which is added: 50 ng/ml EGF, 100
ng/ml
FGF10, 50 nM A8301 and 10 pM DAPT.
In some embodiments, the differentiation medium comprises or consists of a
basal
medium (for example comprising Advanced DMEM/F12, B27 (50x), n-Acetylcystein
(1 mM)
glutamin/glutamax), Noggin (preferably 100 ng/ml), EGF (preferably 50 ng/ml),
gastrin
(preferably 10nM), TGF-beta inhibitor, such as A83-01 (preferably 50 nM) and a
Notch
inhibitor (for example DAPT/DBZ) (preferably 10 pM).
In a particularly preferred embodiment, the differentiation medium comprises
or consists of
a basal medium comprising an EGF receptor activator (e.g. EGF), an FGF
(preferably a
FGF receptor 4 activator, e.g. FGF19), an HGF receptor activator (e.g. HGF), a
Notch
inhibitor, and a TGF-beta inhibitor, more preferably EGF, FGF, HGF, a Notch
inhibitor, and
a TGF-beta inhibitor.
Preferably, the differentiation medium further comprises a glucocorticoid, for
example,
dexamethasone or cortisone. In some embodiments, dexamethasone is used at a
concentration of between 10 nM to 10 pM.
In some embodiments, the differentiation medium additionally comprises
gastrin.
Accordingly, in a preferred embodiment, the differentiation medium comprises
or consists
of a basal medium comprising EGF, FGF, HGF, a Notch inhibitor, a TGF-beta
inhibitor, a
glucocorticoid and gastrin.
Any suitable EGF receptor activator as described herein may be used, and is
preferably
EGF.
The FGF used in the differentiation medium is an FGF able to bind to FGF
receptor 4
(FGFR4), and is preferably FGF19 (preferably from Peprotech). In some
embodiments, no
more than one FGF is used. In other embodiments, two or more FGF are used,
e.g. 2, 3
or more. In some embodiments, FGF is substituted with a compound that
activates the
FGFR4 pathway. The FGF in the differentiation medium is preferably FGF19.
Any suitable HGF receptor activator as described herein may be used, and is
preferably
HGF.

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Any suitable Notch inhibitor may be used. In some embodiments the Notch
inhibitor is a
gamma-secretase inhibitor, for example DAPT, dibenzazepine (DBZ),
benzodiazepine
(BZ) or LY-411575. One or more Notch inhibitors may be used, for example, 2,
3, 4 or
more.
Any suitable TGF-beta inhibitor, as described herein may be used. Non-limiting
examples
of suitable TGF-beta inhibitors are small molecule inhibitors, for example
selected from the
group consisting of: A83-01, SB-431542, SB-505124, SB-525334, LY 364947, SD-
208,
SJN 2511. Advantageously, in some embodiments, the TGF beta inhibitor is
present at 10
times higher concentration than in W02012/168930. Accordingly, in some
embodiments,
a TGF beta inhibitor (e.g. A83-01) is added to the DM at a concentration of
between 250-
750 nM, for example, 400-600 nM or about 500nM. Use of TGF beta inhibitor at
this
higher concentration in the context of a differentiation medium for liver
which also
comprises EGF, FGF, HGF and a Notch-inhibitor leads to better expression of
hepatocyte
markers which is indicative of better differentiation. Using culture methods
not involving
forskolin (e.g. in WO 2012/014076), only about 30% of the cells in the liver
organoids
express hepatocyte markers. For the differentiated liver organoids of the
present invention,
a greater percentage of cells express hepatocyte markers. For example, in some

embodiments, the differentiating organoids comprise more than about 30%, more
than
about 40%, more than about 50%, more than about 60% cells expressing
hepatocyte
markers. However, in some embodiments, a TGF beta inhibitor is absent from the
differentiation medium and the specific examples of differentiation media
described herein
can be adapted accordingly to omit the TGF beta inhibitor.
In one embodiment, the TGF beta inhibitor is A83-01 and/or the Notch inhibitor
is DAPT.
Accordingly, in a preferred embodiment, the differentiation medium comprises
EGF,
FGF19, HGF, DAPT and A83-01. More preferably, the differentiation medium
comprises
EGF, FGF19, HGF, DAPT and A83-01 and further comprises dexamethasone and/or
gastrin.
In some embodiments, the differentiation medium further comprises a BMP
activator, for
example BMP7, BMP4 or BMP2. Preferably, the BMP activator is BMP7.
For example, in some embodiments, the differentiation medium comprises EGF,
gastrin,
HGF, FGF19, A8301, DAPT, BMP7 and dexamethasone. For example, it may comprise
about 50 ng/ml EGF, about 10 nM gastrin, about 25 ng/ml HGF, about 100 ng/ml
FGF19,
about 500 nM A8301, about 10 pM DAPT, about 25 ng/ml BMP7 and about 30uM
Dexamethasone.

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In some embodiments, the differentiation medium comprises one or more receptor
tyrosine
kinase inhibitor (preferably EGF, HGF and FGF19), a TGF-beta inhibitor
(preferably
A8301), a Notch inhibitor (preferably DAPT) and a BMP activator (preferably
BMP7).
In another embodiment, the differentiation medium additionally comprises
Oncostatin M.
5 In some embodiments, the differentiation medium does not comprise
Rspondin and/or
Wnt. In some embodiments, the differentiation medium does not comprise a Wnt
agonist.
In some embodiments, the differentiation medium does not comprise Wnt. In some

embodiments, the differentiation medium does not comprise Nicotinamide.
In some embodiments, retinoic acid is absent from the differentiation medium.
A cAMP
10 pathway activator is preferably absent from the differentiation medium.
In some
embodiments, the differentiation medium does not comprise a BMP inhibitor. In
some
embodiments, the differentiation medium does not comprise PGE2 and/or AA. In
some
embodiments, the differentiation medium does not comprise a prostaglandin
pathway
activator.
15 The invention provides a differentiation medium as described herein, use
of a
differentiation medium as described herein, for example in the methods and
uses
described herein, methods of using a differentiation medium as described
herein, and cells
and organoids obtainable/obtained by culturing using a differentiation medium
as
described herein.
20 There is provided a method of culturing epithelial stem cells using a
differentiation medium
as described herein. Preferably, the method involves culturing expanded
epithelial stem
cells using a differentiation medium as described herein. The epithelial stem
cells may
have been expanded by any suitable method. Preferably, they have been expanded
using
one or more expansion media according to the invention. Alternatively, they
may have
25 been expanded using expansion media as described in W02012/168930 or
W02012/014076.
In some embodiments, the method comprises culturing the epithelial stem cells
in the
expansion medium for 5 or more days, for example 7, 9, 10 or 14 or more days
or at least
one month or at least two months and then culturing the resulting cells in the
differentiation
30 medium. However, the cells may be cultured in the expansion medium for
longer if
desired.
As explained herein, use of an expansion medium of the invention
advantageously allows human epithelial stem cells to be cultured long-term and
so the
cells may be cultured as described herein before being cultured in
differntiation medium.
In some embodiments, the cells are cultured in the differentiation medium for
5-14 days,
35 for example, 7-12 days, more preferably 9-11 days.

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The method may comprise changing the medium for fresh medium during the course
of
the culturing because the components of the medium are used up during
culturing. It will
be clear to the skilled person how often the medium needs to be changed for
fresh
medium. In some embodiments, the medium is changed every other day, but it is
also
envisaged that it may be changed every day or every two days or as required.
The methods for culturing epithelial stem cells and/or obtaining an organoid
in expansion
or differentiation medium are carried out in vitro.
Following culturing in expansion or differentiation medium, the method may
further
comprise obtaining and/or isolating one or more epithelial stem cells or an
organoid. For
example, following culture of the stem cells, it may be useful to remove one
or more stem
cells, differentiated cells and/or one or more organoids cultured in the
expansion or
differentiation medium from the culture medium for use in subsequent
applications.
Differentiated organoids and populations of cells
Organoids can be cultured in a differentiation medium, as described above,
such that they
differentiate into functional cell types (e.g. see Example 15). The invention
further provides
a differentiated organoid or a population of differentiated epithelial cells.
In one embodiment, the invention provides a differentiated organoid or a
population of
epithelial cells obtainable or obtained by a method of the invention, which
comprises
culturing epithelial stem cells in a differentiation medium of the invention.
Illustrative examples of organoids generated using the differentiation medium
and methods
of the invention are given in the accompanying figures.
It can be seen that differentiated organoids according to the invention may
possess a
cystic structure, with on the outside, a layer of cells with at least one bud
and a central
lumen. In some embodiments, they are elongated in shape. The organoids may
have a
section which is formed of multiple layers; such cells often tend to have
their nuclei more
central to the cells, i.e. not polarized. The cells in the multilayer section
may organise
themselves to include a gap, or lumen between the cells. They may include
cells with
polarised nuclei and small cytoplasm. In some embodiments, the differentiated
organoids
have a single layered stratified epithelium.
Although not limiting, the organoids in the outer part of the extracellular
matrix tend to be
larger than the organoids in the centre of the extracellular matrix, perhaps
because they
have better access to the necessary growth factors.
A differentiated organoid of the invention preferably has a three dimensional
structure, very
similar to that of the expansion organoid (see features described above in
section about

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expansion organoids and populations of cells, which also apply here). In some
embodiments, the epithelial cells exist in distinct dividing domains and
differentiating
domains. In some embodiments, all differentiated cell types of the normal in
vivo tissue are
present in said organoid. Differentiated cell types express differentiation
markers which are
known in the art. Differentiated cell types can be identified using such
markers by methods
known in the art (see below).
A differentiated organoid according to the present invention may comprise a
population of
cells of at least 1x103 cells, at least 1 x 104 cells, at least 1x106 cells,
at least 1x106 cells,
at least 1x107 cells or more. In some embodiments, each organoid comprises
between
approximately 1x103 cells and 5x103 cells; generally, 10-20 organoids may be
grown
together in one well, for example of a 24 well plate.
It is clear to the skilled person that an organoid of the invention is not a
naturally occurring
tissue fragment and/or does not comprise a blood vessel. For example, in the
case of a
liver organoid of the invention, does not comprise a naturally occurring liver
lobule or a
naturally occurring bile duct. Similarly, an intestinal organoid of the
invention does not
comprise a naturally occurring crypt or a naturally occurring villus.
The differentiation medium described herein preferably induces or promotes a
specific
differentiation of cells during at least five days of culture. Differentiation
may be measured
by detecting the presence of a specific marker associated with the particular
tissue
lineage, e.g. the liver lineage, as defined herein. Differentiation may be
measured by
detecting the presence of a specific marker associated with the tissue
lineage, e.g. the
liver lineage, as defined herein. Depending on the identity of the marker, the
expression of
said marker may be assessed by RTPCR or immuno-histochemistry after at least
5, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16 or more days of culture in a differentiation
medium as defined
herein.
The term "expressed" is used to describe the presence of a marker within a
cell. In order to
be considered as being expressed, a marker must be present at a detectable
level. By
"detectable level" is meant that the marker can be detected using one of the
standard
laboratory methodologies such as PCR, blotting or FACS analysis. A gene is
considered to
be expressed by a cell of the population of the invention if expression can be
reasonably
detected after 30 PCR cycles, which corresponds to an expression level in the
cell of at
least about 100 copies per cell. The terms "express" and "expression" have
corresponding
meanings. At an expression level below this threshold, a marker is considered
not to be
expressed. The comparison between the expression level of a marker in a cell
of the
invention, and the expression level of the same marker in another cell, such
as for

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example an embryonic stem cell, may preferably be conducted by comparing the
two cell
types that have been isolated from the same species. Preferably this species
is a mammal,
and more preferably this species is human. Such comparison may conveniently be

conducted using a reverse transcriptase polymerase chain reaction (RT-PCR)
experiment.
The differentiated organoid of the invention preferably comprises at least 50%
viable cells,
more preferred at least 60% viable cells, more preferred at least 70% viable
cells, more
preferred at least 80% viable cells, more preferred at least 90% viable cells.
Viability of
cells may be assessed using Hoechst staining or Propidium Iodide staining in
FACS. The
viable cells preferably possess corresponding in vivo functions or
characteristics. For
example, viable liver cells preferably possess hepatic functions or
characteristics or
hepatocytes.
Also provided is a differentiated organoid or a differentiated population of
liver epithelial
cells of the invention in a differentiation medium of the invention.ln one
embodiment, there
is provided an organoid in a differentiation medium, for example as described
herein.
In an embodiment, a differentiated organoid is an organoid which is still
being cultured
using a method of the invention and is therefore in contact with an
extracellular matrix.
Preferably, a differentiated organoid is embedded in a non-mesenchymal
extracellular
matrix.
The organoid or population of epithelial stem cells may be from any mammalian
tissue, but
is preferably from a human. In some embodiments, it is from a mouse, rabbit,
rat, guinea
pig or other non-human mammal.
Culture media
The invention provides a cell culture medium as described herein. A cell
culture medium
(for example, the expansion medium or differentiation medium) that is used in
a method of
the invention comprises any suitable basal medium, subject to the limitations
provided
herein. Basal media for cell culture typically contain a large number of
ingredients, which
are necessary to support maintenance of the cultured cells. Suitable
combinations of
ingredients can readily be formulated by the skilled person, taking into
account the
following disclosure. A basal medium for use in the invention will generally
comprises a
nutrient solution comprising standard cell culture ingredients, such as amino
acids,
vitamins, lipid supplements, inorganic salts, a carbon energy source, and a
buffer, as
described in more detail in the literature and below. In some embodiments, the
culture
medium is further supplemented with one or more standard cell culture
ingredient, for
example selected from amino acids, vitamins, lipid supplements, inorganic
salts, a carbon
energy source, and a buffer.

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The skilled person will understand from common general knowledge the types of
culture
media that might be used as the basal medium in the cell culture mediums of
the invention.
Potentially suitable cell culture media are available commercially, and
include, but are not
limited to, Dulbecco's Modified Eagle Media (DMEM), Minimal Essential Medium
(MEM),
Knockout-DMEM (KO-DMEM), Glasgow Minimal Essential Medium (G-MEM), Basal
Medium Eagle (BME), DMEM/Ham's F12, Advanced DMEM/Ham's F12, lscove's Modified

Dulbecco's Media and Minimal Essential Media (MEM), Ham's F-10, Ham's F-12,
Medium
199, and RPM! 1640 Media.
For example, the basal medium may be selected from DMEM/F12 and RPM! 1640
supplemented with glutamine, insulin, Penicillin/streptomycin and transferrin.
In a further
preferred embodiment, Advanced DMEM/F12 or Advanced RPM! is used, which is
optimized for serum free culture and already includes insulin. In this case,
said Advanced
DMEM/F12 or Advanced RPM! medium is preferably supplemented with glutamine and

Penicillin/streptomycin. AdDMEM/12 (Invitrogen) supplemented with N2 and B27
is also
preferred. Preferably, the basal medium is advanced-DMEM/F12.
In some embodiments, the basal medium comprises Advanced DMEM F12, hepes,
penicillin/streptomycin, Glutamin, NAcetyl Cystein, B27, N2 and Gastrin. In
some
embodiments, culture is initiated with a basal medium comprising N2 and
Gastrin and
penicillin/streptomycin but these are later withdrawn. For example, in some
embodiments,
N2 and Gastrin and penicillin/streptomycin are present in an EM1 medium of the
invention
but not in an EM2 or DM. For example, in some embodiments, N2 and Gastrin and
penicillin/streptomycin are present in an EM1 and EM2 medium of the invention
but not in
a DM. In particularly preferred embodiments, the basal medium is Advanced
DMEM/F12
or a DMEM variant supplemented with penicillin/streptomycin, N2, B27,
glutamine and
gastrin.
In some embodiments, the basal culture medium comprises or consists of
Advanced
DMEM/F12 supplemented with penicillin/streptomycin, 10mM HEPES, Glutamax, lx
N2,
lx B27 (all from lnvitrogen) and 1 mM N-acetylcysteine (Sigma)).
It is furthermore preferred that said cell culture medium is supplemented with
a purified,
natural, semi-synthetic and/or synthetic growth factor and does not comprise
an undefined
component such as fetal bovine serum or fetal calf serum. Various different
serum
replacement formulations are commercially available and are known to the
skilled person.
Where a serum replacement is used, it may be used at between about 1% and
about 30%
by volume of the medium, according to conventional techniques.

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As will be apparent to the skilled reader, the preferred culture methods of
the invention are
advantageous because feeder cells are not required. Feeder cell layers are
often used to
support the culture of stem cells, and to inhibit their differentiation. A
feeder cell layer is
generally a monolayer of cells that is co-cultured with, and which provides a
surface
5 suitable for growth of, the cells of interest. The feeder cell layer
provides an environment in
which the cells of interest can grow. Feeder cells are often mitotically
inactivated (e.g. by
irradiation or treatment with mitomycin C) to prevent their proliferation. The
use of feeder
cells is undesirable, because it complicates passaging of the cells (the cells
must be
separated from the feeder cells at each passage, and new feeder cells are
required at
10 each passage). The use of feeder cells can also lead to contamination of
the desired cells
with the feeder cells. This is clearly problematic for any medical
applications, and even in a
research context, complicates analysis of the results of any experiments
performed on the
cells. As noted elsewhere herein, the culture media of the invention are
particularly
advantageous because they can be used to culture cells without feeder cell
contact, i.e.
15 the methods of the invention do not require a layer of feeder cells to
support the cells
whose growth is being sponsored.
Accordingly, the compositions of the invention may be feeder cell-free
compositions. A
composition is conventionally considered to be feeder cell-free if the cells
in the
composition have been cultured for at least one passage in the absence of a
feeder cell
20 layer. A feeder cell-free composition of the invention will normally
contain less than about
5%, less than about 4%, less than about 3%, less than about 2%, less than
about 1%
feeder cells (expressed as a % of the total number of cells in the
composition) or
preferably no feeder cells at all.
The culture media used in the invention may comprise serum, or may be serum-
free
25 and/or serum-replacement free, as described elsewhere herein. Culture
media and cell
preparations are preferably GMP processes in line with standards required by
the FDA for
biologics products and to ensure product consistency.
A culture medium of the invention will normally be formulated in deionized,
distilled water.
A culture medium of the invention will typically be sterilized prior to use to
prevent
30 contamination, e.g. by ultraviolet light, heating, irradiation or
filtration. The culture medium
may be frozen (e.g. at -20 C or -80 C) for storage or transport. The medium
may contain
one or more antibiotics to prevent contamination. The medium may have an
endotoxin
content of less that 0.1 endotoxin units per ml, or may have an endotoxin
content less than
0.05 endotoxin units per ml. Methods for determining the endotoxin content of
culture
35 media are known in the art.

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A preferred cell culture medium is a defined synthetic medium that is buffered
at a pH of
7.4 (preferably with a pH 7. 2 - 7.6 or at least 7.2 and not higher than 7.6)
with a
carbonate-based buffer, while the cells are cultured in an atmosphere
comprising between
% and 10% 002, or at least 5% and not more than 10% 002, preferably 5 % 002.
5 The invention also provides a composition or cell culture vessel
comprising cells and/or
organoids according to any one of the aspects of the invention described
above, and a
culture medium according to any one of the aspects of the invention described
above. For
example, such a composition or cell culture vessel may comprise any number of
cells or
organoids cultured according to a method of the invention, in a culture medium
as
described above.
According to a still further aspect of the invention, there is provided a
hermetically-sealed
vessel containing a culture medium of the invention. In some embodiments, the
culture
medium is an expansion medium. In some embodiments, the culture medium is a
differentiation medium. Hermetically-sealed vessels may be preferred for
transport or
storage of the culture media, to prevent contamination. The vessel may be any
suitable
vessel, such as a flask, a plate, a bottle, a jar, a vial or a bag.
Compositions and other forms of the invention
The invention provides a composition comprising a culture medium according to
the
invention and stem cells. The invention also provides a composition comprising
a culture
medium according to the invention and organoids. Furthermore, the invention
provides a
composition comprising a culture medium according to the invention and an
extracellular
matrix.
The invention also provides a composition comprising a culture medium of the
invention,
an extracellular matrix and epithelial stem cells of the invention. The
invention also
provides a composition comprising a culture medium of the invention, an
extracellular
matrix and one or more organoids of the invention. The invention also provides
a culture
medium supplement that can be used to produce a culture medium as disclosed
herein. A
'culture medium supplement' is a mixture of ingredients that cannot itself
support stem
cells, but which enables or improves stem cell culture when combined with
other cell
culture ingredients. The supplement can therefore be used to produce a
functional cell
culture medium of the invention by combining it with other cell culture
ingredients to
produce an appropriate medium formulation. The use of culture medium
supplements is
well known in the art.
The invention provides a culture medium supplement that comprises an inhibitor
according
to the invention. The supplement may contain any inhibitor (or combination of
inhibitors)

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52
disclosed herein. The supplement may also contain one or more additional cell
culture
ingredients as disclosed herein, e.g. one or more cell culture ingredients
selected from the
group consisting of amino acids, vitamins, inorganic salts, carbon energy
sources and
buffers.
A culture medium or culture medium supplement may be a concentrated liquid
culture
medium or supplement (e.g. a 2x to 250x concentrated liquid culture medium or
supplement) or may be a dry culture medium or supplement. Both liquid and dry
culture
media or supplements are well known in the art. A culture medium or supplement
may be
lyophilised.
A culture medium or supplement of the invention will typically be sterilized
prior to use to
prevent contamination, e.g. by ultraviolet light, heating, irradiation or
filtration. A culture
medium or culture medium supplement may be frozen (e.g. at -20 C or -80 C) for
storage
or transport. In some embodiments, the culture medium may be stored as a
liquid (e.g. at
approximately 4 C). In some embodiments, the culture medium may be split and
stored as
two components: a frozen component (e.g. at between approximately -20 C and
approximately -80 C) and a liquid component (e.g. at approximately 4 C). In
particular,
temperature-sensitive or time-sensitive degradable material is preferably
included in the
frozen component, whereas less sensitive material (for example DMEM or FCS)
can be
stored in the liquid form and thus included in the liquid component for
storage and
shipping.
The invention also provides a hermetically-sealed vessel containing a culture
medium or
culture medium supplement of the invention. Hermetically-sealed vessels may be
preferred
for transport or storage of the culture media or culture media supplements
disclosed
herein, to prevent contamination. The vessel may be any suitable vessel, such
as a flask,
a plate, a bottle, a jar, a vial or a bag.
The invention also provides a kit comprising a culture medium, culture medium
supplement
and/or a composition of the invention. In some embodiments, the kit further
comprises at
least one other additional component, for example selected from the list
comprising: an
ECM (for example, MatrigelTm), a population of cells and an organoid.
Uses of organoids and populations of cells
The invention provides the use of an organoid of the invention or cells
derived from said
organoid in drug screening, (drug) target validation, (drug) target discovery,
toxicology and
toxicology screens, personalized medicine, regenerative medicine and/or as ex
vivo
cell/organ models, such as disease models.

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Cells and organoids cultured according to the media and methods of the
invention are
thought to faithfully represent the in vivo situation. This is true both for
expanded
populations of cells and organoids grown from normal tissue and for expanded
populations
of cells and organoids grown from diseased tissue. Therefore, as well as
providing normal
ex vivo cell/organ models, the organoids of the invention can be used as ex
vivo disease
models.
Organoids of the invention can also be used for culturing of a pathogen and
thus can be
used as ex vivo infection models. Examples of pathogens that may be cultured
using an
organoid of the invention include viruses, bacteria, prions or fungi that
cause disease in its
animal host. Thus an organoid of the invention can be used as a disease model
that
represents an infected state. In some embodiments of the invention, the
organoids can be
used in vaccine development and/or production.
Diseases that can be studied by the organoids of the invention thus include
genetic
diseases, metabolic diseases, pathogenic diseases, inflammatory diseases etc,
for
example including, but not limited to: cystic fibrosis, inflammatory bowel
disease (such as
Crohn's disease), carcinoma, adenoma, adenocarcinoma, colon cancer, diabetes
(such as
type I or type II), Barrett's esophagus, Gaucher's disease, alpha-1-
antitrypsin deficiency,
Lesch-Nyhan syndrome, anaemia, Schwachman-Bodian-Diamond syndrome,
polycythaemia vera, primary myelofibrosis, glycogen storage disease, familial
hypercholestrolaemia, Crigler-Najjar syndrome, hereditary tyrosinanaemia,
Pompe
disease, progressive familial cholestasis, Hreler syndrome, SCID or leaky
SCID, Omenn
syndrome, Cartilage-hair hypoplasia, Herpes simplex encephalitis, Scleroderma,

Osteogenesis imperfecta, Becker muscular dystrophy, duch*enne muscular
dystrophy,
Dyskeratosis congenitor etc.
For instance, Example 17, shows that liver organoids are suitable disease
models for
alpha-1 antitrypsin (A1AT) deficiency and Alagille Syndrome (see further
comments
below).
Traditionally, cell lines and more recently iPS cells have been used as ex
vivo cell/organ
and/or disease models (for example, see Robinton et al. Nature 481, 295,
2012). However,
these methods suffer a number of challenges and disadvantages. For example,
cell lines
cannot be obtained from all patients (only certain biopsies result in
successful cell lines)
and therefore, cell lines cannot be used in personalised diagnostics and
medicine. iPS
cells usually require some level of genetic manipulation to reprogramme the
cells into
specific cell fates. Alternatively, they are subject to culture conditions
that affect karotypic
integrity and so the time in culture must be kept to a minimum (this is also
the case for

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human embryonic stem cells). This means that iPS cells cannot accurately
represent the in
vivo situation but instead are an attempt to mimic the behaviour of in vivo
cells. Cell lines
and iPS cells also suffer from genetic instability.
By contrast, the organoids of the invention provide a genetically stable
platform which
faithfully represents the in vivo situation (e.g. see Examples 7 and 15). The
organoids of
the invention can also be expanded continuously, providing a good source of
genetically
stable cells. In particular, an expanding population can be "split", meaning
that the
organoid is split apart and all cells of the organoid are divided into new
culture dishes or
flasks. The divided cells are removed from the organoid and can then
themselves be
cultured and expanded to produce new organoids containing further expanded
populations
that can then be split again. Splits are also referred to herein as
"passages". An organoid
of the invention may be cultured for 1 or more passages, for example, 1, 2, 3,
4, 5, 6, 7, 8,
9, 10, 15, 20, 25, 30 or more passages, for example, 20-30 passages, 30-35
passages,
32-40 passages or more. In some embodiments, an expanding cell population or
organoid
is split once a month, once every two weeks, once a week, twice a week, three
times a
week, four times a week, five times a week, six times a week or daily. Thus
the organoids
of the invention can provide an ongoing source of genetically stable cellular
material. In
some embodiments, the expanding organoids of the invention comprise all
differentiated
cell types that are present in the corresponding in vivo situation. In other
embodiments, the
organoids of the invention may be differentiated to provide all differentiated
cell types that
are present in vivo. Thus the organoids of the invention can be used to gain
mechanistic
insight into a variety of diseases and therapeutics, to carry out in vitro
drug screening, to
evaluate potential therapeutics, to identify possible targets (e.g. proteins)
for future novel
(drug) therapy development and/or to explore gene repair coupled with cell-
replacement
therapy.
In some embodiments, the organoids have less than 5000, less than 4000, less
than 3000,
less than 2000, less than 1500, less than 1250, less than 1000, or less than
800 base
substitutions per culture after 13 weekly passages, as determined by whole
genome
sequencing analysis (e.g. in Example 15). In some embodiments, the organoids
are free
from chromosomal aberrations. In some embodiments, the organoids contain less
than 50,
less than 20, less than 10, less than 5, less than 2, less than 1, or zero
copy number
variations (CNVs). In some embodiments, the organoids have a mutational
frequency that
is less than 10-fold, less than 5-fold or less than 2-fold higher than the
mutational
frequency of germ line cells. In some embodiments, the organoids have a
mutational
frequency that is less than 10-fold, less than 5-fold or less than 2-fold
lower than the
mutational frequency of germ line cells. In some embodiments, the organoids
have the

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same mutational frequency as germ line cells have. In some embodiments, the
organoids
have a mutational frequency that is between 10-fold higher and 10-fold lower,
or between
5-fold higher and 5-fold lower, or between 2-fold higher and 2-fold lower than
the
mutational frequency of germ line cells. In some embodiments, mutational
frequency is
5 determined by whole genome sequencing analysis of base substitutions or
of copy number
variations. The comparator germ line cell is preferably from the same organism
as the
organoid, e.g. in the context of these embodiments, a human organoid is
preferably
compared to a human germ line cell.
The organoids of the invention can be frozen and thawed and put into culture
without
10 losing their genetic integrity or phenotypic characteristics and without
loss of proliferative
capacity. Thus the organoids can be easily stored and transported. Thus in
some
embodiments, the invention provides a frozen organoid.
For these reason the organoids or expanded populations of cells of the
invention can be a
tool for drug screening, target validation, target discovery, toxicology and
toxicology
15 screens and personalized medicine.
Accordingly, in a further aspect, the invention provides the use of an
organoid or cell
derived from said organoid according to the invention in a drug discovery
screen, toxicity
assay or in medicine, such as regenerative medicine. For example, any one of
the small
intestinal, colon, pancreatic, gastric, liver or prostate organoids may be
used in a drug
20 discovery screen, toxicity assay or in medicine, such as regenerative
medicine.
Mucosa! vaccines
An additional important use of the organoids is in the development of mucosa!
vaccinations. Mucosal vaccines are vaccines that are administered via the
mucosa. This
can be any mucosal surface such as via the nose, mouth, or rectum. They can be
25 administered via an inhaler, a spray or other external aids. This has
several clear benefits
over injections such as that no medical staff are needed for administering the
vaccine,
which may be important, for example in developing countries.
In the intestine, M cells (or "microfold cells") are cells found in the
follicle-associated
epithelium of the aggregated lymphoid nodules of the ileum. They transport
organisms and
30 particles from the gut lumen to immune cells across the epithelial
barrier, and thus are
important in stimulating mucosa! immunity. They have the unique ability to
take up antigen
from the lumen of the small intestine via endocytosis or phagocytosis, and
then deliver it
via transcytosis to dendritic cells (an antigen presenting cell) and
lymphocytes (namely T
cells) located in a unique pocket-like structure on their basolateral side.

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Organoids can in some cases develop into M cells when stimulated with RANK
ligand (e.g.
see figure 49 of W02012/169830). Therefore, in some embodiments of the
invention, the
expanded cell population comprises M cells. In some embodiments of the
invention, an
organoid comprises M cells. In some embodiments, there is provided a method
for
obtaining M cells or an organoid comprising M cells, wherein the method
comprises
stimulating an organoid with RANK ligand.
The efficiency of mucosal vaccines can be substantially increased when they
are targeted
to M cells. Therefore, the expanded stem cell population or organoid of the
invention can
be used for testing the ability of M cells to take up pathogens or antigens
and to present
them to the immune system. Therefore, in some embodiments the invention
provides the
use of an organoid of the invention in drug screening, for example in vaccine
development
and/or vaccine production. For example, in some embodiments the organoid may
be used
for the development or production of vaccines against viral, bacterial, fungal
or other
parasitic infections, for example (but not limited to) cholera, Respiratory
syncytial virus
(RSV), Rotavirus and HIV. In a particular embodiment, the invention provides
organoids
that have been differentiated in a culture medium of the invention comprising
RANKL, for
use in mucosal vaccine development.
Drug screening
For preferably high-throughput purposes, said organoid of the invention is
cultured in
multiwell plates such as, for example, 96 well plates or 384 well plates.
Libraries of
molecules are used to identify a molecule that affects said organoids.
Preferred libraries
comprise antibody fragment libraries, peptide phage display libraries, peptide
libraries (e.g.
LOPAPTM, Sigma Aldrich), lipid libraries (BioMol), synthetic compound
libraries (e.g. LOP
ACTM, Sigma Aldrich) or natural compound libraries (Specs, TimTec).
Furthermore, genetic
libraries can be used that induce or repress the expression of one of more
genes in the
progeny of the stem cells. These genetic libraries comprise cDNA libraries,
antisense
libraries, and siRNA or other non-coding RNA libraries. The cells are
preferably exposed to
multiple concentrations of a test agent for a certain period of time. At the
end of the
exposure period, the cultures are evaluated. The term "affecting" is used to
cover any
change in a cell, including, but not limited to, a reduction in, or loss of,
proliferation, a
morphological change, and cell death. Said organoid of the invention can also
be used to
identify drugs that specifically target epithelial carcinoma cells, but not
said organoid of the
invention.
The ability to obtain a useful organoid of the invention in short time periods
(days) shows
that the organoids would be highly useful for testing individual patient
responses to specific

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drugs and tailoring treatment according to the responsiveness. In some
embodiments,
wherein the organoid is obtained from a biopsy from a patient, the organoid is
cultured for
less than 21 days, for example less than 14 days, less than 13 days, less than
12 days,
less than 11 days, less than 10 days, less than 9 days, less than 8 days, less
than 7 days
(etc).
The organoids are also useful for wider drug discovery purposes (e.g. see
W02013/093812 which describes screening for drugs for cystic fibrosis or
cholera. This
publication describes disease models for cystic fibrosis and cholera).
Therefore, in some
embodiments, the organoids of the invention could be used for screening for
cystic fibrosis
drugs. Equally, the drug screening methods of the invention may use any
organoid
disease model. Other examples of these are provided in the present application
such as
A1AT and AGS disease models. However, drug screening methods are not limited
to use
with organoid disease models but can be used with any of the organoids
described herein.
It will be understood by the skilled person that the organoids of the
invention would be
widely applicable as drug screening tools for infectious, inflammatory and
neoplastic
pathologies of the human gastrointestinal tract and other diseases of the
gastrointestinal
tract and infectious, inflammatory and neoplastic pathologies and other
diseases of other
tissues described herein including pancreas, liver and prostate. In some
embodiments the
organoids of the invention could be used for screening for cancer drugs.
In some embodiments, the organoids of the invention can be used to test
libraries of
chemicals, antibodies, natural product (plant extracts), etc for suitability
for use as drugs,
cosmetics and/or preventative medicines. For instance, in some embodiments, a
cell
biopsy from a patient of interest, such as tumour cells from a cancer patient,
can be
cultured using culture media and methods of the invention and then treated
with a a
chemical compound or a chemical library. It is then possible to determine
which
compounds effectively modify, kill and/or treat the patient's cells. This
allows specific
patient responsiveness to a particular drug to be tested thus allowing
treatment to be
tailored to a specific patient. Thus, this allows a personalized medicine
approach.
The added advantage of using the organoids for identifying drugs in this way
is that it is
also possible to screen normal organoids (organoids derived from healthy
tissue) to check
which drugs and compounds have minimal effect on healthy tissue. This allows
screening
for drugs with minimal off-target activity or unwanted side-effects.
Drugs for any number of diseases can be screened in this way. For example the
organoids
of the invention can be used for screening for drugs for cystic fibrosis,
Barrett's esophagus,
carcinomas, adenocarcinomas, adenomas, inflammatory bowel disease (such as
Crohn's

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disease), liver disease etc. The testing parameters depend on the disease of
interest. For
example, when screening for cancer drugs, cancer cell death is usually the
ultimate aim.
For cystic fibrosis, measuring the expansion of the organoids in response to
the drugs and
stimuli of CFTR is of interest. In other embodiments, metabolics or gene
expression may
be evaluated to study the effects of compounds and drugs of the screen on the
cells or
organoids of interest.
Therefore, the invention provides a method for screening for a therapeutic or
prophylactic
drug or cosmetic, wherein the method comprises:
culturing an expanded cell population (for example, an organoid) of the
invention,
for example with a culture medium of the invention, optionally for less than
21 days;
exposing said expanded cell population (for example, an organoid) of the
invention
to one or a library of candidate molecules;
evaluating said expanded cell populations (for example, organoids) for any
effects,
for example any change in the cell, such as a reduction in or loss of
proliferation, a
morphological change and/or cell death;
identifying the candidate molecule that causes said effects as a potential
drug or
cosmetic; and optionally
providing said candidate molecule, e.g. as a drug or cosmetic.
In some embodiments, computer- or robot-assisted culturing and data collection
methods
are employed to increase the throughput of the screen. In some embodiments,
the
organoid is derived from a patient biopsy. In some embodiments, the candidate
molecule
that causes a desired effect on the cultured expanded cell population (for
example, an
organoid) is administered to said patient.
Accordingly, in one aspect, there is provided a method of treating a patient
comprising:
(a) obtaining a biopsy from the diseased tissue of interest in the patient;
(b) culturing the biopsy to obtain an organoid;
(c) screening for a suitable drug using a screening method of the invention;
and
(d) treating said patient with the drug obtained in step (c).
In some embodiments, the drug or cosmetic is used for treating, preventing or
ameliorating
symptoms of genetic diseases, metabolic diseases, pathogenic diseases,
inflammatory
diseases etc, for example including, but not limited to: cystic fibrosis,
inflammatory bowel
disease (such as Crohn's disease), carcinoma, adenoma, adenocarcinoma, colon
cancer,
diabetes (such as type I or type II), Barrett's esophagus, Gaucher's disases,
alpha-1-
antitrypsin deficiency, Lesch-Nyhan syndrome, anaemia, Schwachman-Bodian-
Diamond

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syndrome, polycythaemia vera, primary myelofibrosis, glycogen storage disease,
familial
hypercholestrolaemia, Crigler-Najjar syndrome, hereditary tyrosinanaemia,
Pompe
disease, progressive familial cholestasis, Hreler syndrome, SCID or leaky
SCID, Omenn
syndrome, Cartilage-hair hypoplasia, Herpes simplex encephalitis, Scleroderma,
Osteogenesis imperfecta, Becker muscular dystrophy, duch*enne muscular
dystrophy,
Dyskeratosis congenitor etc.
In some embodiments, the invention provides methods for screening for drugs
for
regenerative medicine, e.g. drugs for regenerating the liver.
Target discovery
In some embodiments, the organoids of the invention can be used for target
discovery.
Cells of the organoids originating from healthy or diseased tissue may be used
for target
identification. The organoids of the invention may be used for discovery of
drug targets for
cystic fibrosis, inflammatory bowel disease (such as Crohn's disease),
carcinoma,
adenoma, adenocarcinoma, colon cancer, diabetes (such as type I or type II),
Barrett's
esophagus Gaucher's disease, alpha-1-antitrypsin deficiency, Lesch-Nyhan
syndrome,
anaemia, Schwachman-Bodian-Diamond syndrome, polycythaemia vera, primary
myelofibrosis, glycogen storage disease, familial hypercholestrolaemia,
Crigler-Najjar
syndrome, hereditary tyrosinanaemia, Pompe disease, progressive familial
cholestasis,
Hreler syndrome, SCID or leaky SCID, Omenn syndrome, Cartilage-hair
hypoplasia,
Herpes simplex encephalitis, Scleroderma, Osteogenesis imperfecta, Becker
muscular
dystrophy, duch*enne muscular dystrophy, Dyskeratosis congenitor etc. Organoids

cultured according to the media and methods of the invention are thought to
faithfully
represent the in vivo situation. For this reason they can be a tool to find
novel (molecular)
targets in specific diseases.
To search for a new drug target, a library of compounds (such as siRNA) may be
used to
transduce the cells and inactivate specific genes. In some embodiments, cells
are
transduced with siRNA to inhibit the function of a (large) group of genes. Any
functional
read out of the group of genes or specific cellular function can be used to
determine if a
target is relevant for the study. A disease-specific read out can be
determined using
assays well known in the art. For example, cellular proliferation is assayed
to test for
genes involved in cancer. For example, a Topflash assay as described herein,
may be
used to detect changes in Wnt activity caused by siRNA inhibition. Where
growth reduction
or cell death occurs, the corresponding siRNA related genes can be identified
by methods
known in the art. These genes are possible targets for inhibiting growth of
these cells.
Upon identification, the specificity of the identified target for the cellular
process that was

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studied will need to be determined by methods well known in the art. Using
these methods,
new molecules can be identified as possible drug targets for therapy.
Target and drug validation screens
Patient-specific organoids obtained from diseased and/or normal tissue can be
used for
5 target validation of molecules identified in high throughput screens. The
same goes for the
validation of compounds that were identified as possible therapeutic drugs in
high
throughput screens. The use of primary patient material expanded in the
organoid culture
system can be useful to test for false positives, etc from high throughput
drug discovery
cell line studies.
10 In some embodiments, the organoid of the invention can be used for
validation of
compounds that have been identified as possible drugs or cosmetics in a high-
throughput
screen.
Toxicity assay
Said expanded stem cell population (for example, organoid of the invention),
such as liver,
15 intestinal organoids or pancreatic organoids, can further replace the
use of cell lines such
as Caco-2 cells in toxicity assays of potential novel drugs or of known or
novel food
supplements.
Toxicology screens work in a similar way to drug screens (as described above)
but they
test for the toxic effects of drugs and not therapeutic effects. Therefore, in
some
20 embodiments, the effects of the candidate compounds are toxic.
Culturing pathogens
Furthermore, an organoid of the invention can be used for culturing of a
pathogen, such as
a norovirus which presently lacks a suitable tissue culture or animal model.
Regenerative medicine and transplantation
25 The invention provides the use of organoids in regenerative medicine
and/or
transplantation. The invention also provides methods of treatment wherein the
method
comprises transplanting an organoid into an animal or human.
The organoids can be transplanted into an animal or human and fully
differentiate into
functional cells in vivo (for example, see Example 16). Alternatively, the
organoids can be
30 differentiated in vitro, e.g. using the differentiation media and
methods of the invention,
prior to transplantation into a human or animal (for example, see Example 16).
Organoids of the invention, such as liver, intestinal organoids or pancreatic
organoids are
useful in regenerative medicine, for example in treatment of cirrhosis of the
liver, post-

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radiation and/or post-surgery repair of the intestinal epithelium, in the
repair of the
intestinal epithelium in patients suffering from inflammatory bowel disease
such as Crohn's
disease and ulcerative colitis, and in the repair of the intestinal epithelium
in patients
suffering from short bowel syndrome. Further use is present in the repair of
the intestinal
epithelium in patients with hereditary diseases of the small intestine/colon.
Cultures
comprising pancreatic organoids are also useful in regenerative medicine, for
example as
implants after resection of the pancreas or part thereof and for treatment of
diabetes such
as diabetes I and diabetes II.
In an alternative embodiment, the organoids or cells isolated from the
organoids are are
reprogrammed into related tissue fates such as, for example, pancreatic cells
including
pancreatic beta-cells or hepatocytes or ductal cells for the liver. The
culturing methods of
the present invention will enable to analyse for factors that trans-
differentiate the closely
related epithelial stem cell to a pancreatic cell, including a pancreatic beta-
cell or a
hepatocyte.
It will be clear to a skilled person that gene therapy can additionally be
used in a method
directed at repairing damaged or diseased tissue. Use can, for example, be
made of an
adenoviral or retroviral gene delivery vehicle to deliver genetic information,
like DNA and/or
RNA to stem cells. A skilled person can replace or repair particular genes
targeted in gene
therapy. For example, a normal gene may be inserted into a nonspecific
location within the
genome to replace a nonfunctional gene. In another example, an abnormal gene
sequence
can be replaced for a normal gene sequence through hom*ologous recombination.
Alternatively, selective reverse mutation can return a gene to its normal
function. A further
example is altering the regulation (the degree to which a gene is turned on or
off) of a
particular gene. Preferably, the epithelial stem cells of an organoid or
derived from an
organoid are ex vivo treated by a gene therapy approach and are subsequently
transferred
to the mammal, preferably a human being in need of treatment.
Since small biopsies taken from adult donors can be expanded without any
apparent limit
or genetic harm, the technology may serve to generate transplantable
epithelium for
regenerative purposes. The fact that organoids can be frozen and thawed and
put into
culture without losing their 3D structure and integrity and without
significant cell death
further adds to the applicability of organoids for transplantation purposes.
Furthermore, in
some embodiments, organoids embedded in, or in contact with, an ECM can be
transplanted into a mammal, preferably into a human. In another embodiment,
organoids
and ECM can be transplanted simultaneously into a mammal, preferably into a
human.

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The skilled person will understand that an ECM can be used as a 3D scaffold
for obtaining
tissue-like structures comprising expanded populations of cells or organoids
according to
the invention. Such structures can then be transplanted into a patient by
methods well
known in the art. An ECM scaffold can be made synthetically using ECM
proteins, such as
collagen and/or laminin, or alternatively an ECM scaffold can be obtained by
"decellularising" an isolated organ or tissue fragment to leave behind a
scaffold consisting
of the ECM (for example see Macchiarini et al. The Lancet, Volume 372, Issue
9655,
Pages 2023 - 2030, 2008). In some embodiments, an ECM scaffold can be obtained
by
decellularising an organ or tissue fragment, wherein optionally said organ or
tissue
fragment is from the pancreas, liver, intestine, stomach or prostate.
The invention provides an organoid of the invention or cells derived from said
organoid for
use in transplantation into a mammal, preferably into a human. Also provided
is a method
of treating a patient in need of a transplant comprising transplanting an
organoid of the
invention or cells derived from said organoid into said patient, wherein said
patient is a
mammal, preferably a human.
Advantageously, the invention enables a small biopsy to be taken from an adult
donor and
expanded without any apparent limit or genetic harm and so the technology
provided
herein may serve to generate transplantable epithelium for regenerative
purposes.
The invention provides a method of treating an insulin-deficiency disorder
such as diabetes
in a patient, or a patient having a dysfunctional pancreas, comprising
transplanting a
pancreatic organoid of the invention or cells from a pancreatic organoid of
the invention
into the patient. The invention also provides a method for treating a liver
disease or
condition in a patient, wherein said method comprises transplanting a liver
organoid, or
cells from a liver organoid of the invention, into a patient.
In some embodiments, the cells or organoid do not express or secrete insulin
upon
transplantation into the patient but differentiate within the patient such
that they secrete
insulin. For example, the ability to secrete insulin may not be detectable
immediately upon
transplantation, but may be present by about one month after transplantation,
for example,
by 6 weeks, 2 months or 3 months after transplantation.
The patient is preferably a human, but may alternatively be a non-human
mammal, such
as a cat, dog, horse, cow, pig, sheep, rabbit or mouse.
Thus, included within the scope of the invention are methods of treatment of a
human or
non-human animal patient through cellular therapy. Such cellular therapy
encompasses
the application of the stem cells or organoids of the invention to the patient
through any
appropriate means. Specifically, such methods of treatment involve the
regeneration of

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damaged tissue. In accordance with the invention, a patient can be treated
with allogeneic
or autologous stem cells or organoids. "Autologous" cells are cells which
originated from
the same organism into which they are being re-introduced for cellular
therapy, for
example in order to permit tissue regeneration. However, the cells have not
necessarily
been isolated from the same tissue as the tissue they are being introduced
into. An
autologous cell does not require matching to the patient in order to overcome
the problems
of rejection. "Allogeneic" cells are cells which originated from an individual
which is
different from the individual into which the cells are being introduced for
cellular therapy,
for example in order to permit tissue regeneration, although of the same
species. Some
degree of patient matching may still be required to prevent the problems of
rejection. Thus
in some embodiments the transplantation involves autologous cells. In some
embodiments, the transplantation involves allogeneic cells.
Generally the cells or organoids of the invention are introduced into the body
of the patient
by injection or implantation. Generally the cells will be directly injected
into the tissue in
which they are intended to act. Alternatively, the cells will be injected
through the portal
vein. In some embodiments, the cells are injected into an artery. In some
embodiments,
the cells are injected intrasplenically. For humans, injection through the
portal vein or into
an artery is preferred. A syringe containing cells of the invention and a
pharmaceutically
acceptable carrier is included within the scope of the invention. A catheter
attached to a
syringe containing cells of the invention and a pharmaceutically acceptable
carrier is
included within the scope of the invention.
The skilled person will be able to select an appropriate method and route of
administration
depending on the material that is being transplanted (i.e. population of
cells, single cells in
cell suspension, organoids or fragments of organoids) as well as the organ
that is being
treated.
As discussed above, organoids or cells of the invention can be used in the
regeneration of
tissue. In order to achieve this function, cells may be injected or implanted
directly into the
damaged tissue, where they may multiply and eventually differentiate into the
required cell
type, in accordance with their location in the body. Alternatively, the
organoid can be
injected or implanted directly into the damaged tissue. Tissues that are
susceptible to
treatment include all damaged tissues, particularly including those which may
have been
damaged by disease, injury, trauma, an autoimmune reaction, or by a viral or
bacterial
infection. In some embodiments of the invention, the cells or organoids of the
invention
are used to regenerate the colon, small intestine, pancreas, oesophagus or
gastric system.

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For example, in one embodiment, the cells or organoids of the invention are
injected into a
patient using a Hamilton syringe.
The skilled person will be aware what the appropriate dosage of cells or
organoids of the
invention will be for a particular condition to be treated.
In one embodiment the organoids or cells of the invention, either in solution,
in
microspheres or in microparticles of a variety of compositions, will be
administered into the
artery irrigating the tissue or the part of the damaged organ in need of
regeneration.
Generally such administration will be performed using a catheter. The catheter
may be one
of the large variety of balloon catheters used for angioplasty and/or cell
delivery or a
catheter designed for the specific purpose of delivering the cells to a
particular local of the
body. For certain uses, the cells or organoids may be encapsulated into
microspheres
made of a number of different biodegradable compounds, and with a diameter of
about 15
pm. This method may allow intravascularly administered cells or organoids to
remain at
the site of damage, and not to go through the capillary network and into the
systemic
circulation in the first passage. The retention at the arterial side of the
capillary network
may also facilitate their translocation into the extravascular space.
In another embodiment, the organoids or cells may be retrograde injected into
the vascular
tree, either through a vein to deliver them to the whole body or locally into
the particular
vein that drains into the tissue or body part to which the cells or organoids
are directed. For
this embodiment many of the preparations described above may be used.
In another embodiment, the cells or organoids of the invention may be
implanted into the
damaged tissue adhered to a biocompatible implant. Within this embodiment, the
cells
may be adhered to the biocompatible implant in vitro, prior to implantation
into the patient.
As will be clear to a person skilled in the art, any one of a number of
adherents may be
used to adhere the cells to the implant, prior to implantation. By way of
example only, such
adherents may include fibrin, one or more members of the integrin family, one
or more
members of the cadherin family, one or more members of the selectin family,
one or more
cell adhesion molecules (CAMs), one or more of the immunoglobulin family and
one or
more artificial adherents. This list is provided by way of illustration only,
and is not intended
to be limiting. It will be clear to a person skilled in the art, that any
combination of one or
more adherents may be used.
In another embodiment, the organoids or cells of the invention may be embedded
in a
matrix, prior to implantation of the matrix into the patient. Generally, the
matrix will be
implanted into the damaged tissue of the patient. Examples of matrices include
collagen
based matrices, fibrin based matrices, laminin based matrices, fibronectin
based matrices

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and artificial matrices. This list is provided by way of illustration only,
and is not intended to
be limiting.
In a further embodiment, the organoids or cells of the invention may be
implanted or
injected into the patient together with a matrix forming component. This may
allow the cells
5 to form a matrix following injection or implantation, ensuring that the
cells or organoids
remain at the appropriate location within the patient. Examples of matrix
forming
components include fibrin glue liquid alkyl, cyanoacrylate monomers,
plasticizers,
polysaccharides such as dextran, ethylene oxide-containing oligomers, block co-
polymers
such as poloxamer and Pluronics, non-ionic surfactants such as Tween and
Triton'8', and
10 artificial matrix forming components. This list is provided by way of
illustration only, and is
not intended to be limiting. It will be clear to a person skilled in the art,
that any
combination of one or more matrix forming components may be used.
In a further embodiment, the organoids or cells of the invention may be
contained within a
microsphere. Within this embodiment, the cells may be encapsulated within the
centre of
15 the microsphere. Also within this embodiment, the cells may be embedded
into the matrix
material of the microsphere. The matrix material may include any suitable
biodegradable
polymer, including but not limited to alginates, Poly ethylene glycol (PLGA),
and
polyurethanes. This list is provided by way of example only, and is not
intended to be
limiting.
20 In a further embodiment, the cells or organoids of the invention may be
adhered to a
medical device intended for implantation. Examples of such medical devices
include
stents, pins, stitches, splits, pacemakers, prosthetic joints, artificial
skin, and rods. This list
is provided by way of illustration only, and is not intended to be limiting.
It will be clear to a
person skilled in the art, that the cells may be adhered to the medical device
by a variety of
25 methods. For example, the cells or organoids may be adhered to the
medical device using
fibrin, one or more members of the integrin family, one or more members of the
cadherin
family, one or more members of the selectin family, one or more cell adhesion
molecules
(CAMs), one or more of the immunoglobulin family and one or more artificial
adherents.
This list is provided by way of illustration only, and is not intended to be
limiting. It will be
30 clear to a person skilled in the art, that any combination of one or
more adherents may be
used.
The organoid or population of epithelial stem cells or population of
differentiated cells
obtained using a method of the invention have a variety of uses. For example,
the
invention provides the use of the organoid or population of epithelial stem
35 cells/differentiated cells as described herein in a drug discovery
screen; toxicity assay;

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research of embryology, cell lineages, and differentiation pathways; gene
expression
studies including recombinant gene expression; research of mechanisms involved
in injury
and repair; research of inflammatory and infectious diseases; studies of
pathogenetic
mechanisms; or studies of mechanisms of cell transformation and aetiology of
cancer.
In one aspect, the invention provides the use of an organoid or population of
epithelial
stem cells/differentiated cells as described herein in a drug discovery
screen, toxicity
assay or in regenerative medicine. Similarly, the invention provides the use
of the progeny
of organoids of the invention for these uses.
Toxicity assays may be in vitro assays using an organoid or part thereof or a
cell derived
from an organoid. Such progeny and organoids are easy to culture and more
closely
resemble primary epithelial cells than, for example, epithelial cell lines
such as Caco-2
(ATCC HTB-37), 1-407 (ATCC CCL6), and XBF (ATCC CRL 8808) which are currently
used in toxicity assays. It is anticipated that toxicity results obtained with
organoids more
closely resemble results obtained in patients. A cell-based toxicity test is
used for
determining organ specific cytotoxicity. Compounds that are tested in said
test comprise
cancer chemopreventive agents, environmental chemicals, food supplements, and
potential toxicants. The cells are exposed to multiple concentrations of a
test agent for
certain period of time. The concentration ranges for test agents in the assay
are
determined in a preliminary assay using an exposure of five days and log
dilutions from the
highest soluble concentration. At the end of the exposure period, the cultures
are
evaluated for inhibition of growth. Data are analysed to determine the
concentration that
inhibited end point by 50 percent (TC50).
For example, according to this aspect of the invention, a candidate compound
may be
contacted with cell or organoid as described herein, and any change to the
cells or in
activity of the cells may be monitored.
For high-throughput purposes, said organoids are cultured in multiwell plates
such as, for
example, 96 well plates or 384 well plates. Libraries of molecules are used to
identify a
molecule that affects said organoids. Preferred libraries comprise antibody
fragment
libraries, peptide phage display libraries, peptide libraries (e.g. LOPAPTM,
Sigma Aldrich),
lipid libraries (BioMol), synthetic compound libraries (e.g. LOP ACTM, Sigma
Aldrich) or
natural compound libraries (Specs, TimTec). Furthermore, genetic libraries can
be used
that induce or repress the expression of one of more genes in the progeny of
the adenoma
cells. These genetic libraries comprise cDNA libraries, antisense libraries,
and siRNA or
other non-coding RNA libraries. The cells are preferably exposed to multiple
concentrations of a test agent for certain period of time. At the end of the
exposure period,

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the cultures are evaluated. The term "affecting" is used to cover any change
in a cell,
including, but not limited to, a reduction in, or loss of, proliferation, a
morphological
change, and cell death. Said organoids can also be used to identify drugs that
specifically
target epithelial carcinoma cells, but not said organoids.
Organoids according to the invention can further replace the use of cell lines
such as
Caco-2 cells in drug discovery screens and in toxicity assays of potential
novel drugs or
known drugs or known or novel food supplements.
Furthermore, such organoids can be used for culturing of a pathogen.
The invention further provides an organoid of the invention or a cell derived
from said
organoid of the invention for use in therapy. In one embodiment, there is
provided an
expansion organoid of the invention or cell derived from said organoid of the
invention for
use in therapy. In another embodiment, there is provided a differentiated
organoid of the
invention or a population of differentiated cells of the invention for use in
therapy. Also
provided is an organoid of the invention or a cell derived from said organoid
for use in
treating a disease or condition as described herein.
Similarly, there is provided a method of treating a disease or condition as
described herein
comprising administering one or more organoids of the invention, or cell
derived from said
organoid.
The inventors have also demonstrated successful transplantation of organoids
into
immunodeficient mice (see example 7 of WO 2012/014076), with transplanted
liver
organoid-derived cells generating both cholangyocytes and hepatocytes in vivo.
Therefore,
in one embodiment the invention provides organoids or organoid-derived cells
of the
invention for transplanting into human or animals.
In some embodiments, the expanded organoids, or cells therefrom, are
transplanted into a
human or animal. These organoids or cells may differentiate in vivo (e.g. see
example 16).
In this embodiment, it may be advantageous to administer factors that promote
differentiation to the human or animal, either locally or systemically. For
example, in some
embodiments, the human or animal may be administered forskolin in before,
during and/or
after transplantation. In an alternative embodiment, the organoids are
differentiated in vitro
prior to transplantation into the human or animal, preferably using a culture
medium and
method described herein.
The use of human organoids for transplantation purposes is advantageous over
the use of
fetal or adult cells (e.g. hepatocytes for the liver or beta cells for the
pancreas) for a
number of reasons. Firstly, the culture methods of the invention provide
unlimited
expansion of cells and hence, an unlimited supply. In particular, the
inventors have shown

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that under the correct culture conditions (e.g. using the expansion culture
medium of the
invention), that Lgr5+ cells can undergo more than 1000 divisions in vitro.
Therefore, Lgr5+
cells can be extracted from the organoids and repassaged providing a continual
self-
renewing source of transplantable cells with differentiation potential. For
example, Lgr5+
cells can be extracted from liver organoids and repassaged providing a
continual self-
renewing source of transplantable hepatocyte- and cholangiocyte-generating
cells with
differentiation potential. By contrast, fetal or adult cells (such as
hepatocytes or beta cells)
which are derived from donor organs only provide a single round of
transplantation.
Furthermore, donor cells can only be kept alive for a few days but lose their
phenotypic
properties. This means the transplants must be made as soon as the donor
becomes
available. Organoid-derived cells, on the other hand, retain their phenotype
over multiple
divisions and over prolonged periods of time meaning that they are ready and
available for
transplantation at any stage. This could also allow the organoid-derived cells
to be used as
a temporary treatment to extend the lifespan of patients for patients on the
waiting list for
transplants. A further advantage of the organoids of the invention is that
they can be frozen
and later be defrosted without loss of function. This enables cell banking,
easy storage and
rapid availability for acute use. This could be useful for example, in the
preparation of an
"off-the-shelf" product, for example, in the case of liver, that might be used
for the
treatment of acute liver toxicity. Organoids can also be grown from cells or
tissue
fragments taken as small biopsies from live donors minimising any ethical
objections to the
treatment. The donor may even be from the patient that is to be treated, which
could
reduce any negative side-effects associated with transplantation of foreign
cells and
organs and reduce the need for immunosuppressive drugs.
In some embodiments, the invention also provides a pharmaceutical formulation
comprising the components of the culture medium described herein and a
pharmaceutically acceptable diluent and/or excipient. For example, there is
provided a
pharmaceutical formulation comprising a Wnt agonist (e.g. Rspondin), a TGF
beta
inhibitor, and a cAMP pathway activator (e.g. forskolin) and a
pharmaceutically acceptable
diluent and/or excipient. There is also provided a pharmaceutical formulation
comprising
one or more of an EGF receptor activator (e.g. EGF), a Wnt agonist (e.g.
Rspondin), an
FGF receptor 2 or FGF receptor 4 activator (e.g. FGF), an HGF receptor
activator (e.g.
HGF), a TGF beta inhibitor and Nicotinamide, and a cAMP pathway activator
(e.g.
forskolin) and a pharmaceutically acceptable diluent and/or excipient. In a
preferred
embodiment, the pharmaceutical formulation does not comprise a basal medium.
In some
embodiments, the pharmaceutical formulation does not comprise an extracellular
matrix. It
is envisaged that such formulations may be suitable for promoting expansion of
stem cells

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in vivo, e.g. for regenerative therapy. Such formulations may be administered
in situ (e.g.
at the site of tissue damage) or systemically. Alternatively, the formulations
may be
formulated so that it is suitable for administration by any administration
routes known in the
art, for example intravenous, subcutaneous, intramuscular administration,
mucosa!,
intradermal, intracutaneous, oral, and ocular. A pharmaceutical formulation
may be thus be
in any form suitable for such administration, e.g. a tablet, infusion fluid,
capsule, syrup, etc.
Accordingly, included within the scope of the invention are methods of
treatment of a
human or non-human animal patient through cellular therapy. The term "animal"
here
denotes all mammalian animals. The patient may be at any stage of development,
including embryonic and foetal stages. For example, the patient may be an
adult, or the
therapy may be for pediatric use (e.g. newborn, child or adolescent). Such
cellular therapy
encompasses the administration of cells or organoids generated according to
the invention
to a patient through any appropriate means. Specifically, such methods of
treatment
involve the regeneration of damaged tissue. The term "administration" as used
herein
refers to well recognized forms of administration, such as intravenous or
injection, as well
as to administration by transplantation, for example transplantation by
surgery, grafting or
transplantation of tissue engineered cell populations derived from cells or
organoids
according to the present invention. In the case of cells, systemic
administration to an
individual may be possible, for example, by infusion into the superior
mesenteric artery, the
celiac artery, the subclavian vein via the thoracic duct, infusion into the
heart via the
superior vena cava, or infusion into the peritoneal cavity with subsequent
migration of cells
via subdiaphragmatic lymphatics, or directly into liver sites via infusion
into the hepatic
arterial blood supply or into the portal vein.
In some embodiments, between 104 and 1013 cells per 100 kg person are
administered per
infusion. Preferably, between about 1-5x104 and 1-5x107 cells may be infused
intravenously per 100 kg person. More preferably, between about 1x104 and
10x106 cells
may be infused intravenously per 100 kg person. In some embodiments, a single
administration of cells or organoids is provided. In other embodiments,
multiple
administrations are used. Multiple administrations can be provided over an
initial treatment
regime, for example, of 3-7 consecutive days, and then repeated at other
times.
It is also possible to reconstitute an organoid from one single cell
expressing Lgr5 as
defined herein. This single cell may have been modified by introduction of a
nucleic acid
construct as defined herein, for example, to correct a genetic deficiency or
mutation. It
would also be possible to specifically ablate expression, as desired, for
example, using
siRNA. Potential polypeptides to be expressed could be any of those that are
deficient in

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metabolic diseases, including, for example, a polypeptide deficiency in
metabolic liver
disease, such as AAT (alpha antitrypsin). For elucidating physiology, we might
also
express or inactivate genes implicated in the Wnt, EGF, FGF, BMP or notch
pathway.
Also, for screening of drug toxicity, the expression or inactivation of genes
responsible for
5 liver drug metabolism (for example, genes in the CYP family) would be of
high interest.
This is particularly relevant when the cells of the invention are liver cells.
In one embodiment, the expanded epithelial stem cells may be reprogrammed into
related
tissue fates such as, for example, liver cells including a hepatocyte and a
cholangiocyte
cell. The culturing methods of the present invention will enable to analyse
for factors that
10 trans-differentiate the closely related epithelial stem cell to a liver
cell, including a
hepatocyte and a cholangiocyte cell.
It will be clear to a skilled person that gene therapy can additionally be
used in a method
directed at repairing damaged or diseased tissue. Use can, for example, be
made of an
adenoviral or retroviral gene delivery vehicle to deliver genetic information,
like DNA and/or
15 RNA to stem cells. A skilled person can replace or repair particular
genes targeted in gene
therapy. For example, a normal gene may be inserted into a nonspecific
location within the
genome to replace a non-functional gene. In another example, an abnormal gene
sequence can be replaced for a normal gene sequence through hom*ologous
recombination. Alternatively, selective reverse mutation can return a gene to
its normal
20 function. A further example is altering the regulation (the degree to
which a gene is turned
on or off) of a particular gene. Preferably, the stem cells are ex vivo
treated by a gene
therapy approach and are subsequently transferred to the mammal, preferably a
human
being in need of treatment. For example, organoid-derived cells may be
genetically
modified in culture before transplantation into patients.
25 Thus, in some embodiments, the organoid or population of epithelial stem
cells is for use in
medicine, e.g. for treating a disorder, condition or disease and/or for use in
regenerative
medicine.
In one preferred embodiment, for example, if an organoid is to be used for
regenerative
medicine, the method may start from epithelial cells or from a tissue fragment
in which the
30 cells or tissue fragment are autologous or allogeneic. "Autologous"
cells are cells which
originated from the same organism into which they are being re-introduced for
cellular
therapy, for example in order to permit tissue regeneration. An autologous
cell does not, in
principle, require matching to the patient in order to overcome the problems
of immune
rejection, and/or reduces the need for immune suppression interventions upon
transplant.
35 "Allogeneic" cells are cells which originated from an individual which
is different from the

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individual into which the cells are being introduced for cellular therapy, for
example in order
to permit tissue regeneration, although of the same species. Some degree of
patient
matching may still be required to prevent the problems of rejection.
Techniques for
minimising tissue rejection will be known to those of skill in the art.
In embodiments in which the organoids and/or cells are transplanted into a
patient, it can
be advantageous to administer the cells in a scaffold. Accordingly, there is
provided a
scaffold comprising one or more organoids of the invention or cells derived
from said
organoids. A scaffold provides a two-dimensional or three dimensional network.
Suitable
synthetic materials for such a scaffold comprise polymers selected from porous
solids,
nanofibers, and hydrogels such as, for example, peptides including self-
assembling
peptides, hydrogels composed of polyethylene glycol phosphate, polyethylene
glycol
fumarate, polyacrylamide, polyhydroxyethyl methacrylate, polycellulose
acetate, and/or co-
polymers thereof (see, for example, Saha et al., 2007. Curr Opin Chem Biol.
11(4): 381-
387; Saha et al., 2008. Biophysical Journal 95: 4426-4438; Little et al.,
2008. Chem. Rev
108, 1787-1796). As is known to a skilled person, the mechanical properties
such as, for
example, the elasticity of the scaffold influences proliferation,
differentiation and migration
of stem cells. A preferred scaffold comprises biodegradable (co)polymers that
are replaced
by natural occurring components after transplantation in a subject, for
example to promote
tissue regeneration and/or wound healing. It is furthermore preferred that
said scaffold
does not substantially induce an immunogenic response after transplantation in
a subject.
Said scaffold is supplemented with natural, semi-synthetic or synthetic
ligands, which
provide the signals that are required for proliferation and/or
differentiation, and/or migration
of stem cells. In a preferred embodiment, said ligands comprise defined amino
acid
fragments. Examples of said synthetic polymers comprise Pluronic F127 block
copolymer
surfactant (BASF), and Ethisorb (Johnson and Johnson). In some embodiments
the
cells are cultured in the scaffold. In other embodiments, they are cultured
and then added
to the scaffold.
The uses of the present invention may use a single organoid or they may use
more than
one organoid, for example, 2, 3, 4, 5, 10, 15, 20, 30, 50, 100, 200 or more
organoids.
Advantageously, the methods of the present invention allow a great number of
organoids
and epithelial stem cells to be generated in a short period of time, because
they result in
exponential growth, thereby ensuring that sufficient cells are available for
use in the
application of interest. Wherever there is reference herein to a "method of
treatment" or
"method for treatment", for example involving the organoids or cells obtained
from the
organoids of the invention, this also refers equally to organoids or cells
"for use in
treatment" and to organoids or cells "for use in the manufacture of a
medicament".

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The invention is exemplified below in relation to the liver and pancreas.
Expansion medium (EM) for liver
The inventors previously described a culture medium suitable for culturing
liver epithelial
stem cells which contained EGF, a Wnt agonist, FGF and Nicotinamide in
W02012/014076 and W02012/168930. It has surprisingly been found that adding a
cAMP pathway activator to the culture medium allows human liver epithelial
stem cells to
be cultured long-term. Although the culture medium described in W02012/168930
and
W02012/014076 could be used for long term culture of mouse cells, it could
only be used
for short term culture of human cells for around 5 passages. In contrast, use
of a culture
medium comprising a cAMP pathway activator allows the cells to be passaged
many more
times, for example, 16 or more (see Figure 2).
Culturing the cells in an expansion medium allows the cells to multiply whilst
retaining their
progenitor cell phenotype. Organoids are formed comprising these progenitor
cells. Use
of the expansion medium is therefore advantageous for providing increased
numbers of
these useful progenitor cells and for obtaining organoids containing these
cells.
Accordingly, the invention provides a liver expansion medium comprising an EGF
receptor
activator (e.g. EGF), a Wnt agonist, an FGF receptor 2 or FGF receptor 4
activator (e.g.
FGF), an HGF receptor activator (e.g. HGF), a TGF beta inhibitor and
Nicotinamide,
characterised in that the expansion medium further comprises a cAMP pathway
activator
and/or a BMP activator.
Expansion Medium (EM1) for liver
In some embodiments, additional components are added at the beginning of the
culture
process to establish the culture. The inventors have found that a liver
expansion medium
as described herein additionally comprising Wnt and a BMP inhibitor (e.g.
Noggin) is ideal
for stimulating initial expansion of cells. The inventors have found that this
medium is
optimal for stimulating initial expansion of cells for the first few days.
Therefore, this first
expansion medium is sometimes referred to herein as EMI.
In alternative embodiments, EM1 is an EM1 as described in W02012/168930 or
W02012/014076. Thus, in some embodiments, EM1 does not comprise a cAMP pathway
activator and/or a BMP activator. In some embodiments, EM1 does not comprise a
BMP
activator. In some embodiments, EM1 comprises a cAMP pathway activator but
does not
comprise a BMP activator.
In some embodiments, the EM1 is used for just 1 passage or 1 week. In
embodiments in
which EM1 comprises a serum component, it is advantageous to culture the cells
in EM1

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for no longer than about a week because the serum component can negatively
affect the
cells. Thus, in some embodiments, an EM1 medium is used for culturing cells
from day 0
to day 10, for example from days 0-7, days 0-6, days 0-5, days 0-4, days 0-3,
days 0-2,
days 0-1, wherein day 0 is the day that the cells are isolated from their
tissue of origin and
day 1 is the subsequent day. In some embodiments, the EM1 medium is used only
for the
first day, first two days or first three to four days of culture. In some
embodiments, EM1
medium is used for only 1 passage, or for no more than 2 or 3 passages.
However, the
cells can be cultured in EM1 for longer periods if required.
Thus, in some embodiments, the Wnt and the BMP inhibitor (e.g. Noggin) are
removed or
not replenished after approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6
days, 7 days
or less, for example 3-4 days.
In some embodiments, the EM1 medium is used subsequent to a freezing step or
any
other transportation step involving a medium or temperature change that does
not combine
with optimal growth. This "EM1" medium is preferred for expanding liver cells
during the
first few days of culture.
Accordingly, the invention provides a liver EM1 as described herein, methods
of using
EM1 as described herein, as well as uses of EM1 as described herein.
More specifically, the invention provides an expansion medium as described
herein, which
additionally comprises Wnt and a BMP inhibitor.
In some embodiments, the EM1 of the invention comprises or consists of a basal
medium
for animal or human cells to which is added EGF, a Wnt agonist (e.g.
Rspondin), FGF (e.g.
FGF10), HGF, a TGF beta inhibitor, Nicotinamide, a BMP inhibitor and Wnt. This
EM1
may further comprise a cAMP pathway activator. In some embodiments, this EM1
further
comprises gastrin. An example of this EM1 medium is termed "ENRFHWNicTi (EGF,
Noggin, Rspondin, FGF, HGF, Wnt (e.g. Wnt 3a), Nicotinamide, TGF beta
inhibitor)
supplemented with a cAMP pathway activator".
Any suitable Wnt may be used in EM1. Wnt3a is a preferred example. Other
examples
include Wnt CM (Barker and Huch, Cell Stem Cell, 2010, 8, 6(1):25-36),
CHIR9901 (e.g.
Stemgent), Wnt3a recombinant protein, or any other GSK3b inhibitor.
Wnt conditioned media (Wnt CM) preferably comprises Advanced DMEM, P/S, B27,
N2
and also FCS. 293T cells transfected with Wnt3A expression plasmid produce
Wnt. The
whole medium is taken after a few days (i.e. with secreted Wnt) and used as
the Wnt
source.

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In some embodiments, EM1 comprises EGF, a Wnt agonist (e.g. any one of
Rspondin 1-
4), FGF (e.g. FGF10), HGF, Nicotinamide, gastrin, a TGF-beta inhibitor, a cAMP
pathway
activator (e.g. forskolin), Noggin and Wnt.
In some embodiments, EM1 is supplemented with a ROCK inhibitor. This is
particularly
advantageous in the case of starting the cultures from a frozen stock or from
a single cell.
Y27632 is the preferred ROCK inhibitor for use in the invention.
Thus, in some embodiments, immediately after initiation of the culture the
expansion
medium is supplemented with one or more (e.g. 2, 3 or all) of a BMP inhibitor
(e.g.
Noggin), Wnt (e.g. Wnt CM, Wnt3a recombinant protein, CHIR9901 or any other
GSK3b
inhibitor), and a ROCK inhibitor (e.g. Y27632).
In some embodiments, the expansion medium is additionally supplemented with
thiazovivin (for example at about 2uM, conveniently available through use of
hES cell
cloning recovery solution (Stemgent)). Use of thiazovivin is advantageous as
it helps
increase the efficiency of organoid establishment. For example, in some
embodiments,
the expansion medium is initially supplemented with one or more (e.g. 2, 3, 4
or all) of
about 25 ng/ml Noggin, about 1 pg/ml Wnt CM (Barker and Huch, 2010, supra),
about 10
pM Y27632 and hES cell cloning recovery solution (Stegent).
Other supplementary compounds that may be added to EM1, i.e. immediately after

initiation, are one or more prostaglandin pathway activators, for example,
arachidonic acid
and/or prostaglandin E2.
Any combination of two or more of the supplementary compounds described herein
as
being suitable for use immediately after initiation may be added to the
expansion medium.
In some embodiments, EM1 comprises EGF, a Wnt agonist (e.g. Rspondin, e.g.
Rspondin
1), FGF (e.g. FGF10), HGF, Nicotinamide, a TGF beta inhibitor (e.g. A83.01),
and a cAMP
pathway activator (e.g. FSK). EM1 is optionally supplemented with one or more
of (e.g. 1,
2 or 3 of): i) N2 and B27 without retinoic acid, ii) N-Acetylcysteine, and
iii) gastrin, and
further comprises one or more of (e.g. 1, 2, 3 or 4 of): i) a BMP inhibitor
(e.g. Noggin), ii)
Wnt (e.g. Wnt CM), iii) a ROCK inhibitor (e.g. Y27632), and iv) thiazovivin
(e.g. hES Cell
cloning recovery solution).
For example, EM1 may comprise about 50 ng/ml EGF, about 1 pg/ml Wnt agonist
(e.g.
Rspondin, e.g. Rspondin 1), about 100 ng/ml FGF (e.g. FGF10), about 25 ng/ml
HGF,
about 10mM Nicotinamide, about 5 pM TGF beta inhibitor (e.g. A83.01), about
10pM
cAMP pathway activator (e.g. FSK), and further comprises one or more of (e.g.
1, 2, 3 or 4
of): i) about 25 ng/ml BMP inhibitor (e.g. Noggin), ii) about 1 pg/ml Wnt
(e.g. Wnt CM), iii)
about 10 uM ROCK inhibitor (e.g. Y27632), and iv) thiazovivin (e.g. hES Cell
cloning

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recovery solution). This EM1 is optionally supplemented with one or more of i)
N2 and B27
without retinoic acid, ii) about 1.25 mM N-Acetylcysteine, and about 10 nM
gastrin.
In a preferred embodiment, EM1 comprises EGF, Rspondin, FGF10, HGF,
Nicotinamide,
A83.01, forskolin, and further comprises one or more of (e.g. 1, 2, 3 or 4
of): i) Noggin, ii)
5 Wnt, iii) a ROCK inhibitor (e.g. Y27632), and iv) thiazovivin (e.g. hES
Cell cloning recovery
solution). In this preferred embodiment, EM1 is optionally supplemented with
one or more
of i) N2 and B27 without retinoic acid, ii) N-Acetylcysteine, and gastrin.
In an exemplified embodiment, EM1 comprises about 50 ng/ml EGF, about 1 pg/ml
Rspondin, about 100 ng/ml FGF10, about 25ng/m1 HGF, about 10mM Nicotinamide,
about
10 5uM A83.01, about 10uM forskolin, N2 and B27 without retinoic acid,
about 1.25 mM N-
Acetylcysteine, about 10 nM gastrin, about 25 ng/ml Noggin, about 1 pg/ml Wnt
CM, about
10 pM Y27632, and thiazovivin (e.g. hES Cell cloning recovery solution).
In some embodiments, the invention provides a culture medium for expanding
liver cells,
comprising or consisting of a basal medium, EGF, FGF10, HGF, any one of
Rspondin 1-4,
15 Noggin, nicotinamide, gastrin, a TGF-beta inhibitor, Wnt-3a, and a cAMP
pathway
activator.
When expanding mouse liver cells, the TGF-beta inhibitor (e.g. A83-01) may be
excluded
from the EM1 described above. When expanding human liver cells, the TGF-beta
inhibitor
is preferably present in EM1.
20 In some embodiments, the cAMP pathway activator is not present in an EM1
of the
invention. Accordingly, the statements provided above may be adapted to remove

reference to the cAMP pathway activator as necessary.
The expansion medium which additionally comprises the additional components
for
initiating the culture is referred to herein as expansion medium 1 ("EMI").
25 As discussed above, the methods may comprise culturing the stem cells in
an expansion
medium of the invention comprising supplementary components (i.e. in EM1) for
the first 1,
2, 3, 4, 5, 6, 7 or less days, preferably for the first 3 or 4 days. After
this, the method may
then comprise culturing the stem cells in an expansion medium which does not
comprise
one or both of a BMP inhibitor (such as Noggin) and Wnt. Accordingly, the
expansion
30 medium preferably does not comprise these supplementary components after
the first 3, 4,
5, 6, 7 or more days, preferably after the first 3 or 4 days.
Expansion medium 2 "EM2" for liver
The expansion medium in which the additional components for initiating the
culture are not
present is referred to herein as expansion medium 2 ("EM2").

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Accordingly, the invention provides an expansion medium of the invention, as
described
herein, which preferably does not comprise one or both of Noggin and Wnt. This

expansion medium may be referred to as "EM2". In some embodiments, EM2 does
not
comprise Noggin. In some embodiments, EM2 does not comprise a BMP inhibitor.
In
some embodiments, EM2 does not comprise Wnt. In some embodiments, EM2 does not
comprise Noggin (and preferably does not comprise a BMP inhibitor) or Wnt.
Accordingly, in some embodiments, the EM2 of the invention comprises or
consists of a
basal medium for animal or human cells to which is added EGF, a Wnt agonist,
FGF, HGF,
a TGF beta inhibitor, and Nicotinamide, and wherein EM2 is characterised in
that it further
comprises a cAMP pathway activator and/or a BMP activator. Preferably, EM2
comprises
a cAMP pathway activator and optionally additionally comprises a BMP
activator.
More preferably, in some embodiments, the EM2 of the invention comprises or
consists of
a basal medium for animal or human cells to which is added EGF, a Wnt agonist,
FGF,
HGF, a TGF beta inhibitor, and Nicotinamide, wherein the EM2 does not comprise
Noggin
(more preferably does not comprise a BMP inhibitor) and/or Wnt3a (more
preferably does
not comprise Wnt), and wherein EM2 is characterised in that it further
comprises a cAMP
pathway activator and/or a BMP activator. Preferably, EM2 comprises a cAMP
pathway
activator and optionally additionally comprises a BMP activator.
As explained above, the inventors have found that this medium may be used for
long-term
expansion of human cells. Use of this EM2 is therefore advantageous compared
to the
EM2 described in W02012/168930 and W02012/014076 which could be used for long
term culture of mouse cells, but which could not be used for long term culture
of human
cells.
In some embodiments, EM2 does not comprise Y27632. In some embodiments, EM2
does not comprise a ROCK inhibitor. Thus, in some embodiments, EM2 is an
expansion
medium of the invention which does not comprise Noggin, Wnt or a Rock
inhibitor.
In some embodiments, EM2 does not comprise any of Noggin, Wnt CM (Barker and
Huch,
2010, supra), a ROCK inhibitor (e.g. Y27632) and hES cell cloning recovery
solution
(Stegent).
In some embodiments, EM2 does not comprise a Notch inhibitor.
In some embodiments, EM2 comprises or consists of a basal medium to which is
added:
EGF, a Wnt agonist, FGF, HGF, Nicotinamide, a TGF beta inhibitor and a cAMP
pathway
activator. The Wnt agonist is optionally Rspondin (e.g. Rspondin 1, 2, 3 or 4)
and the
cAMP pathway activator is optionally forskolin (this medium is elsewhere
referred to as
ERFHNic +TGFbi + FSK).

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Advantageously, EM2 may comprise a BMP activator, for example, selected from
BMP7,
BMP4 and BMP2. BMP7 is preferred. It has surprisingly been found that adding a
BMP
activator such as BMP7 to the culture medium allows the liver cells to be
cultured for a
longer term. The invention therefore provides the use of BMP7 for culturing
epithelial stem
cells, such as liver stem cells. Similarly, the invention therefore provides
the use of a BMP
activator for culturing stem cells, such as liver stem cells. The invention
also provides a
method for culturing epithelial stem cells (particularly liver stem cells)
which uses an
expansion medium as described in W02012/014076 or W02012/168930 to which a BMP

activator such as BMP7 is added.
Advantageously, in some embodiments, BMP7 is present in EM2 but not in EM1. In
some
embodiments, about 25ng/m1 BMP7 is added. In some embodiments, BMP7 is not
present
in the EM2 of the invention and the specific examples described herein can be
adapted
accordingly.
In some embodiments, the EM2 comprises EGF, a Wnt agonist (e.g. Rspondin), FGF
(e.g.
FGF10), HGF, nicotinamide, a TGF beta inhibitor (e.g. A83.01), and a cAMP
inhibitor (e.g.
forskolin). Preferably, EM2 further comprises a BMP activator (e.g. BMP7).
For example, EM2 may comprise about 50 ng/ml EGF, about 1 pg/ml Wnt agonist
(e.g.
Rspondin), about 100 ng/ml FGF (e.g. FGF10), about 25 ng/ml HGF, about 10mM
Nicotinamide, about 5uM TGF beta inhibitor (e.g. A83.01), and about 10 pM cAMP
pathway activator (e.g. FSK) and preferably further comprises about 25 ng/ml
BMP
activator (e.g. BMP7).
In an exemplary embodiment, EM2 comprises EGF, FGF (preferably FGF10), HGF,
Rspondin, Nicotinamide, TGF-beta inhibitor (e.g. A83.01) and forskolin and
preferably
further comprises BMP7.
In a further exemplary embodiment, EM2 comprises about 50 ng/ml EGF, about 100
ng/ml
FGF10, about 25 ng/ml HGF, about 1 pg/ml Rspondin conditioned media, about 10
mM
Nicotinamide, about 5 pM A83.01 and about 10 pM forskolin and preferably
further
comprises about 25 ng/ml BMP7.
EM2 may further be supplemented with one or more of (e.g. 1, 2 or 3 of): i) N2
and B27
without retinoic acid, ii) N-Acetylcysteine, and iii) gastrin.
Thus, in one embodiment, EM2 comprises EGF, Rspondin, FGF (e.g. FGF10), HGF,
Nicotinamide, TGF beta inhibitor (e.g. A83.01), cAMP pathway activator (e.g.
FSK), BMP7,
N2 and B27 without retinoic acid, N-Acetylcysteine, and gastrin.

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For example, in a preferred embodiment EM2 comprises EGF, Rspondin, FGF10,
HGF,
Nicotinamide, A83.01, forskolin, BMP7, N2 and B27 without retinoic acid, N-
Acetylcysteine, and gastrin. In an exemplified embodiment, EM2 comprises about
50
ng/ml EGF, about 1 pg/ml Rspondin, about 100 ng/ml FGF10, about 25 ng/ml HGF,
about
10 mM Nicotinamide, about 5 pM A83.01, about 10 pM forskolin, about 25 ng/ml
BMP7,
N2 and B27 without retinoic acid, about 1.25 mM N-Acetylcysteine, and about 10
nM
gastrin.
The basal medium used in EM2 is preferably supplemented with B27, N2 and N-
Acetylcysteine. In some embodiments, the basal medium is Advanced-DMEM/F12.
However, any other suitable basal medium may be used.
In some embodiments, the medium used as an EM1 cell culture medium comprises
all the
components of an EM2 culture medium of the invention and additionally
comprises Wnt
and a BMP inhibitor, for example, Wnt-3a and Noggin. In some embodiments, EM1
does
not comprise a BMP pathway activator.
Isolation of liver epithelial stem cells for culture
The liver epithelial stem cells to be cultured in the expansion method may be
obtained by
any suitable method.
Preferably they are obtained from a human liver and so are human liver
epithelial stem
cells. However, liver epithelial stem cells from non-human animals are also
envisaged for
use in the invention, for example, non-human mammals such as mouse, rabbit,
rat, pig,
cow, sheep, horse, dog, cat.
In some embodiments, the liver cells for use in the methods of invention are
isolated from
a liver biopsy. In some embodiments, liver cells are isolated by collagenase
digestion, for
example, as described in the examples and in DoreII et al., 2008 (Hepatology.
2008
Oct;48(4):1282-91. Surface markers for the murine oval cell response. Dorrell
C, Erker L,
Lanxon-Cookson KM, Abraham SL, Victoroff T, Ro S, Canaday PS, Streeter PR,
Grompe
M). In some embodiments, collagenase digestion is performed on a liver biopsy.
In some
embodiments, collagenase and accutase digestion are used to obtain the liver
cells for use
in the invention.
In some embodiments, the method comprises culturing a fragment of liver which
comprises liver epithelium. In some embodiments, the epithelial stem cells are
isolated
from a liver fragment or a liver biliary duct, more preferably from biliary
duct tissue. In
some embodiments, the epithelial stem cells are obtained from adult liver
tissue.

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Methods for the isolation of bile duct tissue are known to those of skill in
the art. For
example, biliary duct may be isolated from a liver using collagenase
digestion. Briefly, an
adult liver tissue may be washed in a cold (4-10 C) culture medium, preferably
Advanced-
DMEM/F12 (Invitrogen) and then, the tissue can be chopped into pieces of
around 5mm
and further washed with cold dissociation buffer (collagenase, dispase, FBS in
DMEM
media). The tissue fragments are preferably incubated with the dissociation
buffer for
about 2 h at about 37 C. Then, the tissue fragments can be vigorously
suspended in 10 ml
of cold (4-10 C) isolation buffer with a 10 ml pipette. The first supernatant
containing dead
cells is preferably discarded and the sediment preferably suspended with
dissociation
buffer (e.g. 10-15 ml). After further vigorous suspension of the tissue
fragments the
supernatant is enriched in biliary ducts. A suspension containing biliary
ducts can in this
way be obtained and biliary ducts are collected under the microscope and
retained in cold
media (DMEM + 5-10% FBS). This procedure may be repeated until at least 10-20
biliary
ducts/well are collected. Then, the isolated biliary ducts may be
precipitated. Isolated bile
ducts are preferably seeded in 50u1 of matrigel at an approximate ratio of 20
biliary
ducts/well.
Liver expansion organoids and populations of cells
The invention provides a liver organoid or a population of liver epithelial
stem cells
obtainable or obtained by a method of the invention.
Illustrative examples of liver organoids generated using the expansion medium
and
methods of the invention are given in the accompanying figures. In one
embodiment, an
expansion organoid of the invention has a structure essentially as presented
in Figure 8.
In one embodiment, an expansion organoid of the invention exhibits cell
staining
essentially as presented in Figure 8.
In some embodiments, a liver organoid is a three-dimensional organoid, with a
cystic
structure. Under expansion conditions the organoid may consist of stem cells
and
progenitor cells where two domains are defined: (1) A duct-like domain, formed
by a
single-layer cubical epithelia (positive for the ductal marker Krt19) with
cells lining a central
lumen; and (2) a pseudo-stratified epithelial domain where krt19 positive
cells are
detected. This architecture (areas with single layer epithelia together with
areas of
pseudostratified epithelia) resembles the embryonic liver bud. Under expansion
conditions
fully differentiated cells are not present, although expression of
hepatocyte/hepatoblast-
specific markers can in some embodiments be detected.
In a further embodiment, a liver organoid is a three-dimensional organoid
comprising two
domains: (1) a single-layered epithelium preferably formed by polarized cells
with basal

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nuclei, preferably expressing cytokeratin epithelial markers (for example,
KRT19 and
KRT7), and (2) a pseudo-stratified epithelium, preferably comprising non-
polarized E-
Cadherin+, HNF4a+ and/or some KRT7+ cells. In some embodiments, SOX9 and EPHB2

are detectable in almost all the cells within an organoid. In some
embodiments, LGR5 is
5 expressed in the organoid, preferably within the EPHB2+ cell population.
In some
embodiments the organoid has a duct-like phenotype. In a most preferred
embodiment, a
liver organoid has a combination or all of the features recited in this
paragraph and/or the
paragraphs above.
Preferably, a liver organoid cultured using expansion media of the invention
comprising a
10 TGF beta inhibitor may be cultured for at least 4 weeks, more preferably
at least 5 weeks
at 5 fold expansion a week or two or more population doublings per week (e.g.
for at least
10 doublings, at least 20 doublings, more preferably at least 25 doublings,
for example, at
least 30 doublings). Preferably, a liver organoid cultured using expansion
media of the
invention comprising a prostaglandin pathway activator in addition to a TGF
beta inhibitor
15 may be cultured for at least 7 weeks, more preferably at least 8 weeks
at 2 or more
doublings (e.g. 2-3 doublings) per week (i.e. at least 15 doublings, at least
25 doublings, at
least 30 doublings, at least 32 doublings, at least 35 doublings, e.g. 32-40
doublings or at
least 40 doublings, for example, at least 50 doublings). Thus, preferably, a
liver organoid
of the invention, for example a human liver organoid, is obtained using
expansion media of
20 the invention.
Liver expansion organoids ¨ other cellular markers
In some embodiments, an expansion liver organoid or population of liver
epithelial stem
cells that has been cultured in an expansion medium of the invention comprises
one or
more cells that express one or more of (e.g. one, two or all three of):
progenitor cell
25 markers, ductal markers and hepatocyte markers. In some embodiments, the
markers are
cell surface markers. In other embodiments, the markers are not found on the
cell surface.
Examples of progenitor cell markers are Lgr5 and CD133. Preferably an
expansion liver
organoid comprises cells that express Lgr5, more preferably which express Lgr5
and
CD133. Examples of ductal markers are Sox9, Krt19, Krt7 and 002. Preferably an
30 expansion liver organoid comprises cells that express all of Sox9,
Krt19, Krt7 and 002 but
in some embodiments, the cells may comprise only one, two or three of these
markers.
Examples of hepatocyte markers are Hnf4a and Gapdh. Preferably an expansion
liver
organoid comprises cells that express both of Hnf4a and Gapdh, but in some
embodiments, the cells may comprise only one of these markers. In some
embodiments,
35 the liver organoid or population of liver epithelial stem cells that has
been cultured in an
expansion medium of the invention expresses one or more (e.g. 2, 3, 4, or all
5) of Lgr5,

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CD133, Krt19, Krt7 and Hnf4a In some embodiments, the liver organoid or
population of
liver epithelial stem cells that has been cultured in an expansion medium of
the invention
expresses one or more (e.g. 2, 3, 4, 5, 6 or all 7 ) of Lgr5, CD133, Sox9,
Krt19, 002,
Hnf4a and Gapdh.
In some embodiments, an expansion liver organoid of the present invention
exhibits a
marker profile as shown in Figure 8 as shown by cell staining.
Advantageously, using an expansion medium of the invention produces a human
expansion liver organoid having an increased ductal phenotype and a decreased
hepatocyte phenotype compared to using an expansion medium described in
W02012/014076. Ductal cells are less mature than hepatocytes. Thus,
advantageously,
by including a cAMP pathway activator in the expansion medium, the resulting
cells have
more of a progenitor cell phenotype than when a cAMP pathway activator is
absent. The
ability to generate large numbers of cells having a ductal phenotype opens up
new
possibilities for research and therapies that require use of these ductal
cells.
Accordingly, in some embodiments, a liver organoid of the invention has a
ductal
phenotype when cultured in expansion medium of the invention (e.g. EM1 or
EM2).
Using an expansion method of the present invention, ductal markers are
upregulated
compared to when liver epithelial stem cells are cultured in an expansion
medium
described in WO 2012/014076. Therefore, in some embodiments, the invention
provides a
liver expansion organoid or a population of liver epithelial stem cells in
which ductal marker
expression is higher than in a liver expansion organoid or a population of
liver epithelial
stem cells obtained using the same expansion medium but in the absence of a
cAMP
pathway activator.
Using an expansion method of the present invention, hepatocyte markers are
downregulated compared to when liver epithelial stem cells are cultured in an
expansion
medium described in WO 2012/014076. Therefore, the invention provides a liver
expansion organoid or a population of liver epithelial stem cells in which
hepatocyte
marker expression is lower than in a liver expansion organoid or a population
of liver
epithelial stem cells obtained using the same expansion medium but in the
absence of a
cAMP pathway activator.
In some embodiments, the expansion organoid has less than 50% cells which
express
hepatocyte markers, for example, less than 40%, less than 30%, less than 20%,
less than
10%, less than 5% of the cells. In some embodiments, at least 3% of the cells
in a liver
expansion organoid express hepatocyte markers, for example, at least 5%, at
least 15%,
at least 25%, at least 35%, at least 45%. For example, 5-50% of the cells may
express

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hepatocyte markers. For example, the expansion liver organoid obtained by the
methods
and media of the present invention may comprise approximately 30% hepatocyte
lineage
cells (cells expressing markers characteristic of the hepatocyte lineage).
In some embodiments, the well-known liver transcription factors as HNF1a,
HNF1b and
HNF4a are expressed in expansion organoids.
In some embodiments, a liver expansion organoid or population of liver
epithelial stem
cells cultured in an expansion medium of the invention does not express the
mature
hepatocyte marker albumin.
In one embodiment, a human expanded liver cell population or expansion
organoid of the
invention:
a) expresses at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9), preferably all
of the
following stem cell signature genes: LGR4, LGR5, TACSTD1/Epcam, CD44, SOX9,
SP5,
CD24, PROM1, CDCA7 and ELF3; and/or
b) expresses at least one (e.g. 1, 2, 3, 4), preferably all of the
following
reprogramming genes: KLF4, MYC, POU5F1 and SOX2; and/or
c) expresses at least one (e.g. 1, 2,3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13,
14, 15,
16, 17, 18, 19), preferably all of the following hepatocyte/cholangiocyte
specific genes:
HNF1A, HNF1B, HNF4A, HHEX, ONECUT1, ONECUT2, PROX1, CDH1, FOXA2, GATA6,
FOXM1, CEBPA, CEBPB, CEBPD, CEBPG, GLUL, KRT7, KRT19 and MET; and/or
d) does not express at least one (e.g. 1, 2, 3, 4, 5, 6), preferably all
of the
following hepatocyte/cholangiocyte specific genes: NEUROG2, IGF1R and CD34,
AFP,
GCG and PTF1A, for example, it does not express NEUROG2, IGF1R and CD34;
wherein the expression of the genes is preferably detected by measuring
expression
at the mRNA level, for example, using a microarray.
More preferably a human liver cell population or organoid of the invention has
all of
features a) to d) above.
The human liver cell population or organoids of the invention also preferably
express Lgr5
and/or Tnfrsf19, preferably both. In some embodiments, the human liver cell
population or
organoids, when cultured in expansion medium of the invention express Lgr5
and/or
Tnfrsf19, preferably both. Preferably, expression of Lgr5 and/or Tnfrsf19is
detected by RT
PCR. In some embodiments, Lgr5 and/or Tnfrsf19 are present at much lower
levels of
expression in organoids or cells when cultured in the differentiation medium
compared to
their level of expression organoids or cells when cultured in the expansion
medium (for
example at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at
least 10-fold, at

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83
least 15-fold lower). Previously it was not possible to grow human liver
organoids for a
sufficient length of time to perform expression analysis of the markers.
In one embodiment, a mouse expanded liver cell population or expansion
organoid of the
invention:
a) expresses at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11),
preferably all of
the following stem cell markers: Igr5, Igr4, epcam, Cd44, Tnfrsf19, Sox9, Sp5,
Cd24a,
Prom1, Cdca7 and E1f3; and/or
b) does not express the following stem cell marker: Igr6; and/or
c) expresses at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15,
16, 17, 18, 19), preferably all of the following hepatocyte or cholangiocyte
markers when
grown in expansion medium of the invention: Hnf1a, Hnf1b, Hnf4a, Hhex,
Onecut1,
Onecut2, Prox1, Cdh1, Foxa2, Gata6, Foxm1, Cebpa, Cebpb, Cebpd, Cebpg, Glul,
Krt7,
Krt19 and Met; and/or
d) does not express at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13,
14, 15, 16, 17 ) of the following genes when grown in expansion medium of the
invention:
afp, Ins1, Ins2, Gcg, Ptf1a, Cela1, Cela2a, Cela3b, Neurod1, Neurod2, Neurog1,
Neurog2,
Neurog3, Amy2a4, Igf1r, Igf2 and Cd34; and/or
e) expresses at least one (e.g. 1, 2 or 3) of the following reprogramming
genes: K1f4, Myc and Pou5f1 and/or
f) does not express the following reprogramming gene: Sox2,
wherein the expression of the genes is preferably detected by measuring
expression
at the mRNA level, for example, using a microarray.
More preferably a mouse liver cell population or organoid of the invention has
all of
features a) to f) above.
Also provided is a liver expansion organoid or an expanded population of liver
epithelial
stem cells of the invention in an expansion medium of the invention.
In an embodiment, a liver expansion organoid is a liver organoid which is
still being
cultured using a method of the invention and is therefore in contact with an
extracellular
matrix. Preferably, a liver expansion organoid is embedded in a non-
mesenchymal
extracellular matrix.
Culturing liver organoids in differentiation medium
In one embodiment, the differentiation medium comprises EGF, a TGF-beta
inhibitor, and
a Notch inhibitor. In one embodiment, the TGF-beta inhibitor is A83-01 and/or
the Notch

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inhibitor is DAPT. In one embodiment, the differentiation medium further
comprises
Gastrin. In one embodiment, the differentiation medium further comprises FGF,
HGF or
alternatively both FGF and HGF may be present or absent in the differentiation
medium.
Dexamethasone may also be added, for example at a concentration of between
10nM to
10uM. The liver differentiation medium may optionally include a prostaglandin
pathway
activator, such as PGE2 or AA. However, this component may also be excluded
from the
differentiation medium. In some embodiments, oncostatin M may also be added,
for
example at a concentration range between 1 ng/ml to 1mg/ml, to help
differentiation to
hepatocyte fate.
In some embodiments, the differentiation medium comprises one or more receptor
tyrosine
kinase inhibitor (preferably EGF, HGF and FGF19), a TGF-beta inhibitor
(preferably
A8301), a Notch inhibitor (preferably DAPT) and a BMP activator (preferably
BMP7).
As discussed in WO 2012/168930, Rspondin1 and Nicotinamide both inhibit the
expression of the mature hepatocyte marker CYP3A11 and yet promote the
expression of
the hepatoblast marker albumin. Therefore, to increase differentiation of the
cells to more
mature liver fates, Rspondin and Nicotinamide may be removed from the cell
culture. The
expression of specific biliary transcription factors is highly upregulated in
expansion
cultures containing Rspondin1, indicating that the culture gene expression was
unbalanced
towards a more biliary cell fate. Notch and TGF-beta signalling pathways have
been
implicated in biliary cell fate in vivo. In fact, deletion of Rbpj (essential
to achieve active
Notch signalling) results in abnormal tubulogenesis (Development. 2009
May;136(10):1727-39. Notch signaling controls liver development by regulating
biliary
differentiation. Zong Y, Panikkar A, Xu J, Antoniou A, Raynaud P, Lemaigre F,
Stanger
BZ) and the addition of TGF-beta to liver explants facilitates the biliary
differentiation in
vitro (Genes Dev. 2005 Aug 15;19(16):1849-54. Control of liver cell fate
decision by a
gradient of TGF beta signaling modulated by Onecut transcription factors.
Clotman F,
Jacquemin P, Plumb-Rudewiez N, Pierreux CE, Van der Smissen P, Dietz HC,
Courtoy
PJ, Rousseau GG, Lemaigre FP). Since both Notch and TGF-beta signalling
pathways
were highly upregulated in the liver cultures (see WO 2012/168930), the
inventors
reasoned that inhibition of biliary duct cell-fate might trigger the
differentiation of the cells
towards a more hepatocytic phenotype. It was found that addition of a TGF-beta
inhibitor
(such as A8301) and a Notch inhibitor (such as DAPT) to a differentiation
medium that
preferably does not contain Rspondin or Wnt, enhances the expression of mature

hepatocyte markers and increases the number of hepatocyte-like cells.
Conversely,
withdrawal or exclusion of the TGF-beta inhibitor (such as A8301) and/or the
Notch
inhibitor (such as DAPT) in the differentiation medium can promote
differentiation to the

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biliary fate. In addition, withdrawal or exclusion of the cAMP activator (such
as forskolin)
can promote differentiation e.g. to the biliary fate. In some embodiments, the
invention
provides a differentiation medium with an alteration as described in this
paragraph.
Differentiated liver organoids and populations of cells
5 Organoids can be cultured in a differentiation medium, as described
above, such that they
differentiate into functional cell types (e.g. see Example 15). Thus the
invention provides a
method for producing cells expressing hepatocyte markers, wherein the method
comprises
culturing cells in a differentiation medium of the invention. The invention
further provides a
differentiated liver organoid or a population of differentiated liver
epithelial cells.
10 In one embodiment, the invention provides a differentiated liver
organoid or a population of
liver epithelial cells obtainable or obtained by a method of the invention,
which comprises
culturing liver epithelial stem cells in a differentiation medium of the
invention.
Illustrative examples of organoids generated using the differentiation medium
and methods
of the invention are given in the accompanying figures.
15 A preferred liver differentiated organoid comprises or consists of a
cystic structure with on
the outside a layer of cells with buds and a central lumen. This liver
organoid may have
one or more (e.g. 2, 3, or all 4) of the following characteristics: (a) having
a cell density of
>5x105 cells/cm3, preferably >10x105 cells/cm3; (b) having a thickness
equivalent to 2-30
layers of cells, preferably a thickness equivalent to 2-15 layers of cells;
(c) the cells
20 mutually contact in three dimensions, (d) demonstrate a function
inherent to healthy liver
tissue, (e) have a domain which constitutes the main body of the organoid and
is formed
by a multilayered epithelia with non-polarized cells wherein albumin
expression may be
detected.
Generally, use of the differentiation medium of the present invention allows
organoids and
25 cells to be obtained which express low levels of or no ductal markers.
Expression of
higher levels of hepatocyte markers compared to ductal markers indicates that
the cells
are similar to mature liver cells rather than being similar to liver
progenitor cells.
Accordingly, there is provided a differentiated organoid or a population of
differentiated
liver cells in which hepatocyte markers are upregulated compared to ductal
markers.
30 In some embodiments, the liver organoid or population of liver
epithelial stem cells that has
been cultured in a differentiation medium of the invention expresses one or
more mature
hepatocyte markers. In some embodiments, the liver organoid or population of
liver
epithelial stem cells expresses one or more (e.g. 2 or all 3) of the mature
hepatocyte
markers albumin, Cyp3A4 and zona occuludens (Z01). Preferably, the level of
expression
35 of the mature hepatocyte markers is upregulated compared to the level of
expression in an

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organoid or adult liver stem cell that has been cultured only in an expansion
medium of the
invention without also having been cultured in a differentiation medium of the
invention.
Preferably, the level of upregulation is 3x or more, more preferably 5x, 8x,
15, 30x, 50x,
75x, 90x, or 100x or more. For example, the expression of albumin is
preferably increased
by at least 75 times, more preferably at least 85 times, more preferably at
least 95 times
(e.g. see Figure 10).
In some embodiments, a differentiated liver organoid or population of liver
epithelial cells
has hepatocyte morphology, for example, polygonal cell shapes. In some
embodiments, a
differentiated liver organoid or population of liver epithelial cells has high
expression of
hepatocyte markers, such as one or more or all selected from albumin, tyrosine
aminotransferase (TAT), apolipoproteins, cytochrome enzymes (e.g. CYP3A4) and
complement factors (e.g. 03). In some embodiments, a differentiated liver
organoid or
population of liver epithelial cells, can perform one or more or all of the
following functions:
accumulate glycogen; take up LDL, secrete bile acid salts, and detoxify
ammonia (for
example, see Examples 11 and 16). It is to be understood that all the
hepatocyte-type
features are readily combinable and that a liver organoid of the invention may
exhibit any
combination of the above characteristics. In some embodiments, a
differentiated liver
organoid or population of liver epithelial cells shows stronger hepatocyte
function than an
expansion organoid, as measured by any one or more of the above
characteristics
compared to the reference cell line HepG2.In some embodiments, the liver
organoid or
population of liver epithelial stem cells that has been cultured in a
differentiation medium of
the invention expresses at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16,
17, 18), preferably all of the following hepatocyte specific genes: TTR, ALB,
FAH, TAT,
CYP3A7, AP0A1, HMGCS1, PPARG, CYP2B6, 0YP2018, 0YP209, CYP2J2, CYP3A4,
CYP3A5, CYP3A7, CYP4F8, CYP4V2 and SCARB1.
In some embodiments, at least 10% of the cells in a differentiated liver
organoid express a
hepatocyte surface marker, for example, at least 20%, at least 30%, at least
40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90% of the cells, for
example, about
25%-90%, about 40-80% of the cells.
In some embodiments, the differentiated organoid has less than 10% cells which
express
ductal markers, for example, less than 5% or less than 2% of the cells.
In some embodiments, the differentiated organoids stain positive for keratin
under confocal
microscopy.
In some embodiments, the differentiated liver organoid comprises cells that
express
cholangiocyte markers.

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Preferably, culturing the organoids or cells in a differentiation medium of
the invention
produces differentiated liver organoids and cells which have a liver-like
functional
phenotype. In some embodiments, the liver organoid or population of liver
epithelial stem
cells that has been cultured in a differentiation medium of the invention
expresses
hepatocyte markers is capable of accumulating glycogen. In some embodiments,
it is
capable of uptaking LDL. In some embodiments it has active cytochrome
activity, for
example Cytochrome P450 activity. In some embodiments, it is capable of
secreting
A1AT. In some embodiments, it has Cyp3a activity.
In one embodiment, there is provided a liver organoid in a differentiation
medium
comprising a basal medium for animal or human cells to which is added EGF,
gastrin,
FGF19, DAPT, dexamethasone, HGF and A8301.
Uses of liver organoids and populations of cells
The liver organoid or population of liver epithelial stem cells or population
of differentiated
liver cells obtained using a method of the invention have a variety of uses.
For example,
the invention provides the use of the liver organoid or population of liver
epithelial stem
cells/differentiated cells as described herein in a drug discovery screen;
toxicity assay;
research of liver embryology, liver cell lineages, and differentiation
pathways; gene
expression studies including recombinant gene expression; research of
mechanisms
involved in liver injury and repair; research of inflammatory and infectious
diseases of the
liver; studies of pathogenetic mechanisms; or studies of mechanisms of liver
cell
transformation and aetiology of liver cancer.
In one aspect, the invention provides the use of a liver organoid or
population of liver
epithelial stem cells/differentiated cells as described herein in a drug
discovery screen,
toxicity assay or in regenerative medicine. Similarly, the invention provides
the use of the
progeny of liver organoids of the invention for these uses.
Toxicity assays may be in vitro assays using a liver organoid or part thereof
or a cell
derived from a liver organoid. Such progeny and liver organoids are easy to
culture and
more closely resemble primary epithelial cells than, for example, epithelial
cell lines such
as Caco-2 (ATCC HTB-37), 1-407 (ATCC CCL6), and XBF (ATCC CRL 8808) which are
currently used in toxicity assays. It is anticipated that toxicity results
obtained with liver
organoids more closely resemble results obtained in patients. A cell-based
toxicity test is
used for determining organ specific cytotoxicity. Compounds that are tested in
said test
comprise cancer chemopreventive agents, environmental chemicals, food
supplements,
and potential toxicants. The cells are exposed to multiple concentrations of a
test agent for
certain period of time. The concentration ranges for test agents in the assay
are

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determined in a preliminary assay using an exposure of five days and log
dilutions from the
highest soluble concentration. At the end of the exposure period, the cultures
are
evaluated for inhibition of growth. Data are analysed to determine the
concentration that
inhibited end point by 50 percent (TC50).
For example, induction of cytochrome P450 enzymes in liver hepatocytes is a
key factor
that determines the efficacy and toxicity of drugs. In particular, induction
of P450s is an
important mechanism of troublesome drug-drug interactions, and it is also an
important
factor that limits drug efficacy and governs drug toxicity. Cytochrome P450
induction
assays have been difficult to develop, because they require intact normal
human
hepatocytes. These cells have proven intractable to production in numbers
sufficient to
sustain mass production of high throughput assays.
The invention provides the use of liver organoids or populations of liver
cells according to
the invention for use in regenerative medicine, for example in post-radiation
and/or post-
surgery repair of the liver epithelium, in the repair of the epithelium in
patients suffering
from chronic or acute liver failure or disease. Liver diseases for which the
liver organoid or
cells derived from said organoid may be used include, but are not limited to
Hepatocellular
Carcinoma, Alagille Syndrome, Alpha- 1- Antitrypsin Deficiency, Autoimmune
Hepatitis,
Biliary Atresia, Chronic Hepatitis, Cancer of the Liver, Cirrhosis, Liver
Cysts, Fatty Liver
Disease, Galactosemia Gilbert's Syndrome, Primary Biliary Cirrhosis, Hepatitis
A, Hepatitis
B, Hepatitis C, Primary Sclerosing Cholangitis, Reye's Syndrome, Sarcoidosis,
Tyrosinemia, Type I Glycogen Storage Disease, Wilson's Disease, Neonatal
Hepatitis,
Non-alchoholic SteatoHepatitis, Porphyria, and Hemochromatosis.
In some embodiments, the liver organoids or populations of liver cells are
used for therapy
of genetic conditions. Genetic conditions that lead to liver failure could
benefit from cell-
based therapy in the form of partial or full cell replacement using cells
cultured according
to the media and/or methods of the invention. A non-limiting list of genetic
conditions that
lead to liver failure and which are treatable by the present invention
includes: Progressive
familial intrahepatic cholestasis, Glycogen storage disease type III,
Tyrosinemia,
Deoxyguanosine kinase deficiency, Pyruvate carboxylase deficiency, Congenital
dyserythropoietic anemia, Polycystic Liver Disease Polycystic Kidney Disease,
Alpha-1
antitrypsine deficiency, Ureum cycle defects, Organic acidemiea, lysosomal
storage
diseases, and Fatty Acid Oxydation Disorders. Other conditions that may also
benefit from
cell-based therapy include Wilson's Disease and Hereditary Amyloidosis
(FAP).0ther non-
hepatocyte related causes of liver failure that would require a full liver
transplant to reach
full therapeutic effect, may still benefit from some temporary restoration of
function using
cell-based therapy using cells cultured according to the media and/or methods
of the

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invention. A non-limiting list of examples of such conditions which may be
treatable by the
organoids and/or populations of cells of the present invention includes:
Primary Biliary
Cirrhosis, Primary Sclerosing Cholangitis, Aglagille syndrome, hom*ozygous
Familial
hypercholesterolemia, Hepatitis B with cirrhosis, Hepatitis C with cirrhosis,
Budd-Chiari
syndrome, Primary hyperoxaluria, Autoimmune Hepatitis, and Alcoholic liver
disease.
This is exemplified in Example 17, which shows that liver organoids are
suitable disease
models for alpha-1 antitrypsin (A1AT) deficiency and AlegiIle Syndrome. In
fact, the
inventors thereby provide the first ever experimental model for human AlegiIle
Syndrome.
Thus in some embodiments, the invention provides the use of a liver organoid
as a
disease model, for example, for any of the diseases listed above. In some
embodiments,
the invention provides the use of a liver organoid as a disease model for
alpha-1
antitrypsin (A1AT) deficiency or AlegiIle Syndrome (AGS). In some embodiments,
the
invention provides a disease model for alpha-1 antitrypsin (A1AT) deficiency
or AlegiIle
Syndrome (AGS).
In some embodiments, the disease model for A1AT deficiency comprises A1AT-
Protein
aggregates. In some embodiments, the disease model for A1AT deficiency has
reduced
secretion of the A1AT. This mimics reduced A1AT serum levels in patients. In
some
embodiments, supernatants from the disease model for A1AT deficiency have a
reduced
ability to block elastase activity compared to wild-type, e.g. as detected as
described in the
examples under "Elastase inhibition assay". In some embodiments, the disease
model for
A1AT deficiency shows signs of ER stress, such as phosphorylation of elF2a
and/or a
slight increase in apoptosis in the differentiated state compared to wild
type. In some
embodiments, the disease model for A1AT deficiency is characterised by any
combination
or all of these features. In a preferred embodiment, the disease model is an
organoid as
described herein. In some embodiments, the disease model for AGS has reduced
ability to
differentiate to the biliary fate compared to wild type, as detectable, for
example by failure
to upregulate biliary markers such as KRT19 and KRT7. In some embodiments, in
the
disease model for AGS, biliary cells are reduced in numbers and/or unable to
integrate into
the epithelium, as detectable for example by KRT19 staining. For example, in
this disease
model, biliary cells may round up and undergo apoptosis. In some embodiments,
the
disease model for AGS is characterised by any combination or all of these
features. In a
preferred embodiment, the disease model is an organoid as described herein.
The liver organoids of the invention may be used in a method of treating a
hereditary
disease that involves malfunctioning hepatocytes. Such diseases may be early
onset or
late onset. Early onset disease include metabolite related organ failure (e.g.
alpha-1-
antitrypsin deficiency), glycogen storage diseases (e.g. GSD II, Pompe's
disease),

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tyrosinemia, mild DGUOK, CDA type I, Ureum cycle defects (e.g. OTC
deficiency), organic
academia and fatty acid oxidation disorders. Late onset diseases include
primary
hyperoxaluria, familial hypercholesterolemia, Wilson's disease, Hereditary
Amyloidosis and
Polycystic liver disease. Partial or full replacement with healthy hepatocytes
arising from
5 liver organoids of the invention may be used to restore liver function or
to postpone liver
failure. Accordingly, the invention provides the use of the liver organoids
and/or
populations of liver cells for restoration of liver function or to postpone
liver failure.
The liver organoids of the invention may be used in a method of treating
chronic liver
failure arising due to hereditary metabolic disease or as a result of
hepatocyte infection.
10 Treatment of a hereditary metabolic disease may involve administration
of genetically
modified autologous liver organoids of the invention. Treatment of hepatocyte
infections
may involve administration of allogeneic liver organoids of the invention.
In some
embodiments, the liver organoids are administered over a period of 2-3 months.
The liver organoids of the invention may be used to treat acute liver failure,
for example, as
15 a result of liver intoxication which may result from use of paracetamol,
medication or
alcohol. In some embodiments, the therapy to restore liver function will
comprise injecting
hepatocyte suspension from frozen, ready to use allogenic hepatocytes obtained
from
organoids of the invention. The ability to freeze suitable organoids means
that the
organoids can be available for immediate delivery and so it is not necessary
to wait for a
20 blood transfusion.
In the case of replacement or correction of deficient liver function, it may
be possible to
construct a cell-matrix structure from one or more liver organoids generated
according to
the present invention. Thus in some embodiments, there is provided a cell-
matrix structure
derived from a liver organoid and suitable for use in therapy as described
herein.
25 It is thought that only about 10% of hepatic cell mass is necessary for
adequate function.
This makes implantation of organoid unit compositions into children especially
preferable
to whole organ transplantation, due to the relatively limited availability of
donors and
smaller size of juvenile organs. For example, an 8-month-old child has a
normal liver that
weighs approximately 250g. That child would therefore need about 25g of
tissue. An adult
30 liver weighs-approximately 1500g; therefore, the required implant would
only be about
1.5% of the adult liver. In some embodiments, therefore, the treatment
described in this
section is for children. In other embodiments it is for adults.
In some embodiments, the transplantation step involves a scaffold, such as a
polymer
scaffold. When organoid units according to the invention are implanted,
optionally attached
35 to a polymer scaffold, proliferation in the new host will occur, and the
resulting hepatic cell

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mass replaces the deficient host function. The inventors have shown that it is
possible to
generate mature hepatocytes from adult liver stem cells or liver tissue
fragments
comprising stem cells that are suitable for transplantation into non-human
animals or
humans. Using the expansion culture medium according to the invention, the
inventors
have demonstrated that it is possible to maintain and expand a population of
liver stem
cells. Using the differentiation culture medium according to the invention,
the inventors
have shown that hepatoblasts can be differentiated in vivo to mature
hepatocytes suitable
for transplantation purposes. Hence, the inventors provide a new source of
hepatocytes for
liver regeneration, replacement or correction of deficient liver function.
In some embodiments it is desirable to repopulate/replace 10-20% of a
patient's liver with
healthy hepatocytes arising from a liver organoid of the invention.
In some embodiments, the invention provides the liver organoids or populations
of liver
cells obtained from the expansion and/or differentiation media for use in
therapy. Such
organoids and populations of cells are useful for treating ductal cell
disease. For example,
such organoids and populations of cells may be used to treat diseases of the
ductal tree,
for example diseases caused by a mutations in Jagged1 (JAG1), for example
Alagille
syndrome, or mutations in the transporter ABCB4, for example Low Phospholipid
associated Cholelithiasis. Other non-heriditary cholangiopathies such as
Primary Biliary
Cirrhosis, Primary sclerosis cholangitis and Caroli disease may also be
treated using the
liver organoids or expanded populations of liver cells of the invention. Such
diseases may
benefit from expanding duct-cells in culture. Other diseases that could be
treated with
organoids or expanded populations of liver cells of the invention include
hereditary liver
diseases where bilirubin metabolism is affected. Such diseases would benefit
from
hepatocyte transplants. Examples of known hereditary defects in bilirubin
metabolism are
Crigler¨Najjar syndrome (mutation in UGT1A1 gene), Dubin¨Johnson syndrome
(mutation
in cMOAT), and Rotor syndrome (mutations in SLCO1B1 and SLCO1B3).
Thus, in some embodiments, the liver organoid or population of liver
epithelial stem cells is
for use in treating a liver disorder, condition or disease or for use in
regenerative medicine.
The inventors have found that Lgr5 is not detectable in healthy liver,
although residual Lgr5
may be detected. Thus, the invention further provides a method of diagnosing
liver injury
comprising detecting whether Lgr5 is expressed, wherein the expression of Lgr5
protein
indicates liver injury. The invention also provides a method of monitoring the
repair or
regeneration of the liver by monitoring the expression of Lgr5 in the liver.
Lgr5 expression
may be detected by any suitable method, for example, flow cytometry,
immunohistochemistry or by use of PCR methods. Anti-Lgr5 antibodies suitable
for use in

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such diagnosis are known, e.g. see W02012/140274. Accordingly there is
provided an
anti-Lgr5 antibody for use in diagnosing liver injury.
Pancreas
It has similarly been shown that adding a cAMP pathway activator to culture
medium for
pancreas epithelial stem cells increases the number of passages that are
possible for
human cells (see Example 13). Methods for culturing pancreatic cells are
described in
W02010/090513.
Accordingly, there are provided methods and culture media for culturing
epithelial stem
cells, organoids and populations of cells obtainable using the methods and
media
described herein and uses of said organoids and populations of cells, wherein
the
epithelial stem cells are derived from the pancreas.
For example, there is provided a method for culturing epithelial stem cells,
for example to
obtain a pancreatic organoid, wherein said method comprises:
culturing one or more epithelial stem cells from pancreas in contact with an
extracellular matrix in the presence of an expansion medium, the expansion
medium
comprising a basal medium for animal or human cells to which is added:
one or more receptor tyrosine kinase ligands, an Rspondin, Nicotinamide, and a
TGF-beta
inhibitor; anda cAMP pathway activator.
In some embodiments, the culture medium further comprises one or more
components
selected from a BMP inhibitor (e.g. Noggin), a prostaglandin pathway
activator, Wnt,
Gastrin, B27 and N-acetylcholine. The paragraphs elsewhere in the application
that
discuss these components e.g. in relation to the liver embodiments, apply
equally to the
pancreatic embodiments.
In some embodiments, the culture medium further comprises a p38 inhibitor
(e.g.
SB202190).
In a preferred embodiment, the culture medium comprises EGF (e.g. about 50
ng/ml),
FGF10 (e.g. about 100 ng/ml), Rspondin (e.g. about 1 pg/ml), Nicotinamide
(e.g. about 10
mM), A8301 (e.g. about 500 nM), Forskolin (e.g. about 10 pM) Noggin (e.g.
about 10%
CM), Wnt (e.g. about 1 pg/ml), Gastrin (e.g. about 10 nM) and B27 lx, NAc
(e.g. about
1.25 mM), and optionally PGE2 (e.g. about 1 pM) and optionally a p38 inhibitor
(e.g. about
10 pM).
In some embodiments, the methods for culturing epithelial stem cells to obtain
a pancreatic
organoid, further comprise the step of differentiating the cells using
differentiation media
and methods described elsewhere in this application.

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The invention further provides a pancreatic organoid or cell obtained from the
pancreatic
organoids for use in the treatment of diabetes (e.g. diabetes type I or type
II), pancreatitis,
pancreatic cancer or cystic fibrosis, whereby the treating optionally
comprises
transplantation of the organoid or cells obtained from the pancreatic organoid
into a patient
in need thereof. In some embodiments, the transplanted cells are insulin
secreting cells. In
other embodiments, the cells are progenitor cells that mature further after
transplantation
into insulin secreting cells.
Other tissues
It is also envisaged that adding a cAMP pathway activator to culture medium
for other
epithelial stem cells, including intestine (e.g. small intestine or colon),
stomach, prostate,
lung, breast, ovarian, salivary gland, hair follicle, skin, oesophagus or
thyroid will also
increase the number of passages possible for human cells. Thus the methods,
media,
organoids and populations of cells, and uses of said organoids and populations
of cells,
also apply to these tissues.
Definitions
As used herein, the verb "to comprise" and its conjugations is used in its non-
limiting
sense to mean that items following the word are included, but items not
specifically
mentioned are not excluded. In addition the verb "to consist" may be replaced,
if
necessary, by "to consist essentially of" meaning that a product as defined
herein may
comprise additional component(s) than the ones specifically identified, said
additional
component(s) not altering the unique characteristic of the invention. In
addition a method
as defined herein may comprise additional step(s) than the ones specifically
identified, said
additional step(s) not altering the unique characteristic of the invention. In
addition,
reference to an element by the indefinite article "a" or "an" does not exclude
the possibility
that more than one of the element is present, unless the context clearly
requires that there
be one and only one of the elements. The indefinite article "a" or "an" thus
usually means
"at least one".
As used herein, the term "about" or "approximately" means that the value
presented can
be varied by +/-10%. The value can also be read as the exact value and so the
term
"about" can be omitted. For example, the term "about 100" encompasses 90-110
and also
100.
All patent and literature references cited in the present specification are
hereby
incorporated by reference in their entirety.
The following examples are offered for illustrative purposes only, and are not
intended to
limit the scope of the present invention in any way.

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DESCRIPTION OF FIGURES
The invention will now be described further with references to the following
figures in
which:
Figure 1: (A) Expression profile for TGF-beta in human and mouse liver
organoids. (B)
Expression profile for TGF-beta inhibitors in human and mouse liver organoids.
(C) Image
showing growth of human liver organoids with and without a TGF-beta inhibitor.
(D) Graph
indicating the percentage of colony formation efficiency in human organoid
cultures with
and without a TGF-beta inhibitor.
Figure 2: (A) Image showing growth of human liver organoids in culture medium
with and
without FSK and BMP7. (B) Graph showing number of passages in a culture medium
comprising a TGF-beta inhibitor (A8301), BMP7 and/or forskolin (FSK).
Figure 3: (A) Graph showing number of cells per well (indicating proliferation
potential) at
each passage in weeks 0-4. (B) Graph showing number of cells per well at each
passage
in weeks 21-25. (C) Table showing mean doubling time for early and late
passaged cells
(up to the twelfth passage at day 103). (D) Image showing EdU incorporation
the third and
twelfth passage.
Figure 4: Images showing human liver organoids cultured with various different
cAMP
agonists (8Br-cAMP, NKH477, Cholera toxin and forskolin) at passage 0 and
passage 8.
Figure 5: (A) Images showing Lgr5 expression in human liver culture. (B) Flow
cytometry
analysis of Lgr5 positive cells cultured with and without forskolin. (C) Graph
showing qPCR
analysis of the expression of LGR5 in 4 week old cultures treated in the
presence or
absence of FSK.
Figure 6: (A) Images showing human liver organoids grown in the complete
medium
comprising FSK (EM) or transferred to a medium without Rspo (-Rspo), without
FSK (-
FSK) and without Rspo including the porcupine inhibitor (-Rspo + IWIP). (B)
Graphs
showing organoids per well 7 days after removal of Rspondin (-Rspo) or FSK (-
FSK).
Figure 7: (A) Images of stained chromosomes at passage 1 and passage 15. (B)
Graph
showing percentage of cells with normal/abnormal chromosome count at 2 weeks
and at
more than 3 months of culture.
Figure 8: (A) Images showing immunofluorescence staining of expression markers
of the
ductal (Sox9, Krt19 and Krt7) and hepatocyte (Hnf4a) lineages. (B) RT-PCR
results on a
gel showing expression of progenitor (Lgr5 and CD133), ductal (Sox9, Krt19 and
0C2),
and hepatic markers (Hnf4a).

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Figure 9: Images of growing organoids from single cells from human liver
cultures.
Magnifications: 40x (days 0-10), 4x (day 20 onwards).
Figure 10: (A) lmmunofluorescence staining of mature hepatocyte markers in
differentiated
organoids, showing albumin (ALB, red) and zona occuludens (ZO-1, green)
positive cells.
5 (B) Graph showing expression of albumin and Cytochrome p450 3A4 isoform
upon
differentiation by qPCR analysis. Graphs indicate mean SEM of 3 independent
experiments in 3 independent donor derived cultures. EM, expansion medium
including
FSK. DM, differentiation medium. HuLi, whole lysate from human liver.
Figure 11: (A) Images showing glycogen storage determined by PAS (Periodic-
Acid Schiff)
10 staining in cultures grown in EM or DM for 11 days. Magnification, 10x.
(B) Images
showing LDL uptake analysed using Dil-ac-LDL fluorescent substrate (red) in
cultures
maintained in EM or DM (right) for 11 days. Scale bar, 25 pm. (C) Graph
showing Cyp3a4
expression in cells cultured in DM for 11 days. Results are expressed as RLU
per ml per
million cells. HEK293T cells and HepG2 cells were used as negative and
positive controls
15 respectively. Triplicates for each condition were analysed. Results are
shown as mean
SEM of 2 independent experiments in 4 independent donor-derived cultures.
Figure 12: Table 2: List of compounds tested in the culture medium and effect
on organoid
expansion/differentiation.
Figure 13: Representative image of sample 0366 cultured in 10 different
conditions from
20 PO until P6.
Figure 14: Human liver organoids express Lgr5 and markers of the ductal and
hepatocyte
lineages. Gene expression was analyzed by RT-PCR (A) and immunofluorescence
(B) in
human liver cultures grown in our defined expansion medium as described in
Experimental
Procedures. (A) Gene expression was analyzed at early (EP) and late (LP)
passages.
25 Human liver cultures expressed progenitor (LGR5, 50X9), ductal (KRT19,
50X9) and
hepatocyte (HNF4A) markers but they do not express albumin (ALB) while in
expansion
medium. Results are indicated as 2-dCt (28ACT). Values represent mean SEM of
3
independent experiments in 5 independent donor derived cultures. 28ACT were
calculated
using the housekeeping gene GAPDH as reference gene for normalization. (B-F)
Confocal
30 images of a human liver organoid showing that the organoids are formed
by epithelial
derived structures positive for ECAD and the hepatocyte marker HNF4 (B), and
the ductal
markers (KRT19, C; KRT7, D) and 50X9 (E). Nuclei were counterstained with
Hoechst.
(F) Confocal images of a human liver organoid showing that the organoids are
formed by
epithelial derived cells positive for EPCAM. The stem cell marker Lgr5 was
restricted to a
35 subset of cells within the organoid, while the Wnt target gene EPHB2 was
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expressed, but co-localized with LGR5, as expected. (G) Representative image
of RT-PCR
analysis of indicated genes in 2 independent human liver donor-derived
organoid cultures
maintained in Expansion medium (EM) for 2 months in culture. Note expression
of
progenitor marker PROM1 and ductal marker 002 (ONECUT2). (H) Heat map of genes
>2
fold differentially expressed between human liver tissue and organoid in
expansion
medium. Dark, upregulated. Light, downregulated, (I) Representative image of
RT-PCR
analysis of indicated genes in 1 donor derived culture maintained under
complete
expansion medium (EM) or after withdrawal of Rspondin (Rspo) or Forskolin
(FSK). (J)
Representative image of RT-PCR analysis of CYP3A4 in 1 donor derived culture
maintained under complete differentiation medium (DM, complete) for 11 days,
or after
withdrawal of the indicated components, DAPT and/or Dexamethasone (Dexa).
Figure 15: Human liver culture of ductal origin. (A) Cyp3A4 activity of
Percoll purified
primary human hepatocytes after 4 days in culture in comparison to HepG2
cells. (B)
EpCAM marks bileducts in human liver sections. Hepatocytes are EpCAM negative.
(C)
sorting strategy to purify EpCAM+ ductal cells and Hepatocytes. In the first
step, singlets
were gated to avoid contamination by cell aggregates. Subsequently, large
debris and
erythrocytes were excluded. From this population, EpCAM+ Pl- (viable) cells
were sorted
as the ductal population. For hepatocyte sorting large EpCAM- cells were
selected. (D)
Organoid formation efficiency of sorted ductal and hepatocyte populations
after 14 days.
Organoids bigger than 100 pm were scored. (E) EpCAM+ sort derived organoids at
passage 0 and passage 6. (F-G) Organoid formation efficiency of unsorted,
Percoll purified
hepatocytes (F) and the respective percentage of residual EpCAM+ cells (G).
Figure 16: Chromosomal integrity of human liver organoids. (A) Representative
karyotyping image of organoids cultured for 16 days (P1) and 90 days (P14)
illustrating a
normal chromosomal count (n=46). No major chromosomal aberrations were
observed in
any of the samples analyzed (n=15). Detailed chromosomal counts for different
donors are
shown in supplemental Figure S4. (B) Read-depth analysis of whole genome
sequencing
data over the different chromosomes for the biopsy (upper panel) and organoid
culture A
(lower panel) that were derived from donor 2. Read-depth was corrected for GC
content
and normalized for genome coverage. Dotted lines indicate log2 values
associated with a
gain or deletion. (C) Copy number analysis of a region at chromosome 3 that
was found to
harbor a heterozygous gain in culture A of donor 2. Left panels indicate read-
depth
analysis of the indicated region in 5kb bins, corrected for GC content and
normalized for
genome coverage, of the biopsy (upper panel) and organoid culture (lower
panel). Right
panels show the variant allele frequencies of informative non-reference single
nucleotide
polymorphisms (SNPs) in the indicated region for the biopsy (upper panel) and
organoid

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culture (lower panel). (D) Summary of the copy number analysis of the
different organoid
cultures of the two donors. Somatic CNVs were exclusively observed in culture
A derived
from donor 2, which were already present in the parental culture.
Figure 17: Human liver organoids are genetically stable after months of
expansion in
culture. Genetic stability of human liver cultures was analyzed in clonally
grown cultures
that had been expanded for > 3 months (-120 days) in our complete human liver
medium.
(A) Human liver biopsies were dissociated into single cells and clonal
cultures were
obtained by seeding sorted cells at a ratio of 1 cell per well. As
illustrated, cells quickly
proliferated and expanded in culture. DIC images of growing single cells from
human liver
cultures. Magnifications: 40x (days 0-10), 4x (day 20-onwards). (B-D) Genetic
stability of
the human liver organoid cultures clonally expanded long-term in vitro. (B)
Schematic
overview of the experimental setup. Two independent donor liver biopsies were
minced
and cultured for one week. Subsequently, single liver stem cells were isolated
and clonally
expanded to obtain two independent organoid cultures per donor (culture A and
culture B).
These cultures were subjected to long-term expansion after which a second
clonal
expansion step was performed. The resulting organoid cultures were subjected
to whole
genome sequencing (WGS) analysis. To obtain all somatic variation present in
the
cultures, variants were filtered for presence in the original biopsy. To
determine the effect
of long-term culturing on genomic stability, somatic variation was filtered
for presence in
earlier passages. (C) Number of somatic base substitution observed in the
different
organoid cultures. The pie-chart indicates the percentage of the genome that
was
surveyed per donor. The right panels indicate the absolute numbers of base
substitution
observed in the surveyed part of the genome. Indicated are the total number of
somatic
base substitutions per culture and the number induced by long-term culturing.
(D) Effect of
somatic base substitutions on protein-coding DNA. Left panels indicate the
total number of
somatic base substitutions per donor and the left panel indicates the part
that affects
protein-coding DNA.
Figure 18: Upon differentiation liver organoid cultures upregulate hepatocyte
genes.
Human liver cultures were expanded for at least 1 month in culture and
transferred to our
differentiation medium as described in Experimental Procedures. (A) Scheme of
the
experimental plan. (B-C) Expression of hepatocyte genes was determined by
immunofluorescence (B) or qPCR (C) 11 days later. (B) lmmunofluorescence
staining
showing albumin (ALB, dark) and zona occuludens (ZO-1, light) positive cells
all over the
organoid, indicating that the cells start expressing hepatocyte markers. (C)
qPCR analysis
indicated that both, albumin and Cytochrome p450 3A4 isoform were highly
expressed
upon differentiation. Graphs indicate mean SEM of 3 independent experiments
in 3

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independent donor derived cultures. EM, expansion medium including FSK. DM,
differentiation medium, Tissue, whole lysate from human liver. **, p<0,01 when
comparing
EM vs DM. (D) Whole genome transcriptome analysis of human liver cultures
grown in our
expansion medium (EM) or after being cultured 11 days in our defined
Differentiation
medium (DM). Heat map indicates cluster of genes highly expressed in liver
tissue and in
organoid cultures upon differentiation. Of note, this cluster contains genes
essential for
liver function, as the indicated in dark. Light, downregulated; Dark,
upregulated.
Figure 19: Differentiated liver organoids exhibit hepatocyte functions in
vitro and in vivo. To
test whether the cells could have differentiated towards functional
hepatocytes in vitro, we
determined the ability of the cultures to retain some hepatocyte functions in
vitro, upon
differentiation. (A), Glycogen accumulation was determined by PAS (Periodic-
Acid Schiff)
staining in organoids grown in EM or DM for 11 days. PAS positive staining was

exclusively observed in the organoids after Differentiation (DM), indicating
that the cells
exhibit capacity to accumulate glycogen. Magnification, 10x. (B) LDL uptake
was analysed
using Dil-ac-LDL fluorescent substrate in cultures maintained in EM (left) or
DM (right) for
11days. Only cultures maintained in DM incorporated the substrate. Nuclei were
counter-
stained with DRAQ5. Scale bar, 25 pm. (C) Albumin production during 24h was
measured
in the supernatant of liver organoids. Results are expressed as mean SEM of
2
independent experiments in 4 independent donor-derived cultures. (D) CYP3A4
activity
was measured as described in methods in cultures kept in DM for 11 days.
Results are
expressed as RLU per ml per million cells. HEK293T cells and HepG2 cells were
used as
negative and positive controls respectively. Note that organoids upon DM
exhibit similar
the CYP3A4 activity as fresh isolated hepatocytes. Triplicates for each
condition were
analyzed. Results are shown as mean SEM of 2 independent experiments in 4
independent donor-derived cultures. (E), Midazolam metabolism is performed
exclusively
by functional CYP3A3/4/5 enzymes. 3 different organoid cultures from 2
different donors
and HepG2 cells were plated and cultured for 11 days as described, then
midazolam was
added to the medium (5E M) and after 24 hours, concentrations of 1-0H
midazolam and 1-
OH midazolam glucuronide were determined as described in methods. Duplicates
for each
condition and donor were analyzed. Results are shown as mean SEM of 2
independent
experiments. (F) Bile acid production was measured as described in methods.
Results are
shown as SEM of 2 independent experiments in 2 independent donor-derived
cultures.
Duplicates for each condition and donor were analyzed. (G) Ammonia elimination
was
measured as described in methods. Results are shown as SEM of n=3
independent
experiments in 2 independent donor-derived cultures and are expressed as
nM/h/million
cells. (H) Retrorsine/CCI4 treated Balbc/nude mice were transplanted with 1-
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liver organoid cells and sacrificed after 120 days. The presence of foci of
human Albumin
positive, but human KRT19 negative hepatocytes proves successful engraftment
and
differentiation in mouse liver. (I) Average serum levels of human Albumin in
mouse
circulation after transplantation. Results are shown as SEM of 2 vehicle
control animals,
2 primary hepatocyte transplanted mice and 6 human liver organoid transplanted
animals.
**, p<0,01 and *, p<0,05 when comparing EM vs DM.
Figure 20: Transplantation of human liver organoids into damaged mouse liver.
(A) Control
staining for human specific Albumin (hAlbumin) and Kertatin-19 (hKrt19)
antibodies.
hAlbumin recognises human but not mouse hepatocytes, whereas hKrt19 stains
human
but not mouse bile ducts. (B) Liver sections of mice sacrificed 2 hours or 2
days after
human liver organoid cell transplantation stained for hKrt19. After 2 hours
human cells are
mostly seen in blood vessels in and around portal veins, whereas cells start
to engraft in
the tissue 2 days after the transplant. (C) Example singlet or doublet human
Albumin
positive hepatocytes observed in the liver of human liver organoid
transplanted Balbc/nude
mice. (D) Human serum Albumin levels of individual transplanted mice over 120
days. (E)
Average human serum alpha-1-antitrypsin levels of transplanted mice over 120
days.
Results are shown as SEM of 2 vehicle control animals and 3 human liver
organoid
transplanted animals.
Figure 21: Human A1AT deficiency liver cultures as an in vitro disease model.
(A)
Representative pictures of A1AT deficient patient derived liver organoids at
Passage 2 and
Passage 11 (4x magnification). (B) ELISA measurement of Albumin secretion in
supernatant from donor and A1AT deficient patient organoids in EM or after 11
days in
DM. Patients and donors show a similar level of albumin release. Results are
expressed
as mean SEM of 2 independent experiments. (C) A1AT deficient patient
organoids were
differentiated for 11 days and incubated with Dil-Ac-LDL as described in
materials and
methods. Fluorescence microscopy shows robust LDL uptake in patient organoids.
Scale
bar, 50 pm (D) Fold induction of Albumin and CYP3A4 mRNA levels after 11 days
of
differentiation of donor and A1AT deficient patient organoids. Results are
expressed as
mean SEM of 2 independent experiments (E-H) lmmunohistochemistry for A1AT on
liver
tissue (E/G) and liver derived organoids from a healthy donor (F) and a
representative
A1AT deficient patient (H). Arrows indicate A1AT protein aggregates in patient
derived
liver tissue (G) and organoids (H). Scale bar, 20 pm. (I) ELISA measurement of
A1AT
secretion in supernatants from Donor and patient organoids after 11 days of
differentiation.
Results are expressed as mean SEM of 2 independent experiments. (J)
Enzymatic
measurement of Elastase inhibition by supernatants of differentiated donor and
patient
derived organoid cultures (as described in materials and methods).
Supernatants from all 3

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patients show reduced inhibition of Elastase activity. Results are expressed
as mean
SEM of 2 independent experiments (K) Western blot of total lysates from donor
and A1AT
deficient patient organoids after 11 days of differentiation. Increased elF2a
phosphorylation at Ser51 was detected in the 3 patients. Representative image
is shown.
Pat., patient.
Figure 22: Organoids from A1AT deficiency and AGS patients mimic disease
phenotypes
in vitro. (A) SERPIN1A Sanger Sequencing of Donor #1 and a1AT Patient #1.
Chromatograms of 3 Al AT-deficient patients (PiZZ) and 1 donor with wildtype
SERPINA1
(PiMM). The hom*ozygous G to A mutation causes an amino acid change from
glutamic
acid to lysine at position 342. (B) Clustering analysis of the different
donors (1-5) and a1AT
Patient (A1AT_pat) organoids and tissues. Note that, regarding differentiation
ability, the
behaviour of a1AT Patient derived organoids resembles donor derived organoids.
i.e.
organoids in EM cluster cluster with donor EM organoids and a1AT-D organoids
cultured
in DM cluster with donor derived organoids cultured in DM conditions. (C)
histological
staining for cleaved caspase-3 in d nor and a1AT Patient derived organoids
differentiated
in DM for 11 days. (D) quantification of apoptotic cells in wildtype and a1AT
Patient
derived organoids in EM and after differentiation in DM. Results are shown as
SEM of 6
random sections of organoids per 2 independent donors and patients. (E) gRT-
PCR of
Lgr5 and ductal markers (Krt19 and Krt7) in EM and after ductal
differentiation. AGS
patients fail to upregulate ductal markers upon differentiation. (F)
lmmunofluorescence of
differentiated wildtype and AGS patient organoids. Krt19 positive cells in AGS
patient
organoids do not integrate into the epithelium and show signs of apoptosis
(arrows). EM,
expansion medium. DM, differentiation medium, ductal diff, ductal
differentiation medium
(see text). AGS, AlegiIle syndrome.
Examples
Example 1: TGF-beta (TGFb) inhibition increases Human liver orpanoid formation

efficiency
Human liver tissue was digested using collagenase dissociation and liver cells
isolated as
described in Material and Methods. Cells were cultured in mouse liver medium
containing
Egf, Rspo, Fgf10, Hgf and Nicotinamide (ERFHNic) and 2-4 weeks after, RNA was
isolated and analysed for expression of TGFb signaling pathway regulators.
(Figure 1A)
Expression profile showed that human liver organoids express high levels of
TGFb while
(Figure 1B) TGFb inhibitors (SMAD6, SMAD7) or sequesters (LTBP2, LTBP3) were
almost
absent in the human liver cultures when cultured under mouse medium
conditions. Graphs
represent the absolute value as obtained from the microarray. Note that the
data is

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represented in logarithmic scale. (Figure 1C-D) Human liver cells isolated by
collagenase
dissociation were dissociated to single cell, counted and 3000 or 10000 cells
were seeded
per well in a 48we11 plate. Mouse liver culture medium (ERFHNic) or the same
medium
supplemented with A8301 (+A) was overlaid and organoids were allowed to grow.
Organoids numbers were counted 15 days after seeding. Treatment with A8301
significantly increased organoid formation efficiency. (Figure 1C) DIC images
of organoids
treated with mouse liver medium supplemented (right panel) or not (left panel)
with the
ALK5/6 inhibitor A8301. (Figure 1D) Graph indicating the % of colony formation
efficiency
in cultures seeded in the presence or absence of A8301. Experiments were
performed in
triplicate. Five different donor derived cultures were counted. Results are
expressed as
mean SEM of 5 independent experiments. The organoid efficiency results shown
in
Figure 1D illustrate that many more organoids are formed when a TGF beta
inhibitor is
present in the medium compared to when it is absent.
Example 2: FSK and BMP7 are useful for the long-term culture of human liver
organoids
Human liver cells isolated by collagenase dissociation were dissociated to
single cell,
counted and 3000 or 10000 cells were seeded per well in a 48 well plate. Mouse
liver
culture medium (ERFHNic) or medium supplemented with A8301 or A8301 and BMP7
or
A8301 and BMP7 and Forskolin (FSK) was overlaid as indicated and organoids
were
allowed to grow. The cultures were split every week 7-10 days at a ratio of
1:4 -1:6 dilution.
All the cultures started to grow and proliferate however, the cultures grown
in mouse
medium or medium supplemented with A8301 only or BMP7 and A8301 arrested
proliferation after some weeks in culture and could not be expanded any
further as
indicated in the graph. Supplementing the culture medium with A8301 combined
with FSK
significantly increased the expansion efficiency of the cultures which have
been able to
grow for >18 passages at a split ratio of 1:4-1:6 every 7-10 days for >5
months. The
results are shown in Figure 2.
Example 3: Under FSK supplemented medium, cells maintain their proliferation
potential over time
To quantify the proliferation capacity of the human liver cultures, expansion
ratios, in vitro
growth curves and EdU incorporation, at early and late passages, were analysed
in human
liver cultures grown in complete medium (ENRFHNic supplemented with A8301 and
FSK
as described in methods). (Figure 3A-C) Cell numbers were counted by Trypan
blue
exclusion at the indicated time points, in at least 3 independent human liver
cultures
(independent donor material). The cultures followed an exponential growth
curve within

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each time window analysed. Graphs illustrate the number of cells counted per
well at each
passage from passage P1-P4 (Figure 3A), P16-P18 (Figure 3B). The doubling
time, or
amount of time the culture needs to double its original size was calculated as
follows:
doubling time = In(2)/growth rate for each time window analysed. Note that the
doubling
time was essentially maintained once the culture had started to expand from
day 16
onwards, indicating that the expansion potential is maintained within the time
period
analysed. (D) Similar EdU incorporation was detected in early and late
passages, again
indicating that the cells maintain their proliferation potential in vitro
after long-term
culturing.
Example 4: Other cAMP activators also maintain the human liver cultures for
long
term
Human liver organoids were seeded in mouse medium supplemented with A8301 and
with
one of the cAMP activators as indicated in Figure 4. Cholera toxin was used at
a
concentration of 100 ng/ml. Only the cultures that were treated with cAMP
activators were
able to be expanded >2 months (P8). The results are shown in Figure 4.
Example 5: Human liver cultures treated with FSK express high levels of LGR5
Lgr5 expression was analysed in cultures grown in our defined culture medium
by
immunofluorescence and flow cytometry analysis. (Figure 5A) Confocal image of
a human
liver organoid showing that the organoids are formed by epithelial derived
cells positive for
EPCAM (blue). The stem cell marker Lgr5 (green) was restricted to a subset of
cells within
the organoid, while the Wnt target gene EPHB2 (red) was broadly expressed, but

colocalized with LGR5 as expected. Flow cytometry analysis of LGR5 positive
cells is
shown in Figure 5B. Staining was performed on single cells isolated from a
culture that had
been cultured for >4 weeks in the presence or absence of FSK. Only in the
presence of
FSK Lgr5 cells could be readily detected as a 1-3% of the culture population.
Experiment
was performed in 2 independent human liver donor cultures. (Figure 5C) qPCR
analysis of
the expression of LGR5 in 4 week old cultures treated in the presence or
absence of FSK.
LGR5 expression levels are 2-3x upregulated upon treatment with FSK.
Example 6: Wnt signaling and cAMP are important for the growth of the cultures

Expanding human liver organoids grown in complete medium as described in
Methods
were maintained in that medium (EM) or transferred to a medium without Rspo (-
Rspo),
without FSK (-FSK) and without Rspo including the porcupine inhibitor (-Rspo +
IWIP).
Organoid numbers were counted 1 week later. The results show that both, cAMP
and Wnt
are essential signaling pathways to maintain the human liver culture in vitro.
(Figure 6A)
DIC images of the cultures treated with the different compounds as indicated
in the figure.

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(Figure 6B) Graphs indicating the number of organoids in the presence/absence
of the
compounds in 2 independent human liver cultures.
Example 7: Human liver cultures maintain chromosome numbers over time
Genetic stability of the human liver organoids cultured for long-term. (Figure
7A)
Representative image of a chromosome spread illustrating a normal count (n=46)
of a
metaphase of a cell cultured for 16 days (P1) or cultured for 100 days (P15).
The table
illustrates the % of cells with chromosomal counts as indicated.
Example 8: Human liver organoids express markers of the ductal and hepatocvte

lineages
Gene expression was analysed by immunofluorescence (A) or RT-PCR (B) analysis
in
human liver cultures grown in our defined complete expansion medium as
described in
methods. (Figure 8A) Human liver cultures expressed progenitor (LGR5, CD133)
ductal
(KRT19 and KRT7) and hepatocyte (HNF4A) markers.
Further analysis confirmed that the stem cell markers PROM1 and LGR5, as well
as ductal
(S0X9, 002) and hepatocyte markers (HNF4a) were readily expressed (Figure 14A
and
14G and H). Histologically, liver organoids displayed a duct-like phenotype
characterized
by two types of epithelia: 1) a single-layered epithelium formed by polarized
cells with
basal nuclei, expressing cytokeratin epithelial markers (KRT19 and KRT7), and
2) a
pseudo-stratified epithelium with non-polarized E-Cadherin+, HNF4a+ and some
KRT7+
cells (Figure 14B-D). SOX9 (Figure 14E) and EPHB2 (Figure 14F) were detectable
in
almost all the cells within an organoid while LGR5 was detectable within the
EPHB2+
population (Figure 14F).
Example 9: Human organoids in complete media grow from single isolated cells
Human liver cultures grown for at least 2 months in our defined medium were
dissociated
to single cell and sorted as described in methods. Cells were seeded at a
ratio of 1 cell per
well. Cells quickly proliferated and expanded. DIC images of growing single
cells from
human liver cultures. Magnifications: 40x (days 0-10), 4x (day 20-on).
Example 10: Upon Differentiation, Liver cultures upregulate HEPATOCYTE
specific
genes
Human liver cultures were expanded for at least 1 month in culture and
transferred to our
differentiation medium as described in Methods. Expression of hepatocyte genes
was
determined by immunofluorescence (Figure 10A) or qPCR (Figure 10B) 11 days
later.
(Figure 10A) lmmunofluorescence staining showing albumin (ALB, red) and zona
occuludens (Z0-1, green) positive cells all over the organoid, indicating that
the cells start

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expressing the mature hepatocyte markers. (Figure 10B) qPCR analysis indicated
that
both, albumin and Cytochrome p450 3A4 isoform were highly expressed upon
differentiation. Graphs indicate mean SEM of 3 independent experiments in 3
independent donor derived cultures. EM, expansion medium including FSK. DM,
differentiation medium, HuLi, whole lysate from human liver.
Example 11: Liver cultures accumulate glycogen, uptake LDL and maintain
cytochrome activity, in vitro
To test whether the cells could have differentiated towards functional
hepatocytes in vitro,
we determined the ability of the cultures to accumulate glycogen, uptake LDL
and have
active cytochrome activity. (Figure 11A), Glycogen accumulation was determined
by PAS
(Periodic-Acid Schiff) staining in organoids grown in EM or DM for 11 days.
PAS positive
staining (pink) was exclusively observed in the organoids after
Differentiation (DM),
indicating that the cells have recovered the capacity to accumulate glycogen.
Magnification, 10x. (Figure 11B) LDL uptake was analysed using Dil-ac-LDL
fluorescent
substrate (red) in cultures maintained in EM (left) or DM (right) for 11days.
Only cultures
maintained in DM incorporated the substrate (red). Nuclei were counter-stained
with
DRAQ5. Scale bar, 25 pm. (C), Cyp3a4 is expressed exclusively in mature
hepatocytes. It
has an important role in the detoxifying function of the liver. CYP3A4
activity was
measured as described in methods in cultures kept in DM for 11 days. Results
are
expressed as RLU per ml per million cells. HEK293T cells and HepG2 cells were
used as
negative and positive controls respectively. Triplicates for each condition
were analysed.
Results are shown as mean SEM of 2 independent experiments in 4 independent
donor-
derived cultures.
Example 12: METHODS
Human liver organoid culture
Liver cells were isolated by collagenase digestion as follows: tissue (0.5-
1cm3) was
minced, rinsed 2x with DMEM (Gibco) 1%FCS and incubated with the digestion
solution
(2.5 mg/ml collagenase D (Roche) + 0.1 mg/ml DNase I (Sigma) in EBSS (Hyclone,

Thermoscientific), for 20-40 at 37 C. The digestion was stopped by adding cold
DMEM
1`)/0FCS and the suspension was then filtered through a 70 um Nylon cell
strainer and spun
5 min at 300-400g. The pellet was resuspended in DMEM 1`)/0FCS and kept cold.
Any
material retained on the strainer was further digested for 10 min in Accutase
(Innovative
Cell Technologies) at 37 C. Then, the digestion was stopped and the cells were
collected
as before. The different fractions (collagenase and accutase) were mixed and
washed with
cold Advanced DMEM/F12 and spun at 300-400g for 5 min. The cell pellet was
mixed with

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Matrigel (BD bioscience) and 3000-10000 cells were seeded per well in a
48we11/plate.
After Matrigel had solidified, culture medium was added. Culture media was
based on
AdDMEM/F12 (Invitrogen) supplemented with N2 and B27 without retinoic acid
(both from
Gibco), 1.25 mM N-Acetylcysteine (Sigma), 10 nM gastrin (Sigma) and the growth
factors:
50 ng/ml EGF (Peprotech), 10% RSPO1 conditioned media (home-made), 100 ng/ml
FGF10 (Peprotech), 25ng/m1 HGF, 10mM Nicotinamide (Sigma), 5uM TGF-beta
inhibitor
(A83.01 (Tocris)) and 10uM FSK (Tocris). For the establishment of the culture,
the first 3
days after isolation the medium was supplemented with 25ng/m1 Noggin
(Peprotech), 30%
Wnt CM (home-made prepared as described in (Barker and Huch 2010)), 10 uM Rock
inhibitor (Y27632) and hES Cell cloning Recovery solution (Stemgent). Then,
the medium
was changed into a medium without Noggin, Wnt, Y27632, hES Cell cloning
Recovery
solution (Stemgent) while 25ng/m1 BMP7 (Peprotech) were supplemented on top.
One
week-10 days organoids were removed from the Matrigel, mechanically
dissociated into
small fragments, and transferred to fresh Matrigel. Passage was performed in
1:4-1:8 split
ratio once per week for at least 6 months. To prepare frozen stocks, organoid
cultures
were dissociated and mixed with Recovery cell culture freezing medium (Gibco)
and froze
following standard procedures. When required, the cultures were thawed using
standard
thawing procedures, embedded in Matrigel and cultured as described above. For
the first 3
days after thawing, the culture medium was supplemented with Y-27632 (10 pM,
Sigma
Aldrich).
Single cell (clonal) culture
For clonogenic assays, single cell suspensions from established cultures were
dissociated
with TriplE express (gibco). Propidium iodide staining was used to label dead
cells and
FSC: Pulse-width gating to exclude cell doublets (MoFlow, Dako). Cells were
embedded in
Matrigel and seeded in 96 well plates at a ratio of 1 cell/well. Cells were
cultured as
described above with medium supplemented with Y-27632 (10 pM, Sigma Aldrich)
for the
first 4 days. Passage was performed in split ratios of 1:4-1:8 once per week
for at least 8
months. All phase contrast pictures were acquired using a Leica DMIL
microscope and a
DFC420C camera.
Hepatocyte differentiation
To enhance hepatocyte cell fate, liver organoids were seeded and kept 2-4 days
under the
liver expansion conditions explained above. Then, medium was changed to
AdDMEM/F12
medium supplemented with 1%N2 and 1% B27 without retinoic acid (both from
Gibco) and
containing EGF (50 ng/ml), gastrin (10nM, Sigma), HGF (25ng/ml, Peprotech),
FGF19
(100 ng/ml), A8301 (500 nM, Tocris Bioscience), DAPT (10 uM, Sigma), BMP7
(25ng/m1)

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and Dexamethasone (30uM). Medium was changed every other day for a period of 9-
11
days.
Hepatocyte functional studies
To assess glycogen storage and LDL uptake, liver organoids grown in EM or DM
for 11
days were stained by Periodic acid-Schiff (PAS, Sigma) and Dil-Ac-LDL
(biomedical
technologies), respectively, following manufacturer's instructions. To
determine albumin
and A1AT secretion, liver organoids were differentiated as described. Culture
medium was
changed every other day and culture supernatant was collected was collected
24h after
the last medium change. HepG2 and HEK293T cells (ACCC) were cultured for 24h
in the
same medium without growth factors and were used as positive and negative
control
respectively. The amount of albumin and A1AT in culture supernatant was
determined
using a human specific Albumin or human specific A1AT ELISA kit (both from
Assay Pro).
To measure Cyp3a activity the cultures were differentiated as described and
the day of the
experiment the cells were removed from the matrigel and cultured with the
Luciferin-PFBE
substrate (50 pM) in Hepatozyme medium supplemented with 10% FBS (Gibco). As
controls, HepG2 and HEK293Tcells were cultured for 24h in DMEM 10%FBS and the
day
of the experiment transferred to Hepatozyme medium supplemented with 10% FBS
(Gibco) and Luciferin-PFBE substrate (50 pM). Cytochrome P450 activity was
measured
8h later using the P450-Glo Assay Kit (Promega) according to manufacturer's
instructions.
In vitro growth curves
Expansion ratios were calculated from human liver cultures as follows: 3x103
cells were
grown in our defined medium for 7 or 10 days. Then, the cultures were
dissociated by
incubation with TrypLE Express (Gibco) until single cells. Cell numbers were
counted by
trypan blue exclusion at the indicated time points. From the basic formula of
the
exponential curve y(t) = y0 x e(growth rate x t ) (y = cell numbers at final
time point; y0 =
cell numbers at initial time point; t = time) we derived the growth rate.
Then, the doubling
time was calculated as doubling time = In(2)/growth rate for each time window
analyzed.
Karyotyping
Organoid cultures in exponential growing phase were incubated for 16 hours
with 0.05
pg/ml colcemid (Gibco). Then, cultures were dissociated into single cells
using TrypLE
express (Gibco) and processed using standard karyotyping protocols.
Chromosomes from
100 metaphase-arrested cells were counted.

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lmmunohistochemistry and immunoflorescence
Tissues and organoids were fixed o/n with formalin or 4% PFA respectively,
washed and
transferred to tissue cassettes and paraffin blocks using standard methods.
Tissue
sections (4 pM) were prepared and stained with antibodies, H&E or PAS using
standard
techniques. The antibodies and dilutions used are listed in Supplementary
Table I. Stained
tissues were counterstained with Mayer's Hematoxylin. Pictures were taken with
a Nikon
E600 camera and a Leica DFDC500 microscope (Leica). For whole mount
immunofluorescence staining, organoids were processed as described in Barker
et al,
(Barker et al, 2010). Nuclei were stained with Hoechst33342 (Molecular
Probes).
Flow cytometry analysis
Exponentially growing organoids were cultured for at least 5 days in the
presence or
absence of FSK. Then, organoids were dissociated into single cells using
Accutase,
resuspended in DMEM + 2% FBS and incubated with Lgr5 antibody (AP2745d,
Abgent) for
45 min. A1exa488-conjugated donkey anti-rabbit Ig was used as secondary
antibody
(Molecular Probes). Cells were analyzed with a BD FACS Calibur (Becton-
Dickinson);
FSC: propidium iodide was used to label dead cells for exclusion and pulse-
width gating to
exclude cell doublets.
RT-PCR and qPCR analysis
RNA was extracted from organoid cultures or freshly isolated tissue using the
RNeasy Mini
RNA Extraction Kit (Qiagen), and reverse-transcribed using reverse-transcribed
using
Moloney Murine Leukemia Virus reverse transcriptase (Promega). All targets
were
amplified (40 cycles) using gene-specific primers and MilQ syber green (Bio-
Rad). Data
were analyzed using BioRad CFX manager. cDNA was amplified in a thermal cycler

(GeneAmp PCR System 9700; Applied Biosystems, London, UK) as previously
described
(Huch et al, 2009).
Image analysis
Images of cultivated cells were acquired using either a Leica DMIL microscope
and a
DFC420C camera or an EVOS FL system (Life Technologies). lmmunofluorescence
images were acquired using a confocal microscope (Leica, 5P5) or a confocal
microscope
(Leica, 5P8). Images were analyzed and processed using Leica LAS AF Lite
software
(Leica 5P5 confocal).
Data analysis
All values are represented as mean standard error of the mean (S.E.M.). Man-
Whitney
non-parametric test was used. p<0.05 was considered statistically significant.
In all cases

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data from at least 3 independent experiments was used. All calculations were
performed
using SPSS package.
Table 2: List of tested compounds
List of all the compounds tested for their capacity to enhance human liver
culture
proliferation, long-term maintenance or differentiation (Figure 12). Human
liver cultures
were seeded in ERFHNic medium supplemented with A8301 and the compound
indicated
on the list. Seeding efficiency and capacity to expand long-term the cultures
was
evaluated.
Example 13: FSK-supplemented media is advantageous for expansion of pancreatic

organoids
Pancreatic ductal cells from healthy human control sample (0366) were cultured
in 10
different conditions from passage 0. Samples were passaged once a week. Red
crosses
indicated death of the culture. Arrowheads indicated culture is growing. The
graph
indicates that a culture medium comprising forskolin allows passaging of
pancreatic
organoids beyond five weeks, even in the "Wnt" medium which could not be
passaged
beyond five weeks in the absence of forskolin. Therefore, forskolin is also
advantageous
for the growth and expansion of pancreatic organoids. FRSK (Forskolin 10uM);
Nic
[medium containing B27 lx, NAc (1.25 mM), Egf (50ng/m1), Gastrin (10nM), Fgf10

(10Ong/m1), Noggin (10% CM), Rspo (10%CM) and Nicotinamide (10mM)]; PGE2
[medium
containing B27 lx, NAc (1.25 mM), Egf (50ng/m1), Gastrin (10nM), Fgf10
(10Ong/m1),
Noggin (10% CM), Rspo (10`)/0CM), Nicotinamide (10mM), A8301 (500nM) and PGE2
(1uM)]; Wnt [medium containing B27 lx, NAc (1.25 mM), Egf (50ng/m1), Gastrin
(10nM),
Fgf10 (10Ong/m1), Noggin (10% CM), Rspo (10`)/0CM), Nicotinamide (10mM), A8301

(500nM) and Wnt (50`)/0CM)]; Wnt+PGE2 [medium containing B27 lx, NAc (1.25
mM), Egf
(50ng/m1), Gastrin (10nM), Fgf10 (10Ong/m1), Noggin (10% CM), Rspo (10%CM),
Nicotinamide (10mM), A8301 (500nM), Wnt (50`)/0CM) and PGE2 (1uM)]; Complete
[medium containing B27 lx, NAc (1.25 mM), Egf (50ng/m1), Gastrin (10nM), Fgf10

(10Ong/m1), Noggin (10% CM), Rspo (10%CM), Nicotinamide (10mM), A8301 (500nM),

Wnt (50%CM), PGE2 (1uM) and p38i (10uM)].
Example 14: Human liver organoid cultures initiate from ductal cells
To assess the cell-of-origin of our cultures, we FACS-purified hepatocytes and
duct cells
from 3 independent human hepatocyte isolations instead of liver biopsies.
Hepatocyte
isolations by collagenase perfusion yield high numbers of fresh, viable and
functional
human hepatocytes that are used for hepatocyte transplantation infusions
(Gramignoli et
al., 2012) (Figure 15A). We employed EpCAM to differentially sort hepatocytes
(EpCAM-)

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from ductal cells (EpCAM+, bile duct and canal of herring ductal /progenitor
cells, Figure
15B and 150) (Schmelzer et al., 2007; Yoon et al., 2011). Ductal (EpCAM+)
cells
developed into long-term, self-renewing organoid structures with a striking
efficiency of
28.4 3.2% (Figure 15D-E). When crude hepatocyte preparations (not
differentially sorted)
were directly cultured, cells grew into organoid structures with an efficiency
that correlated
directly with the amount of residual EpCAM+ cells in the crude preparation
(Figure 15F-G).
Therefore, we concluded that in our culture system ductal cells and not
hepatocytes revert
to a bi-potential progenitor state (i.e. epithelial stem cells in the context
of the invention).
Example 15: Human liver cultures established from single human liver cells are

genetically stable
Genetic stability is a concern for the future application of cells that have
undergone
derivation and expansion in culture (Lund et al., 2012). Adult stem cells may
have evolved
to minimize the risk of accumulating somatic mutations (Cairns, 1975). Indeed,
karyotyping
of clonal human liver organoids cultured for 3 months revealed that the cells
maintain
normal chromosome numbers over time (Figure 16A). The ability to repeatedly
generate
clonal cultures from single liver stem cells allowed us to isolate sufficient
DNA for whole
genome sequencing (WGS) analysis and subsequent characterization of the
mutational
load present in the cultured cells after several months of in vitro expansion
(Figure 17A).
From two donors, we obtained biopsy samples, which we dissociated and cultured
in bulk
for 7 days. Subsequently, we isolated single cells by flow cytometry and
established 2
independent clonal lines for each of the two livers (cultures A and B). After
3 months of
expanding these cultures, a second cloning step was performed. The combined
procedure
allowed us to determine all the genomic variation that had accumulated in a
single cell
during life, derivation, and 3 months of culturing (Figure 17B).
We observed 720 ¨ 1424 base substitutions per cultures of which only a small
part was
introduced during the 3 months culture, which is equivalent to 13 weekly
passages (63 ¨
139; Figure 170). The majority of the base substitutions were therefore
incorporated
during life or introduced during organoid derivation. Interestingly, we
observe twice as
many base substitutions in both cultures derived from donor 1 compared to the
cultures
derived from donor 2 (Figure 170). This is most probably the result of the
high age of
donor 1 (74 years) compared to donor 2 (30 years), suggesting that the
majority of the
somatic base substitutions we observed were acquired during life.
How do these numbers compare to published data? It has been reported that iPS
cells
contain 1,058 ¨ 1,808 de novo base substitutions per line (determined at
passage
numbers between 15 and 25) when compared to their parental somatic cells
(Cheng et al.,

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2012). Of note, these numbers do not include the variation acquired in vivo in
the parental
somatic cells, which we did determine here for the clonal liver organoid
cultures. We
therefore conclude that liver organoid cultures accumulate in the order of 10-
fold fewer
base substitutions during in vitro expansion compared to iPS cells. Of the
total number of
base substitutions only few were located in protein coding DNA (7 ¨ 9 base
substitutions
per culture; Figure 17D). With the exception of one synonymous mutation in
culture A from
donor 2, all mutations were already present in the early passage clonal
cultures, indicating
that they were incorporated during life or organoid derivation and not during
3 months-
expansion. None of the mutated genes occurs in COSMIC databases. In iPS cells,
it has
been reported that an average of 6 base substitutions per line affect protein
coding DNA
(Cheng et al., 2012; Gore et al., 2011) which were reported to be enriched for
genes
mutated or being drivers in cancers (Gore et al., 2011). Next, we checked for
evidence of
chromosomal aberrations in the WGS data of the different liver organoid
cultures. In line
with our karyotyping analysis, we did not observe any chromosomal aberration
(Figure
16B). We observed 2 copy number variants (CNVs), heterozygous gains, in one of
the liver
organoid cultures (Figures 16C). In the other cultures, we did not detect any
CNV (Figure
16D). Moreover, these 2 CNVs were already present in the early passage
cultures and
therefore did not result from long-term culturing, suggesting they were either
acquired in
vivo or during organoid derivation. ES cell cultures routinely show abnormal
karyotypes
(Baker et al., 2007) and iPS cells have been reported to harbor considerable
amounts of
somatic CNVs (Hussein et al., 2011; Laurent et al., 2011) (Martins-Taylor et
al., 2011;
Mayshar et al., 2010) (Abyzov et al., 2012), complicating their clinical use.
Example 16: Differentiation into functional hepatocvtes in vitro and upon
transplantation
Similar to what we had observed with the mouse liver organoid cultures under
expansion
conditions, the human counterparts failed to express markers of mature
hepatocytes, such
as Albumin or CYP3A4 (Figure 14A and Figure 18C, EM bars). Therefore, we
defined a
human differentiation medium (DM) by combining our acquired knowledge on mouse

hepatocyte differentiation with known hepatocyte differentiation-promoting
compounds.
Removal of the growth stimuli R-spo and FSK directly resulted in the up-
regulation of
Albumin and CYP3A4 gene expression (Figure 141). To this medium, we then added
the
Notch inhibitor DAPT, FGF19 and dexamethasone (Figure 14J). When testing
compounds
to improve our culture conditions, we noticed that BMP7 slightly facilitated
the expression
of hepatocyte markers ALB and CYP3A4, without compromising the proliferation
ability of
the culture itself. Therefore, 5-7 days prior to the start of differentiation,
we supplemented
the expansion medium (EM) with 25ng/m1 BMP7, which was then maintained during
the

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differentiation step (Figure 18A). Using this combination of growth factors
(BMP7, FGF19,
HGF and EGF), small molecule inhibitors (DAPT and A8301) and Dexamethasone,
the
cells acquired pronounced hepatocyte morphologies, including polygonal cell
shapes, as
made visible by ZO-1 staining (Figure 18B). We subsequently examined the level
of
maturity of the differentiated cells by using gene expression profiling,
immunofluorescence
and various biochemical assays.
Gene expression profiles proved that the differentiated cultures expressed
high levels of
hepatocyte markers (Figure 18D). Hepatocyte specific genes such as ALB,
several
cytochrome enzymes, Apolipoproteins (APOB) and several complement factors (03)
were
readily expressed upon differentiation in all 4 donors analyzed (Figure 18D).
We confirmed
these results by qPCR and RT-PCR analysis for selected genes (ALB, several
cytochromes, and TAT) (Figure 180 and Figure 14K) and found that the
differentiated
cultures express levels of cytochrome CYP3A4 expression similar to that of
human liver
tissue. A 100-1000x fold increase in Albumin expression was also detected on
the DM-
treated cultures, although the expression levels were still 1000x lower when
compared to
freshly isolated human liver material. lmmunofluorescence visualized cells
with high levels
of ALB and MRP4 within the organoids (Figure 18B). Similar results were
obtained with
cultures derived from EpCAM+ sorted cells (Figure 14L-M).
We next assessed the ability of the hepatocyte cells to retain hepatocyte
function in vitro.
lmmunohistochemistry analysis indicated that the cells could accumulate
glycogen (Figure
19A) and take up LDL (Figure 19B). Biochemical analyses demonstrated that the
differentiated cells secreted high levels of Albumin into the medium (Figure
190).
Cytochrome family members, such as Cyp3a4, are expressed exclusively in mature

hepatocytes. They play an important detoxifying function for exogenous
molecules in the
liver (Casciano, 2000). Upon differentiation, the cultures exhibited similar
p450-3A4 activity
as fresh isolated hepatocytes (Figure 19D, compare to Figure 15A). We also
observed that
the differentiated cultures hydroxylated midazolam, another indication of
functional
CYP3A3/4/5 activity (Wendel et al., 1994), and glucuronidated hydroxy-
midazolam,
thereby showing evidence of both phase I and ll detoxifying reactions (Figure
19E). We
then assessed the ability of the cultured cells to synthetize bile acids, a
hallmark of
hepatocyte function. Upon differentiation, bile acid salts were readily
secreted into the
medium (Figure 19F). Finally, the cultures also exhibited the ability to
detoxify ammonia at
similar levels to HepaRG cells (Figure 19G). In all cases, the expanded human
liver
organoids showed stronger hepatocyte functions when compared to the
standard/reference cell line HepG2 cells (Figure 19).

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To test the ability of the cultures to engraft in damaged tissue and to fully
differentiate into
functional hepatocytes in vivo, we treated Balb/c nude mice with CCI4-
retrorsine to induce
acute liver damage. As shown by others, this treatment is permissive for the
engraftment
of hepatocytes (Guo et al., 2002; Schmelzer et al., 2007). Using human-
specific antibodies
(Figure 20A), we initially detected Krt19 positive, ductal-like cells at 2h
and d2 after
transplantation, distributed throughout the liver parenchyma (Figure 20B). At
later time
points, we observe Albumin+, Krt19- human cells as singlets or doublets or,
more rarely, in
larger hepatocyte foci in the mouse liver (Figure 19H and Figure 200). This
agreed with
the non-chronic nature of our damage model, which provides no stimulus for
expansion of
the transplant after the initial engraftment. We detected human Albumin and
human alpha-
1- antitrypsin in the circulation of recipient mice within 7-14 days (Figure
191 and Figure
DIE), at a level that remained stable for more than 60 days in 5/6 mice and
for more
than 120 days in 2/5 animals. While transplantation of primary human
hepatocytes initially
yielded higher levels of human Albumin in mouse circulation (Figure 191), the
levels
15 approximated those of transplanted organoids within a month. Presence of
human albumin
and human alpha-1-antitrypsin in mouse serum proved, together with Albumin and
Krt19
stainings, that transplanted cells differentiated into human hepatocytes in
vivo.
Transplantation method:
We used a modified version of the protocol used by Guo et al. (Guo et al.,
2002). In short,
20 female BALB/c nude mice (around 7 weeks of age) were pretreated with two
injections of
70 mg/kg Retrorsine (Sigma) at 30 and 14 days before transplantation. One day
prior to
transplantation, mice received 0.5 ml/kg 0014 and 50 mg/animal anti-asialo GM1
(Wako
pure chemical industries) via IP injection. Furthermore, animals received 7.5
ug/ml FK506
in drinking water until the end of the experiment, due to the reported
positive effects on
liver regeneration (He et al., 2010). On the day of transplantation, mice were
anaesthetized
and suspensions of 1-2x106 human liver organoid cells derived from 4
independent donors
(p6 to p10) were injected intrasplenically. Transplanted mice received weekly
injections of
50 mg/animal anti-asialo GM1 (Wako pure chemical industries) to deplete NK
cells. To
monitor the transplantation state, blood samples were taken in regular
intervals from the
tail vein and analyzed for the presence of human albumin and human a1-
antitrypsin using
respective human specific ELISAs (Assaypro).
Karyotyping and Genetic stability analysis:
Organoid cultures in exponential growing phase were incubated for 16 hours
with 0.05
ug/m1 colcemid (Gibco). Then, cultures were dissociated into single cells
using TrypLE
express (Gibco) and processed using standard karyotyping protocols.

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DNA libraries for WGS analysis were generated from 1 ug of genomic DNA using
standard
protocols (Illumine). The libraries were sequenced with paired-end (2 x 100
bp) runs using
Illumine HiSeq 2500 sequencers to a minimal depth of 30 x base coverage
(average depth
of ¨ 36.9 x base coverage). As reference sample, liver biopsies was sequenced
to equal
depth for the different donors. The data for the whole genome sequencing were
deposited
to the EMBL European Nucleotide Archive, accession number ERP005929.
Immunohistochemistry, immunofluorescence and Image analysis:
Tissues and organoids were fixed o/n with formalin or 4% PFA respectively, and
stained
and imaged by methods known in the art.
Microarray methods:
For the expression analysis of human liver cultures, total RNA was isolated
from liver
biopsies or from organoids cultures grown in our defined medium, using Qiagen
RNAase
kit following manufacturer's instructions. Five hundred ng of total RNA were
labeled with
low RNA Input Linear Amp kit (Agilent Technologies, Palo Alto, CA). Universal
human
Reference RNA (Agilent) was differentially labeled and hybridized to the
tissue or cultured
samples. A 4X 44 K Agilent Whole Human Genome dual colour Microarray (G4122F)
was
used. Labeling, hybridization, and washing were performed according to Agilent

guidelines.
Example 17: Organoids from human patients model disease pathogenesis in vitro
Encouraged by the establishment of a culture medium that allows the long-term
expansion
of genetically stable liver cells, we explored whether our culture system
would be suitable
for disease modeling. A1AT deficiency is an inherited disorder that
predisposes to chronic
obstructive pulmonary disease and chronic liver disease (Stoller and
Aboussouan, 2005).
Alpha-1 antitrypsin is a protease produced in the liver, which functions to
protect the lung
against proteolytic damage from neutrophil elastase. The most frequent
mutation causing
a severe phenotype is the Z allele, which involves a substitution of glutamic
acid with
lysine at position 342 (G1u342Lys) in the SERPINA1 gene, which causes
accumulation of
misfolded a1-antitrypsin in the endoplasmic reticulum of hepatocytes. The ZZ
mutant
phenotype is characterized by a ¨80% reduction of the protein in plasma, which
subsequently causes lung emphysema (Stoller and Aboussouan, 2005).
We obtained human liver biopsies from 3 patients diagnosed with A1AT
deficiency who
were undergoing liver transplantation. Biopsies were divided into samples for
histological
characterization, RNA isolation, DNA isolation and for expansion in culture.
We confirmed
that all 3 patients carried the hom*ozygous Z allele (PiZZ), by Sanger
sequencing of the
SERPINA1 locus (Figure 22A). The isolated cells rapidly grew into 3-D
structures

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generating organoids that closely resembled the organoids derived from healthy
biopsies
(Figure 21A) and were grown for >4 months in culture at a 1:5 split
ratio/week, similar as
the cultures derived from healthy/donor biopsies.
We then confirmed the ability of the A1AT-D derived cultures (PiZZ cultures)
to
differentiate into functional hepatocytes in vitro. Gene expression analysis
demonstrated
that the cells differentiated normally. When submitted to hierarchical
clustering analysis,
differentiated organoids derived from A1AT-deficient patients clustered
together with
differentiated organoids derived from healthy donor biopsies (Figure 22B). Of
note,
functional tests revealed that the differentiated cells from A1AT patients
secrete high levels
of Albumin and take up LDL similar to healthy donor-derived organoid cultures
(Figure
21B-D).
We then analyzed the ability of the cultured cells to mimic the pathology of
the disease in
vitro. Functional, healthy hepatocytes secrete A1AT protein into the
bloodstream to inhibit
neutrophil elastase mainly in the lungs (Figure 21E). In A1AT-deficiency, the
molecular
pathogenesis of the liver disease relates to the aggregation of the protein
within the
endoplasmic reticulum of hepatocytes (Lawless et al., 2008). A1AT-Protein
aggregates
were readily observed within the cells of the differentiated organoids derived
from the
A1AT-D patient (Figure 21H), similar to what was found in the original biopsy
(Figure 21G),
while these aggregates were essentially absent from the organoids derived from
healthy
donor-material (Figure 21F). A1AT ELISA confirmed reduced secretion of the
protease
inhibitor from PiZZ organoids (Figure 211), which mimics the reduced A1AT
serum levels in
patients. Likewise, supernatants from differentiated ZZ mutant organoids
showed a
strongly reduced ability to block elastase activity (Figure 21J).
Advanced stages of A1AT deficiency are characterized by liver injury and
cirrhosis due to
combined effects of uncontrolled protease activity and apoptotic loss of
functional
hepatocytes (Fairbanks and Tavill, 2008). Protein misfolding and resulting ER
Stress are
the primary causes that drive hepatocytes from PiZZ individuals to eventual
apoptosis
(Lawless et al., 2008). Differentiated liver organoids from A1AT-D patients
mimicked the in
vivo situation and showed signs of ER stress, such as phosphorylation of elF2a
(Figure
21K) and a slight increase in apoptosis in the differentiated state (Figure
220 and D).
Using a biopsy from a patient suffering from AlegiIle syndrome (AGS), we
tested whether
structural defects of the biliary tree can also be modeled. AGS is a rare
genetic disorder
caused by mutations in the Notch signaling pathway, which results in partial
to complete
biliary atresia (Kamath et al., 2013). Patient organoids could be expanded at
normal rates
and showed no obvious difference to donor in the undifferentiated state.
However, upon

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differentiation to the biliary fate by withdrawal of R-spondin, Nicotinamide,
TGFbi and FSK
from the culture medium, AGS patient organoids failed to upregulate biliary
markers such
as KRT19 and KRT7, while donor (wildtype, wt) organoids readily did (Figure
22E).
Staining for KRT19 revealed that biliary cells were reduced in numbers and
were unable to
integrate into the epithelium. Rather, they rounded up and underwent apoptosis
inside the
organoid (Figure 22F). This finding is in line with AGS mouse models, which
show that
Jagged-1/Notch2 is dispensable for biliary lineage specification, but required
for biliary
morphogenesis (Geisler et al., 2008; McCright et al., 2002). Thus, AGS liver
organoids
mimic the patient phenotype and constitute the first human 3D model system to
study
AlegiIle syndrome.
Methods for A1 AT-D functional experiments:
Enzymatic Elastase inhibition assay: For measurement of the inhibitory action
of a1-
antitrypsin in organoid supernatants, donor and patient organoids were
differentiated for 11
days. Culture medium was changed every 2-3 days and culture supernatant was
collected
24h after the last medium change. For the assay, 160 ul of supernatant are
mixed with 20
ul of a 2 mg/ml N-Succinyl-Ala-Ala-Ala-p5 nitroanilide (Sigma) 100 mM Tris pH
8.0 solution
in a clear-bottom 96-well plate. After addition of 6x10-4 U of Elastase
(porcine pancreas,
Sigma) in 100 mM Tris pH 8.0, the increase in absorbance at 410 nm is measured

continuously over 30 minutes. Elastase inhibition by supernatants is measured
as the
decreased inclination of absorbance over time in comparison to uninhibited
controls (plain
medium) and compared to a dilution series of purified human a1-antitrypsin
(Zemaira) in
medium.
Detection of elF2a phosphorylation: Donor and a1-antitrypsin deficient patient
organoids
were differentiated for 11 days. Culture medium was changed every 2-3 days and
organoids were lysed in Lysis buffer (50 mM Tris pH 7.5, 50 mM NaCI, 0.5%
Triton-X100,
0.5% NP40 substitute, 5 mM EGTA, 5 mM EDTA, lx Complete protease inhibitor
(Roche),
lx PhosStop (Roche)). Using standard techniques lysates were resolved by SDS-
Page
and blotted on PVDF membranes (Millipore).
Example 18: The method works across multiple donors
To generalize our findings across multiple donors, we obtained 12 additional
healthy
human donor liver biopsies and cultured them in our improved human liver
medium. Under
our improved conditions (ERFHNic + Tgfbi + FSK), all 12 human liver-derived
cultures
grew exponentially, with a consisting doubling time of ¨60h independent of the
age of the
culture (2 weeks or 3 months). EdU incorporation confirmed that the cells
maintained their
proliferative state in vitro 3 months after the initiation of culture. Of
note, cultures grown

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under these culture conditions could be readily frozen and thawed. Overall,
these results
support the fact that the combination of Wnt signaling and cAMP activation,
combined with
Tgf-6 inhibition, successfully sustains long-term expansion of human liver
progenitors in
vitro.
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Baker, D.E., Harrison, N.J., Maltby, E., Smith, K., Moore, H.D., Shaw, P.J.,
Heath, P.R., Holden, H.,
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Cairns, J. (1975). Mutation selection and the natural history of cancer.
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