U.S. patent application number 17/325693 was filed with the patent office on 2021-10-21 for method for inducing human cholangiocyte differentiation.
The applicant listed for this patent is INSERM (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE), UNIVERSITE DE RENNES, UNIVERSITE PARIS - SUD. Invention is credited to Anne CORLU, Noushin DIANAT, Anne DUBART KUPPERSCHMITT, Anne WEBER BENAROUS.
Application Number | 20210324338 17/325693 |
Document ID | / |
Family ID | 1000005695847 |
Filed Date | 2021-10-21 |
United States Patent
Application |
20210324338 |
Kind Code |
A1 |
DUBART KUPPERSCHMITT; Anne ;
et al. |
October 21, 2021 |
METHOD FOR INDUCING HUMAN CHOLANGIOCYTE DIFFERENTIATION
Abstract
The invention relates to a method for inducing human
cholangiocyte differentiation of progenitor cells called
hepatoblasts. More specifically, the invention relates to a method
for differentiating hepatoblasts to cholangiocytes by culturing
said hepatoblasts with a particular medium having interleukin-6
(IL-6) activity. The differentiation method can specifically induce
cholangiocyte dilferentiation from hepatoblasts, and the human
cholangiocytes differentiated according to the invention may be
useful for drug discovery for treatment of cholangiopathies and
bioengineered livers.
Inventors: |
DUBART KUPPERSCHMITT; Anne;
(Villejuif, FR) ; WEBER BENAROUS; Anne;
(Villejuif, FR) ; CORLU; Anne; (Rennes Cedex,
FR) ; DIANAT; Noushin; (Villejuif, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSERM (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE
MEDICALE)
UNIVERSITE PARIS - SUD
UNIVERSITE DE RENNES |
Paris
Orsay
Rennes |
|
FR
FR
FR |
|
|
Family ID: |
1000005695847 |
Appl. No.: |
17/325693 |
Filed: |
May 20, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15125596 |
Sep 13, 2016 |
11066649 |
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PCT/EP2015/055811 |
Mar 19, 2015 |
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17325693 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2506/45 20130101;
C12N 2506/02 20130101; A61K 35/407 20130101; C12N 2501/998
20130101; C12N 2501/02 20130101; C12N 2533/54 20130101; C12N
2501/2306 20130101; C12N 2501/119 20130101; C12N 5/0679 20130101;
C12N 2501/727 20130101; C12N 2501/11 20130101; C12N 2501/115
20130101; C12N 2501/12 20130101; C12N 2500/38 20130101; C12N
2500/25 20130101; C12N 2501/305 20130101; C12N 2501/415 20130101;
C12N 2501/16 20130101; C12N 2506/14 20130101; C12N 2501/155
20130101; C12N 2501/395 20130101; C12N 2501/385 20130101; C12N
2501/999 20130101; C12N 5/0672 20130101 |
International
Class: |
C12N 5/071 20060101
C12N005/071; A61K 35/407 20060101 A61K035/407 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 19, 2014 |
EP |
14305387.4 |
Claims
1-11. (canceled)
12. A population of human cholangiocytes produced by a method of
(i) providing a population of human hepatoblasts (hHB); and (ii)
culturing the population of hHB in at least one cholangiocyte
induction medium to specifically induce cholangiocyte
differentiation and produce a population of cholangiocytes, wherein
the step (ii) comprises the steps of: (a) culturing the population
of hHB in a first cholangiocyte induction medium, wherein said
first cholangiocyte induction medium is a chemically defined medium
(CDM) comprising both growth hormone (GH) and epidemal growth
factor (EGF), without human interleukin-6 or a variant thereof, and
without sodium taurocholate hydrate or sodium butyrate; (b) further
culturing the population of hHB produced in step (a) in a second
cholangiocyte induction medium according to step (ii), wherein said
second cholangiocyte induction medium is the CDM of step (ii) to
which is added human interleukin-6 (IL-6) or a variant thereof
having at least 90% amino acid identity to the human IL-6 and at
least 90% of the activity of the human IL-6; and (c) further
culturing the population of hHB produced in step (b) in a third
cholangiocyte induction medium, wherein said third cholangiocyte
induction medium is a CDM of step (a) to which is added sodium
taurocholate hydrate and optionally sodium butyrate, wherein steps
(a), (b) and (c) are performed sequentially (a) to (c), wherein at
least 60% of differentiated cells produced in step (ii) are
cholangiocytes, and wherein said population of cholangiocytes
displays a normal phenotype.
13. (canceled)
14. A method of treating a patient with a cholangiopathy,
comprising providing a population of human cholangiocytes produced
by a method of (i) providing a population of human hepatoblasts
(hHB); and (ii) culturing the population of hHB in at least one
cholangiocyte induction medium to specifically induce cholangiocyte
differentiation and produce a population of cholangiocytes, wherein
the step (ii) comprises the steps of: (a) culturing the population
of hHB in a first cholangiocyte induction medium, wherein said
first cholangiocyte induction medium is a chemically defined medium
(CDM) comprising both growth hormone (GH) and epidemal growth
factor (EGF), without human interleukin-6 or a variant thereof, and
without sodium taurocholate hydrate or sodium butyrate; (b) further
culturing the population of hHB produced in step (a) in a second
cholangiocyte induction medium according to step (ii), wherein said
second cholangiocyte induction medium is the CDM of step (ii) to
which is added human interleukin-6 (IL-6) or a variant thereof
having at least 90% amino acid identity to the human IL-6 and at
least 90% of the activity of the human IL-6; and (c) further
culturing the population of hHB produced in step (b) in a third
cholangiocyte induction medium, wherein said third cholangiocyte
induction medium is a CDM of step (a) to which is added sodium
taurocholate hydrate and optionally sodium butyrate, wherein steps
(a), (b) and (c) are performed sequentially (a) to (c), wherein at
least 60% of differentiated cells produced in step (ii) are
cholangiocytes, and wherein said cholangiocytes display a normal
phenotype, and administering to the patient a pharmaceutically
effective amount of the cholangiocytes.
15. A method of screening for a compound useful in the treatment of
a cholangiopathy comprising the steps of: providing a population of
human cholangiocytes produced by a method of (i) providing a
population of human hepatoblasts (hHB); and (ii) culturing the
population of hHB in at least one cholangiocyte induction medium to
specifically induce cholangiocyte differentiation and produce a
population of cholangiocytes, wherein the step (ii) comprises the
steps of: (a) culturing the population of hHB in a first
cholangiocyte induction medium, wherein said first cholangiocyte
induction medium is a chemically defined medium (CDM) comprising
both growth hormone (GH) and epidemal growth factor (EGF), without
human interleukin-6 or a variant thereof, and without sodium
taurocholate hydrate or sodium butyrate; (b) further culturing the
population of hHB produced in step (a) in a second cholangiocyte
induction medium according to step (ii), wherein said second
cholangiocyte induction medium is the CDM of step (ii) to which is
added human interleukin-6 (IL-6) or a variant thereof having at
least 90% amino acid identity to the human IL-6 and at least 90% of
the activity of the human IL-6; and (c) further culturing the
population of hHB produced in step (b) in a third cholangiocyte
induction medium, wherein said third cholangiocyte induction medium
is a CDM of step (a) to which is added sodium taurocholate hydrate
and optionally sodium butyrate, wherein steps (a), (b) and (c) are
performed sequentially (a) to (c), wherein at least 60% of
differentiated cells produced in step (ii) are cholangiocytes, and
wherein said cholangiocytes display a normal phenotype, contacting
the cholangiocytes produced with a test compound, and determining
the effect of the test compound on said cholangiocytes.
16. The method of claim 15, wherein the chemically defined medium
(CDM) comprises human IL-6.
17. The method of claim 16, wherein the human IL-6 has an amino
acid sequence as shown in Uniprot Accession number P05231 for a
full length human IL-6 protein.
18. The method of claim 15, wherein the variant has 100% of the
activity of human IL-6.
19. The method of claim 15, wherein at least 70% of differentiated
cells are cholangiocytes.
20. The method of claim 15, wherein at least 80% of differentiated
cells are cholangiocytes.
21. The method of claim 14, wherein in the providing step the
chemically defined medium (CDM) comprises human IL-6.
22. The method of claim 21, wherein the human IL-6 has an amino
acid sequence as shown in Uniprot Accession number P05231 for a
full length human IL-6 protein.
23. The method of claim 14, wherein in the providing step the
variant has 100% of the activity of human IL-6.
24. The method of claim 14, wherein in the providing step at least
70% of differentiated cells are cholangiocytes.
25. The method of claim 14, wherein in the providing step at least
80% of differentiated cells are cholangiocytes.
Description
FIELD OF THE INVENTION
[0001] The invention relates to a method for inducing human
cholangiocyte differentiation of progenitor cells called
hepatoblasts. More specifically, the invention relates to a method
for differentiating hepatoblasts to cholangiocytes by culturing
said hepatoblasts with a particular medium having interleukin-6
(IL-6). The differentiation method can specifically induce
cholangiocyte differentiation from hepatoblasts, and the human
cholangiocytes differentiated according to the invention may be
useful for treatment of cholangiopathies.
BACKGROUND OF THE INVENTION
[0002] In the early stages of liver organogenesis, the major
components of hepatic parenchyma are hepatic progenitor cells
(hepatoblasts, HB), which can differentiate into hepatocytes and
cholangiocytes. Cholangiocytes are biliary epithelial cells that
line the intra- and extra-hepatic ducts of the biliary tree. Around
the eighth week of gestation in humans, hepatoblasts in the
vicinity of the portal mesenchyme form a single layer ring of cells
called the "ductal plate", which give rise to cholangiocytes..sup.1
The ductal plate is also assumed to be the compartment of hepatic
stem cells in fetal and neonatal livers, although this is still
controversial..sup.2 4 One of the first pathways that prime biliary
commitment and formation of the ductal plate is the NOTCH pathway:
JAGGED1-positive mesenchymal cells in periportal areas interact
with adjacent NOTCH2-positive hepatoblasts and induce cholangiocyte
differentiation. NOTCH pathway activation induces SOX9 expression,
which is the most specific and earliest marker of biliary cells in
developing liver and controls bile duct morphogenesis..sup.1
Sal-like protein 4 (SALL4) is also reported as a key transcription
factor controlling the lineage commitment of hepatoblasts, not only
by inhibiting their differentiation into hepatocytes but also by
driving their differentiation toward cholangiocytes.
[0003] The main physiological function of cholangiocytes is to
actively regulate bile composition by modification of
hepatocyte-derived bile components through a series of secretory
and reabsorptive events. In addition to their role in the
modification of ductal bile, cholangiocytes participate in the
detoxification of xenobiotics..sup.5 They are also the primary
targets of injury in a variety of cholestatic liver diseases,
ranging from inherited disorders (Alagille syndrome and cystic
fibrosis) or autoimmune cholangitis to primary biliary cirrhosis
which represent the main indication for liver transplantation in
pediatrics..sup.6 Despite the physiological and pathological
importance of cholangiocytes, their limited number (3% of the total
liver mass) and their intrahepatic localization have limited the
development of in vitro cell models in order to gain molecular
insights into their function. At present, few human cholangiocyte
cell lines are available. They are derived from either
cholangiocarcinoma or normal cells immortalized by SV40,.sup.7 and
most of the cells studied in vitro are derived from rat
cholangiocytes..sup.8 However, important dissimilarities exist
between rodent and human models: in particular, only human, but not
rodent, small cholangiocytes express cystic fibrosis transmembrane
conductance regulator (CFTR)..sup.9
[0004] Pluripotent stem cells, which can differentiate into various
cell types and display an infinite ability to proliferate, have
appeared as an alternative and reproducible source of
differentiated cells with therapeutic interest..sup.10 To date,
numerous studies have focused on the development of efficient
protocols to generate hepatocyte-like cells, and several groups
have already generated hepatocyte-like cells from hESCs and hiPSCs
in vitro.
[0005] However the question remains whether hepatic progenitors
generated via different protocols are able to generate
cholangiocytes.
SUMMARY OF THE INVENTION
[0006] In a first aspect, the invention relates to a method for
inducing human cholangiocyte differentiation comprising the steps
of:
[0007] (i) providing a population of human hepatoblasts (hHB);
and
[0008] (ii) culturing the population of hHB in at least one
cholangiocyte induction medium to produce a population of
cholangiocytes, wherein said cholangiocyte induction medium is a
chemically defined medium (CDM) which has interleukin-6
activity.
[0009] In a second aspect, the invention relates to a population of
human cholangiocytes produced by a method of the invention.
[0010] In a third aspect, the invention relates to a population of
human cholangiocytes of the invention, for use in a method of
treatment of the human body.
[0011] In a fourth aspect, the invention relates to a population of
human cholangiocytes of the invention, for use in the treatment of
a patient with a cholangiopathy.
[0012] In a fifth aspect, the invention relates to a method of
screening for a compound useful in the treatment of a
cholangiopathy comprising the steps of:
[0013] (c) contacting a population of cholangiocytes produced by a
method of claim 9 with a test compound, and;
[0014] (d) determining the effect of the test compound on said
cholangiocytes.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present approach recapitulates the key stages of liver
development and enabled to stepwise differentiate hESCs and hiPSCs
into definitive endoderm cells, hepatic progenitors and
cholangiocyte-like cells in a fully defined medium without feeder
cells or serum.
[0016] The inventors thus developed a robust and efficient method
for differentiating pluripotent stem cells, hESC and hiPSC, and
HepaRG-derived hepatic progenitors (hepatoblasts) to differentiate
further along the cholangiocytic lineage and reported a new
approach, which drive differentiation of pluripotent stem cells
toward the cholangiocytic lineage using feeder-free and defined
culture conditions. After differentiation into hepatic progenitors,
pluripotent stem cells were differentiated further into
cholangiocytes using growth hormone, epidermal growth factor,
Interleukin-6 and then sodium taurocholate. These conditions also
allowed us to generate cholangiocytes from HepaRG-derived
hepatoblasts. Pluripotent stem cells- and HepaRG-derived
cholangiocyte-like cells expressed markers of cholangiocytes
including Cytokeratin 7 and Osteopontin, and transcription factors
SOX9 and Hepatocyte nuclear factor 6. The cells also displayed
specific proteins important for cholangiocyte functions including
cystic fibrosis transmembrane conductance regulator, secretin
receptor and nuclear receptors. They formed primary cilia and also
responded to hormonal stimulation by increase of intracellular
Ca2+. The inventors also demonstrated by integrative genomics that
the expression of genes, which signed pluripotent stem cells- or
HepaRG-cholangiocytes separates hepatocytic lineage from
cholangiocyte lineage. When grown in a 3D matrix, cholangiocytes
developed epithelial/apicobasal polarity and formed functional
cysts and biliary ducts.
Methods for Inducing Differentiation of the Invention
[0017] In a first aspect, the invention relates to a method for
inducing human cholangiocyte differentiation comprising the steps
of:
[0018] (i) providing a population of human hepatoblasts (hHB);
and
[0019] (ii) culturing the population of hHB in at least one
cholangiocyte induction medium to produce a population of
cholangiocytes, wherein said cholangiocyte induction medium is a
chemically defined medium (CDM) which has interleukin-6 (IL-6)
activity.
[0020] As used herein, the term "population" refers to a population
of cells, wherein the majority (e.g., at least about 60%,
preferably at least about 70%, more preferably at least about 80%)
of the total number of cells have the specified characteristics of
the cells of interest (e.g. a population of human hepatoblasts
comprises at least about 60%, preferably at least about 70%, more
preferably at least about 80% of cells which have the hepatic
functions and which express the markers typically expressed by
human hepatoblasts listed below).
[0021] As used herein, the terms "hepatoblasts" (HB) or "hepatic
progenitor cells" are used herein interchangeably. They refer to
cells that are capable of expressing characteristic biochemical
markers, including but not limited to Alpha-fetoprotein (AFP),
Albumine (Alb), Cytokeratin 19 (CK19), Ep-CAM and Hepatocyte
nuclear factor 4alpha (HNF4alpha). Such cells can differentiate
into either hepatocytes or cholangiocytes and express markers of
both lineages (i.e. as above-mentioned CK19 which is a specific
marker of cholangiocytes; HNF4 alpha and AFP which are specific
markers of hepatocytes). As described below, HB may be derived from
pluripotent stem cells, from HepaRG, from fetal liver or directly
obtained from other cell types by redirected differentiation.
[0022] As used herein, the term "marker" refers to a protein,
glycoprotein, or other molecule expressed on the surface of a cell
or into a cell, and which can be used to help identify the cell. A
marker can generally be detected by conventional methods. Specific,
non-limiting examples of methods that can be used for the detection
of a cell surface marker are immunocytochemistry, fluorescence
activated cell sorting (FACS), and enzymatic analysis but also
RT-PCR and molecular biology methods to detect mRNA of the
protein.
[0023] As used herein, the term "cholangiocytes" refers to the
epithelial cells of the bile ducts. Cholangiocytes are biliary
epithelial cells, which, like hepatocytes, originate from
hepatoblasts during embryonic development. The morphology of
cholangiocytes as well as their functions varies along the biliary
trees. Cholangiocytes in the small interlobular bile ducts are
cuboidal cells but become columnar and mucus secreting in larger
bile ducts approaching the extrahepatic portion. In the healthy
liver, cholangiocytes contribute to bile secretion via net release
of bicarbonate and water. Several hormones and locally acting
mediators are known to contribute to cholangiocyte
fluid/electrolyte secretion. These include secretin, acetylcholine,
ATP, and bombesin. As the primary bile flows through the bile
ducts, its composition is regulated by the intrahepatic bile duct
epithelium that reabsorbs fluids, aminoacids, glucose and bile
acids, while secreting water and electrolytes and IgA.
Cholangiocytes refer to cells that are capable of expressing
characteristic biochemical markers, including but not limited to
CFTR, G-protein coupled bile acid receptor 1 (TGR5), Aquaporin-1
(AQP1), SOX9, Secretin receptor (SCTR), JAG1, CK7, CK19, CK18,
Osteopontin (OPN), HNF6 and HNF1.beta..
[0024] As used herein, the term "chemically defined medium" (CDM)
refers to a nutritive solution for culturing cells which contains
only specified components, preferably components of known chemical
structure. A chemically defined medium is a scrum-free and
feeder-free medium. As used herein, "serum-free" refers to a
culture medium containing no added serum. As used herein,
"feeder-free" refers to culture medium containing no added feeder
cells. The term feeder-free encompasses, inter alia, situations
where hepatoblasts are passaged from a culture without feeders into
a culture medium without added feeders even if some of the feeders
from the first culture are present in the second culture.
[0025] In one embodiment, the cholangiocyte induction medium
comprises IL-6.
[0026] Accordingly, the invention relates to a method for inducing
human cholangiocyte differentiation comprising the steps of: [0027]
(i) providing a population of human hepatoblasts (hHB) and [0028]
(ii) culturing the population of hHB in at least one cholangiocyte
induction medium to produce a population of cholangiocytes, wherein
said cholangiocyte induction medium is a chemically defined medium
(CDM) comprising IL-6.
[0029] As used herein, the term "Interleukin-6" (IL-6) refers to a
polypeptide involved in the regulation of immune responses such as
lymphocyte differentiation. IL-6 is well known in the art and is
also known as B cell-stimulating factor 2 (BSF-2) or interferon
beta-2 (IFNB2). The term also refers to any of the
naturally-occurring forms of the IL-6, or variants thereof that
maintain IL-6 activity (e.g. within at least 50%, 80%, 90% or 100%
activity compared to wild type IL-6 as measured by methods known in
the art). In some embodiments, variants have at least 90% amino
acid sequence identity across their whole sequence compared to the
naturally occurring IL-6 polypeptide. The naturally occurring human
IL-6 protein has an aminoacid sequence as shown in Uniprot
Accession number P05231.
[0030] In one embodiment, IL-6 is added to the cholangiocyte
induction medium of the invention in a concentration ranging from 1
to 50 ng/ml, preferably at about 10 ng/ml. IL-6 can be purchased
from Miltenyi.
[0031] The population of hepatoblasts (HB) may be cultured for 2 to
15 days, to produce the population of cholangiocytes.
[0032] When the population of human hepatoblasts (HB) is a
population of human pluripotent stem cells-derived HB, said
population of human pluripotent stem cells-derived HB may be
cultured for 2 to 10 days, preferably 6 days, to produce the
population of cholangiocytes. When the population of human
hepatoblasts (HB) is a population of HepaRG-derived HB, said
population of HepaRG-derived HB may be cultured for 2 days.
[0033] The expression of one or more cholangiocyte markers and/or
one or more hepatoblast markers may be monitored and/or detected in
the population of differentiating cells.
[0034] In one embodiment, when the population of human hepatoblasts
(HB) is a population of human pluripotent stem cells-derived HB,
the step (ii) comprises the steps of:
[0035] (a) culturing the population of hHB in a first cholangiocyte
induction medium, wherein said first cholangiocyte induction medium
is a CDM comprising growth hormone (GH); and
[0036] (b) further culturing the population of hHB in the
cholangiocyte induction medium which has IL-6 activity.
[0037] In a preferred embodiment, when the population of human
hepatoblasts (HB) is a population of human pluripotent stem
cells-derived HB, the step (ii) comprises the steps of:
[0038] (a) culturing the population of hHB in a first cholangiocyte
induction medium, wherein said first cholangiocyte induction medium
is a CDM comprising both growth hormone (GH) and epidemal growth
factor (EGF);
[0039] (b) further culturing the population of hHB in the
cholangiocyte induction medium which has IL-6 activity; and
[0040] (c) further culturing the population of hHB in a third
cholangiocyte induction medium, wherein said third cholangiocyte
induction medium in a CDM comprising sodium taurocholate hydrate
and optionally sodium butyrate.
[0041] As used herein, the term "Growth hormone" (GH) refers to a
peptide hormone that stimulates growth and metabolism. GH has not
only a mitogenic but also a developmental effect in liver. It
stimulates the synthesis of liver-specific transcription factors.
The naturally occurring human GH protein has an aminoacid sequence
as shown in Uniprot Accession number P01241.
[0042] In one embodiment, GH is added to the culture medium of the
invention in a concentration ranging from 1 to 100 ng/ml,
preferably at about 50 ng/ml. GH can be purchased from Sigma.
[0043] As used herein, the term "Epidemal growth factor" (EGF)
refers to a growth factor that stimulates cell growth,
proliferation, and differentiation by binding to its receptor EGFR.
The naturally occurring human EGF protein has an aminoacid sequence
as shown in Uniprot Accession number P01133.
[0044] In one embodiment, EGF is added to the culture medium of the
invention in a concentration ranging from 1 to 100 ng/ml,
preferably at about 25 ng/ml. EGF can be purchased from
Peprotech.
[0045] As used herein, the term "Sodium taurocholate hydrate"
refers to the compound having the following formula:
##STR00001##
[0046] In one embodiment, sodium taurocholate hydrate is added to
the culture medium of the invention in a concentration ranging from
1 to 100 .mu.M, preferably at about 10 .mu.M. Sodium taurocholate
hydrate can be purchased from Sigma.
[0047] In a particular embodiment, the population of hHB is
cultured for at least 3 days in each of steps a) to c).
[0048] In a preferred embodiment, the step (ii) (b) comprises the
steps of:
[0049] (x) culturing for 3 days the population of hHB in the
cholangiocyte induction medium which has IL-6 activity;
[0050] (y) further passaging at least one time the population;
and
[0051] (z) further culturing for another 3 days the population of
hHB in the cholangiocyte induction medium which has IL-6
activity.
[0052] In a still preferred embodiment, the population of hHB has
been passaged at least one time before culturing the population in
a cholangiocyte induction medium.
[0053] To maintain proliferating cells in culture for an extended
period of time, it is necessary to subculture the cells (remove
them from one culture vessel, and place them in a new culture
vessel containing fresh medium and a lower cell concentration).
Each time a population of cells is subcultured, it is referred to
as a "passage". In order to move the cells from the first culture
vessel to the second culture vessel, the monolayer of cells have to
be detached from the surface of the first culture vessel.
[0054] Analysis of passaged population can be performed by cell
sorting methods. These methods can be used to determine the
composition of heterogeneous cell populations, and to isolate
specific subpopulations of cells with desirable characteristics
which can then be used to conduct further research, or used
therapeutically in a clinical setting. Cell sorting methods can
only be used effectively on single cells.
[0055] When the culture has grown to confluence, human hepatoblasts
(hHB) are passaged to another culture vessel. The determination to
passage the cells and the techniques for accomplishing such
passaging can be performed in accordance with the culture methods
of invention (e.g., through morphology assessment and dissection
procedures).
[0056] Moreover, adhesion between cells or between cells and
substrate is needed for growth and division of normal cells, and
proteins (extracellular matrix) present between cells or between
the cells and the substrate are also needed. In the method of
inducing differentiation to choloangiocytes according to the
invention, the extracellular matrices described below may be added
in the steps above. Examples of the extracellular matrices include
matrigel, fibronectin, vitronectin, laminin, nidogen, tenascin,
thrombospondin, type-I collagen, type-IV collagen, gelatin, and
synthetic substrates equivalent thereto, and in the invention,
type-1 collagen can be used particularly favorably. Specifically,
they can be used, as they are coated on or added to a container for
culture.
[0057] In one embodiment, the population of hHB is passaged on
type-I collagen.
[0058] In another embodiment, when the population of human
hepatoblasts (HB) is a population of HepaRG-derived HB, the step
(ii) comprises the steps of:
[0059] (a) culturing the population of hHB in the cholangiocyte
induction medium which has IL-6 activity; and
[0060] (b) further culturing the population of hHB in a second
cholangiocyte induction medium, wherein said second cholangiocyte
induction medium in a CDM comprising sodium taurocholate hydrate
and optionally sodium butyrate.
[0061] In a preferred embodiment, when the population of human
hepatoblasts (HB) is a population of HepaRG-derived HB, the step
(ii) comprises the steps of:
[0062] (a) culturing for 2 days the population of hHB in the
cholangiocyte induction medium which has IL-6 activity;
[0063] (b) further culturing for 2 days the population of hHB in a
second cholangiocyte induction medium, wherein said third
cholangiocyte induction medium in a CDM comprising sodium
taurocholate hydrate and optionally sodium butyrate; and
[0064] (c) further culturing for 2 days the population of hHB in a
third cholangiocyte induction medium, wherein said third
cholangiocyte induction medium in a CDM comprising sodium
taurocholate hydrate and sodium butyrate.
[0065] As previously described, the population of hHB is a
population of human pluripotent stem cells-derived HB or
HepaRG-derived HB.
[0066] HepaRG is a well-characterized human hepatoma cell line
which is permanently able to differentiate into mature hepatocytes
from a stage of bipotent progenitors.sup.13 and which is widely
used for pharmaco-toxicology assays..sup.14 Such human hepatoma
cell line has been described in the international patent
application No WO 03/004627 which is hereby incorporated by
reference in its entirety.
[0067] As used herein, the term "pluripotent" refers to cells with
the ability to give rise to progeny that can undergo
differentiation, under appropriate conditions, into cell types that
collectively exhibit characteristics associated with cell lineages
from the three germ layers (endoderm, mesoderm, and ectoderm).
Pluripotent stem cells can contribute to tissues of a prenatal,
postnatal or adult organism. A standard art-accepted test, such as
the ability to form a teratoma in 8-12 week old SCID mice, can be
used to establish the pluripotency of a cell population. However,
identification of various pluripotent stem cell characteristics can
also be used to identify pluripotent cells.
[0068] More specifically, human pluripotent stem cells may express
at least some, and optionally all, of the markers from the
following non-limiting list: SSEA-3, SSEA-4, TRA-I-60, TRA-I-81,
TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-I, OCT4, LIN28, REX1, and
NANOG.
[0069] In a particular embodiment, the human pluripotent stem cells
are human embryonic stem cells (hESCs) or human induced pluripotent
stem cells (hiPSCs).
[0070] As used herein, the term "embryonic stem cells" refers to
embryonic cells, which are capable of differentiating into cells of
all three embryonic germ layers (i.e., endoderm, ectoderm and
mesoderm), or proliferating in an undifferentiated state. Such
cells may comprise cells which are obtained from the embryonic
tissue formed after gestation (e.g., blastocyst) before
implantation of the embryo (i.e., a pre-implantation blastocyst),
extended blastocyst cells (EBCs) which are obtained from a
post-implantation/pre-gastrulation stage blastocyst (see
WO2006/040763) and embryonic germ (EG) cells which are obtained
from the genital tissue of a fetus any time during gestation,
preferably before 10 weeks of gestation.
[0071] The embryonic stem cells may be obtained using well-known
cell-culture methods. For example, human embryonic stem cells can
be isolated from human blastocysts. Human blastocysts are typically
obtained from human in vivo preimplantation embryos or from in
vitro fertilization-derived (IVF) embryos. Alternatively, a single
cell human embryo can be expanded to the blastocyst stage. For the
isolation of human ES cells the zona pellucida is removed from the
blastocyst and the inner cell mass (ICM) is isolated by
immunosurgery, in which the trophectoderm cells are lysed and
removed from the intact ICM by gentle pipetting. The ICM is then
plated in a tissue culture flask containing the appropriate medium
which enables its outgrowth. Following 9 to 15 days, the ICM
derived outgrowth is dissociated into clumps either by a mechanical
dissociation or by an enzymatic degradation and the cells are then
re-plated on a fresh tissue culture medium. Colonies demonstrating
undifferentiated morphology are individually selected by
micropipette, mechanically dissociated into clumps, and re-plated.
Resulting ES cells are then routinely split every 4-7 days. For
further details on methods of preparation human ES cells see
Thomson et al., [U.S. Pat. No. 5,843,780].
[0072] Commercially available stem cells may also be used. Human ES
cells can be purchased from the NIH human embryonic stem cells
registry (http://escr.nih.gov). Non-limiting examples of
commercially available embryonic stem cell lines are BG01, BG02,
BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE32, H9 and WA09.
[0073] As used herein, the term "induced pluripotent stem cell"
refers to a pluripotent stem cell artificially derived from a
non-pluripotent cell. A non-pluripotent cell can be a cell of
lesser potency to self-renew and differentiate than a pluripotent
stem cell. Cells of lesser potency can be, but are not limited to,
somatic stem cells, tissue specific progenitor cells, primary or
secondary cells. iPSCs have been reproducibly obtained by
reprogramming different cell types by forced expression of the
OCT4, SOX2, c-MYC and KLF4 transcription factor cocktail or by an
alternative combination of factors, substituting KLF4 and c-MYC by
NANOG and LIN28. As used herein, the term "reprogramming" refers to
the process of changing the fate of a target cell into that of a
different cell type, caused by the expression of a small set of
factors (or reprogramming factors) in the target cells.
[0074] Methods for generating induced pluripotent stem cells based
on expression vectors encoding reprogramming factors have been
described in the art, see for example WO2007/69666, EP2096169-A1 or
WO2010/042490.
[0075] Expression vectors for ectopic expression of the
reprogramming factors may be, for example, plasmid vector, cosmid
vector, bacterial artificial chromosome (BAC) vector,
transposon-based vector (such as PiggyBac) or viral vector.
[0076] Alternatively, the reprogramming factors, for example, Oct4,
Sox2, Klf4 and c-Myc, or corresponding coding DNA or RNA, are
introduced into the target cells without integration of exogenous
genetic material in the host DNA, i.e. without introduction of the
nucleotide sequence in the cell's genome. An expression vector such
as a plasmid vector can be delivered into said cells for ectopic
expression of the reprogramming factor, in the form of naked DNA.
Alternatively, RNAs coding for said reprogramming factors either
chemically modified or not, can be introduced into the cells to
reprogram them (see for example Warren L, et al, 2010, Cell Stem
Cell. November 5:7(5):618-30). Other expression vectors have been
described for example in WO 2009115295.
[0077] In one embodiment, the hiPSCs are derived from cells
obtained from a healthy subject. In another embodiment, the hiPSCs
are derived from cells obtained from an subject with a
cholangiopathy and the cholangiocytes in the population display a
disease phenotype.
[0078] As used herein, the term "cholangiopathy" refers to diseases
including primary biliary cirrhosis, primary sclerosing
cholangitis, AIDS cholangiopathy, disappearing bile duct syndromes,
Alagille's syndrome, cystic fibrosis, and biliary atresia.
[0079] In one embodiment, the population of human pluripotent stem
cells-derived HB is obtained by a method for inducing hepatic
differentiation comprising the steps of:
[0080] (i) providing a population of human pluripotent stem
cells;
[0081] (ii) culturing the population in a endoderm induction medium
to produce a population of anterior definitive endoderm (ADE)
cells, wherein the endoderm induction medium is a CDM comprising a
WNT agonist, a PI3K inhibitor and a TGF.beta. ligand;
[0082] (iii) culturing the population of ADE cells in a hepatic
induction medium to produce a population of specified hepatic
endodermal cells, wherein the hepatic induction medium is a CDM
comprising a fibroblast growth factor (FGF) and a bone morphogenic
protein (BMP); and
[0083] (iv) culturing the population of specified hepatic
endodermal cells in a hepatic maturation medium to produce a
population of hHB, wherein the hepatic maturation medium is a CDM
comprising a FGF, hepatic growth factor (HGF), EGF and retinoic
acid (RA).
[0084] In a particular embodiment, the population of human
pluripotent stem cells-derived HB is obtained by a method for
inducing hepatic differentiation comprising the steps of:
[0085] (i) providing a population of human pluripotent stem
cells;
[0086] (ii) culturing the population in a endoderm induction medium
to produce a population of anterior definitive endoderm (ADE)
cells, wherein the endoderm induction medium is a CDM comprising
WNT3a, LY294002 and Activin A;
[0087] (iii) culturing the population of ADE cells in a hepatic
induction medium to produce a population of specified hepatic
endodermal cells, wherein the hepatic induction medium is a CDM
comprising FGF2 and BMP4; and
[0088] (iv) culturing the population of specified hepatic
endodermal cells in a hepatic maturation medium to produce a
population of hHB, wherein the hepatic maturation medium is a CDM
comprising FGF4, HGF, EGF and RA.
[0089] As used herein, the term "Anterior Definitive Endoderm"
(ADE) refers to cells which typically express endoderm markers,
such as SOX17, FOXA2, GSC, MIXL11, LHX1, CXCR4, GATA6, EOMES and
HEX. Anterior definitive endoderm (ADE) cells may lack expression
of pluripotency markers or markers associated with ectodermal or
mesodermal lineages. For example the ADE cells may not express at
detectable levels one or more, preferably all, of the following;
OCT4, SOX2, alkaline phosphatase, SSEA-3, NANOG, SSEA-4, TRA-1-60
and KLF-4.
[0090] At step ii), the endoderm induction medium is a CDM
comprising a WNT agonist, a PI3K inhibitor and a TGF.beta.
ligand;
[0091] As used herein, the term "WNT agonist" refers to a compound
(natural or synthetic) which is effective to activate the WNT
signaling.
[0092] In a particular embodiment, the WNT agonist is WNT3a as
described in Marson et al. 2009 and the international patent
application No WO 2009/032194. The naturally occurring human WNT3a
protein has an aminoacid sequence as shown in Uniprot Accession
number P56704.
[0093] For example, the endoderm induction medium may contain 10 to
100 ng/ml WNT agonist, such as WNT3a, preferably about 50
ng/ml.
[0094] Other WNT agonists are well known form the skilled man in
the art. Non-limiting examples of WNT agonists include chemical
compounds belonging to the 5-thiophenepyrimidine class as described
in Wang et al. 2009 and in the international patent application WO
2010/056907 (for instance the
2-chloro-N-(2-morpholinoethyl)-4-(4-(thiophen-2-yl)pyrimidin-2-ylamino)
benzamide, chemical compounds belonging to the aminopyridine class
or to the indirubin structural class (for instance the
6-bromoindirubin-3'-oxime ("BIO") as described in Wang et al.
2009.
[0095] As used herein, the term "PI3K inhibitor" refers to a
compound (natural or synthetic) which is effective to inhibit PI3K
activity. In addition, the inhibitors with a specific activity on
PI3K may be preferred. Inhibitors of PI3K are, in most cases,
compounds that interfere with the binding of ATP in the binding
site of PI3K ATP, thus preventing a more or less specific activity
of these kinases. In some cases, inhibitors of PI3K are allosteric
inhibitors.
[0096] Non-limiting examples of PI3K inhibitors include: LY294002
(Cell Signaling #9901); NVP-BEZ235 (BEZ235) (Novartis); GDC-0941
(Genentech/Roche); GDC-0980 (Genentech); PI-103 (Piramed); XL147
(Exilixis/Sanofi-Aventis); XL418 (Exilixis); XL665 (Exelixis);
LY29002 (Eli Lilly); ZSTK474 (Zenyaku Kogyo); BGT226 (Novartis);
wortmannin; quercetin; tetrodotoxin citrate (Wex Pharmaceuticals);
thioperamide maleate; IC87114; PIK93; TGX-115; deguelin; NU 7026;
OSU03012; tandutinib (Millennium Pharmaceuticals); MK-2206 (Merck);
OSU-03012; triciribine (M.D. Anderson Cancer Center); PIK75;
TGX-221; NU 7441; PI 828; WHI-P 154; AS-604850; AS-041164 (Merck
Serono); AS-252424; AS-605240; AS-604850; compound 15e;
17-P-hydroxywortmannin; PP121; WAY-266176; WAY-266175; BKM120
(Novartis); PKI-587 (Pfizer); BYL719 (Novartis); XL765
(Sanofi-Aventis); GSK1059615 or GSK615 (GlaxoSmithKline); IC486068;
SF1126 (Semafore Pharmaceuticals); CAL-101 (Gilead Sciences);
LME00084; PX-478 (Oncothyrcon); PX-866 (Oncothyrcon); PX-867
(Oncothyrcon), BAY 80-6946 (Bayer), GSK2126458 (GlaxoSmithKline),
INK 117 (Intellikine), IPI-145 (infinity Pharmaceuticals) Palomid
529 (Paloma Pharmaceuticals); ZSTK474 (Zenyaku Kogyo); PWT33597
(Pathway Therapeutics); TG100-115 (TargeGen); CAL263 (Gilead
Sciences); SAR245408 (Sanofi-Aventis); SAR245409 (Sanofi-Aventis);
GNE-477; CUDC-907; and BMK120 (Novartis).
[0097] In one embodiment, the PI3K inhibitor is LY294002 (a
morpholine derivative of quercetin) or
2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one. LY294002 may be
obtained commercially or synthesized as described in U.S. Pat. No.
5,703,075, the content of which is incorporated herein by
reference.
[0098] In another embodiment, the PI3K inhibitor is a prodrug of
LY294002 comprising a reversibly quaternized nitrogen as described
in international patent application WO2004/089925. On example of
such prodrug is SF1226 (Semafore Pharmaceuticals) which is composed
of the PI3K inhibitor LY294002 conjugated to an RGD targeting
peptide.
[0099] For example, the endoderm induction medium may contain 1 to
100 .mu.M PI3K inhibitor, such as LY294002, preferably about 10
.mu.M.
[0100] As used herein, the term "TGF.beta. ligand" refers to any
peptide of the TGF.beta. superfamily. Members of the TGF.beta.
superfamily possess a characteristic structure and are well-known
in the art. The TGF.beta. ligand may be Activin or TGF.beta..
[0101] In one embodiment, the TGF.beta. ligand is Activin (also
referred herein as Activin A). Activin is well known in the art and
is a dimeric polypeptide which exerts a range of cellular effects
via stimulation of the Activin/Nodal pathway (Vallier et al., Cell
Science 118:4495-4509 (2005)). The naturally occurring human WNT3a
protein has an aminoacid sequence as shown in GeneBank Accession
number NP_002183. Activin is readily available from commercial
sources (e.g. Stemgent Inc. MA USA).
[0102] Conveniently, the concentration of Activin in the medium may
be from 10 to 1000 ng/ml, preferably about 100 ng/ml.
[0103] In a particular embodiment, the population of human
pluripotent stem cells is cultured for 3 days in a endoderm
induction medium to produce a population of anterior definitive
endoderm (ADE) cells.
[0104] At step iii), the hepatic induction medium is a CDM
comprising a fibroblast growth factor (FGF) and a Bone morphogenic
protein (BMP)
[0105] As used herein, the term "Family Growth Factor" (FGF) refers
to any naturally occurring compound (e.g. a protein) capable of
stimulating cellular growth, proliferation and cellular
differentiation by binding to one fibroblast growth factor receptor
(FGFR). By binding to one FGFR, the compound increases for example
the tyrosine phosphorylation of said receptor.
[0106] In one embodiment, the FGF is FGF2 (also known as Basic
fibroblast growth factor). The naturally occurring human FGF2
protein has an aminoacid sequence as shown in Uniprot Accession
number P09038.
[0107] Typically, FGF2 is added to the culture medium of the
invention in a concentration ranging from 1 to 50 ng/ml, preferably
at about 20 ng/ml. FGF2 can be purchased from Peprotech.
[0108] As used herein, the term "Bone morphogenetic protein" (BMP)
refers to any growth factors binding to one bone morphogenetic
protein receptors (BMPR). By binding to one BMPR, the growth factor
results in mobilization of members of the SMAD family of
proteins.
[0109] In one embodiment, the BMP is BMP4 (also known as Bone
morphogenetic protein 4). The naturally occurring human BMP4
protein has an aminoacid sequence as shown in Uniprot Accession
number P12644.
[0110] Typically, BMP4 is added to the culture medium of the
invention in a concentration ranging from 1 to 50 ng/ml, preferably
at about 10 ng/ml. BMP4 can be purchased from R&D systems.
[0111] In a particular embodiment, the population of ADE cells is
cultured for 3 days in a hepatic induction medium to produce a
population of specified hepatic endodermal cells.
[0112] At step iv), the hepatic maturation medium is a CDM
comprising a FGF, hepatic growth factor (HGF), EGF and retinoic
acid (RA).
[0113] In one embodiment, the FGF is FGF4 (also known as heparin
secretory transforming protein 1 or Kaposi sarcoma oncogene). The
naturally occurring human FGF4 protein has an aminoacid sequence as
shown in Uniprot Accession number P08620.
[0114] Typically, FGF4 is added to the culture medium of the
invention in a concentration ranging from 1 to 50 ng/ml, preferably
at about 30 ng/ml. FGF4 can be purchased from Peprotech.
[0115] As used herein, the term "Hepatic growth factor" (EGF)
refers to a growth factor which regulates cell growth, cell
motility, and morphogenesis by activating a tyrosine kinase
signaling cascade after binding to the proto-oncogenic c-MET
receptor. The naturally occurring human HGF protein has an
aminoacid sequence as shown in Uniprot Accession number P14210.
[0116] In one embodiment, HGF is added to the culture medium of the
invention in a concentration ranging from 1 to 100 ng/ml,
preferably at about 25 ng/ml. HGF can be purchased from
Peprotech.
[0117] In one embodiment, EGF is added to the culture medium of the
invention in a concentration ranging from 1 to 100 ng/ml,
preferably at about 50 ng/ml. EGF can be purchased from
Peprotech.
[0118] In one embodiment, RA is added to the culture medium of the
invention in a concentration ranging from may be from 10.sup.-8 M
to 10.sup.-6 M, preferably at about 10.sup.-7 M. RA can be
purchased from Sigma.
[0119] In a particular embodiment, the population of specified
hepatic endodermal cells is cultured between 2 and 4 days in a
hepatic maturation medium to produce a population of hHB.
[0120] In another aspect, the invention relates to a method for
obtaining a population of human pluripotent stem cells-derived HB
comprising the steps of:
[0121] (i) providing a population of human pluripotent stem
cells;
[0122] (ii) culturing the population in a endoderm induction medium
to produce a population of anterior definitive endoderm (ADE)
cells, wherein the endoderm induction medium is a CDM comprising a
WNT agonist, a PI3K inhibitor and a TGF.beta. ligand;
[0123] (iii) culturing the population of ADE cells in a hepatic
induction medium to produce a population of specified hepatic
endodermal cells, wherein the hepatic induction medium is a CDM
comprising a fibroblast growth factor (FGF) and a bone morphogenic
protein (BMP); and
[0124] (iv) culturing the population of specified hepatic
endodermal cells in a hepatic maturation medium to produce a
population of hHB, wherein the hepatic maturation medium is a CDM
comprising a FGF, HGF, EGF and retinoic acid (RA).
[0125] In one embodiment, the population of human pluripotent stem
cells-derived HB is obtained by a method for inducing hepatic
differentiation comprising the steps of:
[0126] (i) providing a population of human pluripotent stem
cells;
[0127] (ii) culturing the population in a endoderm induction medium
to produce a population of anterior definitive endoderm (ADE)
cells, wherein the endoderm induction medium is a CDM comprising
WNT3a, LY294002 and Activin A;
[0128] (iii) culturing the population of ADE cells in a hepatic
induction medium to produce a population of specified hepatic
endodermal cells, wherein the hepatic induction medium is a CDM
comprising FGF2 and BMP4; and
[0129] (iv) culturing the population of specified hepatic
endodermal cells in a hepatic maturation medium to produce a
population of hHB, wherein the hepatic maturation medium is a CDM
comprising FGF4, HGF, EGF and RA.
Populations of Cholangiocytes According to the Invention and
Pharmaceutical Compositions Comprising them
[0130] In another aspect, the invention also relates to a
population of cholangiocytes obtainable by a method as defined
above.
[0131] In one embodiment, the cholangiocytes display a normal
phenotype
[0132] In another embodiment, the cholangiocytes display a genetic
mutation, in particular genetic mutation that affects key protein
within cholangiocytes and which is associated with a
cholangiopathy. It should be noted that, the genetic mutation or
defect which is responsible for the disease phenotype may be
corrected in vitro. Various techniques are available to correct
genetic mutations or defects in isolated mammalian cells.
[0133] Genetic Modification of Cholangiocytes of the Invention
[0134] The term "genetically modified" indicates that the
cholangiocytes comprise a nucleic acid molecule not naturally
present in non-modified cholangiocytes, or a nucleic acid molecule
present in a non-natural state in said cholangiocytes (e.g.,
amplified). The nucleic acid molecule may have been introduced into
said cells or into an ancestor thereof (such as iPS which contains
a cholangiopathy associated genetic mutation).
[0135] A number of approaches can be used to genetically modify
cholangiocytes, such as virus-mediated gene delivery,
non-virus-mediated gene delivery, naked DNA, physical treatments,
etc. To this end, the nucleic acid is usually incorporated into a
vector, such as a recombinant virus, a plasmid, phage, episome,
artificial chromosome, etc.
[0136] In a particular embodiment of the invention, the
cholangiocytes cells are genetically modified using a viral vector
(or a recombinant virus). In this embodiment, the heterologous
nucleic acid is, for example, introduced into a recombinant virus
which is then used to infect cholangiocytes. Different types of
recombinant viruses can be used, in particular lentivirus.
[0137] In a preferred embodiment, said lentivirus encodes an
immortalizing protein, such as SV40T, hTERT, CDK4, etc.
[0138] In another preferred embodiment, said lentivirus encodes a
wild-type protein (such a protein usually displaying a disease
associated genetic mutation in patients suffering from a
cholangiopathy) such as wild-type CFTR, PC1 and PC2 proteins. Other
genetic mutations involved in cholangiopathies have been previously
described.sup.46.
[0139] In one embodiment, the nucleic acid sequence encoding the
protein of interest (for instance an immortalizing protein or a
wild-type protein used for correcting a disease phenotype) is
operatively linked to a promoter.
[0140] As used herein, the term "operably linked" means that the
components described are in a relationship permitting them to
function in their intended manner. Thus, a nucleic acid sequence is
"operably linked" when placed into a functional relationship with
another sequence nucleic sequence. For instance, a promoter is
"operably linked" to a coding sequence if the promoter causes the
transcription of the coding sequence. Generally, operably linked
means the linked nucleic acid sequences are contiguous.
[0141] As used herein, the term "promoter" refers to a DNA sequence
that determines the site of transcription initiation for an RNA
polymerase. A promoter may comprise a RNA polymerase III promoter
that can provide high levels of constitutive expression across a
variety of cell types and will be sufficient to direct the
transcription of a distally located sequence, which is a sequence
linked to the 3' end of the promoter sequence in a cell. Suitable
promoters include, for example, constitutive, regulated,
tissue-specific or ubiquitous promoters, which may be of cellular,
viral or synthetic origin.
[0142] In one embodiment, the promoter is a constitutive promoter
such as human elongation factor-1 alpha (EF-1 alpha) etc.
[0143] In another embodiment, the promoter is a
cholangiocyte-specific promoter such as CK7, TGR5, SCTR, etc.
[0144] Importantly, cholangiocytes are the target of disease in a
variety of conditions often known as "cholangiopathies" (also
referred as "cholestatic liver diseases"). These diseases include
primary biliary cirrhosis, primary sclerosing cholangitis, AIDS
cholangiopathy, disappearing bile duct syndromes, Alagille's
syndrome, cystic fibrosis, ciliopathies and biliary atresia. As a
group, cholangiopathies account for approximately 18% of adult
liver transplantations and the majority of pediatric liver
transplantations.
[0145] The invention also provides a pharmaceutical composition
comprising the population of cholangiocytes according to the
invention. The pharmaceutical composition may generally include one
or more pharmaceutically acceptable and/or approved carriers,
additives, antibiotics, preservatives, adjuvants, diluents and/or
stabilizers. Such auxiliary substances can be water, saline,
glycerol, ethanol, wetting or emulsifying agents, pH buffering
substances, or the like. Suitable carriers are typically large,
slowly metabolized molecules such as proteins, polysaccharides,
polylactic acids, polyglycolic acids, polymeric amino acids, amino
acid copolymers, lipid aggregates, or the like. This pharmaceutical
composition can contain additional additives such as mannitol,
dextran, sugar, glycine, lactose or polyvinylpyrrolidone or other
additives such as antioxidants or inert gas, stabilizers or
recombinant proteins (e. g. human serum albumin) suitable for in
vivo administration.
[0146] As used herein, the term "pharmaceutically acceptable"
refers to molecular entities and compositions that do not produce
an adverse, allergic or other untoward reaction when administered
to a mammal, especially a human, as appropriate. A pharmaceutically
acceptable carrier or excipient refers to a non-toxic solid,
semi-solid or liquid filler, diluent, encapsulating material or
formulation auxiliary of any type.
Screening Methods of the Invention
[0147] In another aspect, the cholangiocytes of the invention
obtained from healthy or diseased patients may also be used
advantageously for screening applications in the pharmaceutical
industry. Such screening tests can be used to search for new drugs
with clinical applications or for toxicology tests. The
cholangiocytes according to the invention may for instance be
useful for generating cellular models of cholangiopathies as
described above.
[0148] Accordingly, the invention provides a method of screening
for a compound useful in the treatment of cholangiopathy comprising
the steps of: [0149] (a) contacting a population of cholangiocytes
produced by a method of the invention with a test compound, and;
[0150] (b) determining the effect of the test compound on said
cholangiocytes.
[0151] Test Compounds of the Invention:
[0152] In one embodiment, the test compound may be selected from
the group consisting of peptides, proteins, peptidomimetics, small
organic molecules, aptamers or nucleic acids. For example, the test
compound according to the invention may be selected from a library
of compounds previously synthesised, or a library of compounds for
which the structure is determined in a database, or from a library
of compounds that have been synthesised de novo.
[0153] In a particular embodiment, the test compound may be
selected from small organic molecules. As used herein, the term
"small organic molecule" refers to a molecule of size comparable to
those organic molecules generally used in pharmaceuticals. The term
excludes biological macromolecules (e.g.; proteins, nucleic acids,
etc.); preferred small organic molecules range in size up to 2000
Da, and most preferably up to about 1000 Da.
Therapeutic Methods and Uses
[0154] One major field of application is cell therapy or
regenerative medicine. Regenerative medicine can be used to
potentially cure any disease that results from malfunctioning,
damaged or failing tissue by either regenerating the damaged
tissues in vivo by direct in vivo implanting of a composition
comprising cholangiocytes of the invention.
[0155] Another aspect of the invention thus relates to a population
of cholangiocytes of the invention for use in a method of treatment
of the human body.
[0156] More particularly, an aspect of the invention relates to a
population of cholangiocytes of the invention for use in the
treatment of a cholangiopathy.
[0157] The invention also relates to a method for treating a
cholangiopathy comprising the step of administering a
pharmaceutically effective amount of a population of cholangiocytes
of the invention to a patient in need thereof.
[0158] In the context of the invention, the terms "treating" or
"treatment", as used herein, refer to a method that is aimed at
delaying or preventing the onset of a pathology, at reversing,
alleviating, inhibiting, slowing down or stopping the progression,
aggravation or deterioration of the symptoms of the pathology, at
bringing about ameliorations of the symptoms of the pathology,
and/or at curing the pathology.
[0159] As used herein, the term "pharmaceutically effective amount"
refers to any amount of a population of cholangiocytes according to
the invention (or a pharmaceutical composition thereof) that is
sufficient to achieve the intended purpose.
[0160] Effective dosages and administration regimens can be readily
determined by good medical practice based on the nature of the
pathology of the subject, and will depend on a number of factors
including, but not limited to, the extent of the symptoms of the
pathology and extent of damage or degeneration of the tissue or
organ of interest, and characteristics of the subject (e.g., age,
body weight, gender, general health, and the like).
[0161] For therapy, populations of cholangiocytes and
pharmaceutical compositions according to the invention may be
administered through different routes. The dose and the number of
administrations can be optimized by those skilled in the art in a
known manner.
[0162] In one embodiment, the cholangiocytes of the invention may
be useful for autologous regenerative therapy of a patient
suffering from cholangiopathy in need of regenerative therapy due
to specific disorders or treatments associated to such disorders,
including without limitation, Alagille's syndrome and cystic
fibrosis due to the deficit in one identified gene that can be
replaced in vitro.
[0163] In one embodiment, the invention relates to the
cholangiocytes of the invention for use as a cell therapy product
for implanting into a human patient, as an allogenic graft or,
after genetic correction, as an autologous graft (i.e the cells
have the same genotype as the patient's cells).
[0164] In one embodiment the cholangiocytes of the invention may be
useful for bioengineered livers: either by infusing them together
with other hepatic cells into a decellularized liver, by generation
of vascularized and functional human liver from human iPSCs by
transplantation of liver buds created in vitro (Takebe et al Nature
2013 499), by 3D printing or every other method to generate liver
tissue.
[0165] The invention will be further illustrated by the following
figures and examples. However, these examples and figures should
not be interpreted in any way as limiting the scope of the present
invention.
FIGURES
[0166] FIG. 1: Generation of hepatoblasts from human embryonic stem
cells (hESCs). (A) Protocol to differentiate hESCs into
progenitors. (B) Images showing the sequential morphological
changes that occur to give a polygonal shape after 10 days of
culture in appropriate conditions.
[0167] FIG. 2: hESC-derived hepatoblasts differentiate into
cholangiocytes. Diagram summarizing our cholangiocytic
differentiation protocol. HESC that had been maintained in a
feeder-free condition were differentiated into hepatoblasts before
passaging onto collagen I-treated wells then induced into
cholangiocytic differentiation. Cells were grown 3 days in GH and
EGF then IL-6. At day 17, cells reached confluency and were
replated onto collagen I-treated wells. Cells were further
differentiated for 3 days in IL-6, then for 3 additional days in
sodium taurocholate hydrate.
[0168] FIG. 3: Differentiation of HepaRG-hepatoblasts into
cholangiocytes. Diagram of both protocols for HepaRG cell
differentiation from the progenitor stage into hepatocytes or
cholangiocytes. Scale bar=50 .mu.m.
EXAMPLE
[0169] Material & Methods
[0170] Cell culture: H9 hESCs (WA09, WiCell, Madison) were
maintained on irradiated mouse embryonic fibroblast (MEF) feeder
cells (Globalstem), in DMEM/F12 medium supplemented with 20%
knockout serum replacement, 1 mM L-glutamine, 1% non-essential
amino acids (all from Gibco), 0.1 mM .beta.-mercaptoethanol (Sigma
M3148), and 4 ng/ml FGF2 (R&D systems) at 37.degree. C./5%
CO.sub.2. Cells were passaged once a week with collagenase IV (1
mg/ml Gibco) and plated onto 0.1% gelatin-coated 60 mm culture
dishes (Corning) for amplification before starting the
differentiation. During this period feeder-free hESCs were
maintained as described previously..sup.11 HiPSC line iPS P2 was
already established in the laboratory from human foreskin
fibroblasts (CRL 2097, ATCC). IPS P2 cells were maintained on MEF
feeders in the same medium, and conditions described for hESCs and
were passaged once a week. Prior to differentiation, iPS P2 cells
were plated on culture dishes pre-coated with 2 mg/ml Geltrex (Life
technologies A1413302). hiPSCs were maintained in Nutristem medium
(Stemgent) at 37.degree. C./5% CO.sub.2 for a few passages before
starting differentiation.
[0171] HepaRG cell line was cultured as previously
described..sup.13,14 Four days after seeding, HepaRG displayed
hepatic progenitor features (HepaRG-HB). At this step, when
maintained in classical HepaRG medium during six days cells are
engaged in hepatic lineage (HepaRG-cHep). For complete hepatocyte
differentiation (HepaRG-Hep) HepaRG-HB were maintained for two
weeks in the classical culture medium, and the culture medium was
then supplemented with 2% DMSO for two additional weeks. In this
study, to prevent spontaneous differentiation along hepatocytic
lineage, and to promote cholangiocyte differentiation HepaRG-HB
were cultured in a reduced medium: William's E medium with reduced
fetal bovine serum (2%), insulin (1 .mu.g/ml), and hydrocortisone
hemisuccinate (1 .mu.M).
[0172] hESC/hiPSC differentiation into hepatic progenitor cells:
One day before passage of hESCs/hiPSCs for differentiation, 12-well
plates (Corning, Corning Life Sciences, Dutscher) were coated with
0.1% porcine gelatin (Sigma G1890) for one hour at room
temperature, then with coating medium (DMEM, 10% Fetal bovine serum
(FBS, Hyclone), 1% non-essential amino acids and 2% L-glutamine),
and incubated for 24 h at 37.degree. C. The following day,
feeder-free hESCs were plated on the coated plates in CDM-BSA
medium supplemented with 10 ng/mi Activin A and 12 ng/ml FGF2. To
start differentiation, hESCs/hiPSCs maintenance medium was replaced
by RPMI supplemented with B27 serum-free supplement (Life
technologies 17504-044) and cells were changed daily thereafter.
During the first five days of definitive endoderm differentiation,
100 ng/ml Activin A and 10.sup.-5 M LY294002 (VWR) were added to
the medium. Wnt3a (50 ng/ml) (R&D systems) was also added to
the hiPSC's medium during the first 3 days of differentiation. To
induce the hepatic specification of endoderm, 20 ng/ml FGF2, 10
ng/ml BMP4 (R&D systems) and 50 ng/ml Activin A were added for
3 days. After three days of hepatic specification, the medium was
supplemented with 30 ng/ml FGF4 (R&D systems), 25 ng/ml HGF
(Peprotech), 50 ng/ml EGF (Peprotech) and 10.sup.-7 M Retinoic acid
(Sigma R2625) for two to four days to obtain hepatoblasts.
[0173] Differentiation of hESC/hiPSC-derived hepatoblasts into
cholangiocytes: At days 10-12 of differentiation,
hESC/hiPSC-hepatoblasts were harvested with cell dissociation
buffer (0.1 mg/ml EDTA, 0.5 mg/ml BSA in PBS) and seeded onto
12-well collagen I-coated plates (BD Biosciences) with plating
medium (William's E/Ham F12 1:1, 10% FBS (PAA Laboratories), 1
mg/mi fraction V fatty acid-free BSA (Sigma), 1 mM L-glutamine) for
4 hours. Cells were then incubated overnight with biliary
differentiation medium (BDM) (William's E/Ham F12 1:1, 10.sup.5 M
linoleic acid-Albumin (Sigma L9530), 510.sup.-8 M
3,3',5-Triiodo-L-thyronine (Sigma T2752), 0.2 IU Insulin, 6.104 M
Vitamin C (Boyer), 6.104 M human apo-transferrin (Sigma T5391), 1
mM sodium pyruvate (Gibco)). The next day, cells were incubated
with BDM supplemented with 50 ng/ml human growth hormone (GH, Sigma
H5916) and 25 ng/ml EGF for three days. Cells were then incubated
with 10 ng/ml Interleukin-6 (IL-6 Miltenyi 130-093-929) for another
three days. At day 17, cells were passaged on collagen I-coated
12-well plates as described for the first passage. At day 18, BDM
medium was supplemented with 10 ng/ml IL-6 for three days. Finally,
the cells were incubated in 10 .mu.M sodium taurocholate hydrate
(Sigma 86339) for 2 days. For the transcriptome analysis, 1 .mu.M
Sodium Butyrate (NaBut Sigma 303410) was added to the medium
between days 21 to 23 (2 days). Three independent differentiation
experiments were performed for hESCs and hiPSCs. Phase-contrast
images were taken with a Nikon Eclipse microscope.
[0174] Differentiation of HepaRG progenitors into cholangiocytes:
HepaRG-HB cultured in the reduced medium were treated for 2 days
with IL-6 (10 ng/ml), then for 2 days with sodium taurocholate
hydrate (10 nM) and then for 2 days with sodium taurocholate
hydrate (10 nM) and sodium butyrate (1.8 .mu.M) to prevent
spontaneous differentiation along the hepatocytic lineage.
Independent culture experiments were performcd 4 times. For
proliferation assay, VEGF-165 (20 ng/ml) (Promokine, C-64420) was
added on HepaRG-Chol culture for 2 days. Immunostaining pictures
were analyzed and quantification was assessed with Cell Health
Profiling BioApplication from Cellomics software.
[0175] PCR and quantitative RT-PCR (qRT-PCR): Total RNA was
purified with an RNAeasy kit (Qiagen). cDNAs were obtained by using
QuantiTect kit (Qiagen 204341) or the High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems, Carlsbad, Calif.) according
to the manufacturer's instructions. The level of gene expression
was measured and analyzed by real-time PCR over 40 cycles using
Taqman universal master mix II and Taqman gene expression assays
(Applied Biosystems) using an ABI-7000 real-time PCR system and ABI
7000 software. The expression level of CFTR TGR5 (GPBAR-1), SCTR,
GGT1, SOX9, AQP1 and JAG was determined in relation to RPL13A,
which served as a housekeeping gene. Simple PCR assays were
performed using Platinum Taq DNA polymerase (Invitrogen 10966-034)
according to the manufacturer's instructions.
[0176] For HepaRG, the Real-time PCR was performed with the
fluorescent dye SYBR Green methodology using the SYBR Green PCR
Master Mix (Applied Biosystems). Data were quantified with the
StepOne Plus software v2.2.1.
[0177] Cellular Ca.sup.2+ imaging: At day 17, differentiating
hESC-Chols were plated on glass coverslips coated with type I
collagen and differentiated further into cholangiocyte-like cells
as described above. At day 23 hESC-Chols were loaded with 3 .mu.M
Fura2-AM (Invitrogen, F1201) in BDM medium (37.degree. C. and 5%
CO.sub.2), for 45 minutes. Cells were then washed twice, and
transferred into a perfusion chamber placed on the stage of a Zeiss
inverted microscope (Axiovert 35). Intracellular calcium increases
were measured after stimulation with somatostatin (Eumedica), ATP
(Sigma), or Acetylcholine (Sigma). Calcium imaging was performed as
described previously..sup.41 Fluorescence images were collected by
a charge-coupled device camera (Princeton), digitized and
integrated in real time by an image processor (Metafluor,
Princeton).
[0178] 3D cell culture assays for cyst formation: hESC/hiPSC- or
HepaRG-chol were detached with trypsin and dissociated to reach a
single cell suspension. Cells were then suspended in a mixture of
rat-tail type I collagen (BD Biosciences), Matrigel 40% (BD
Biosciences), HEPES (0.02M) and NaHCO.sub.3 (2.35 mg/ml). A cell
suspension containing 5000 cells was added to 24-well inserts (BD
biosciences 353104) and incubated at 37.degree. C. for 3 hours. For
hESC-, hiPSC-Chol, one ml of BDM medium supplemented with 20 ng/ml
HGF and 10 ng/ml EGF, and for HepaRG-Chol one ml of reduced HepaRG
medium, supplemented with 20 ng/ml HGF and 10 ng/ml EGF was added
on top of the insert as well as in the well, and incubated at
37.degree. C./5% CO.sub.2 up to 2 weeks.
[0179] Immunocytochemistry: Cells were fixed with 4%
Paraformaldehyde for 15 minutes at room temperature and
permeabilized with 0.5% Triton X-100 for 15 minutes, 3% BSA-PBS for
30 minutes at room temperature or with 0.2% saponin and 5% donkey
serum for 45 minutes at room temperature. Primary antibodies were
diluted in 1% BSA-PBS, and incubated overnight at 4.degree. C.
Secondary antibodies were diluted in 1% BSA-PBS and incubated for 1
hour at room temperature. Cell nuclei were stained with DAPI or
Hoechst H33342 (Sigma B2261) and cells were mounted in Fluoromount
medium (Sigma F4680). CO-029 antibody (mouse, 1/100 dilution) was a
kind gift from Dr Claude Boucheix (UMR-S 1004, Villejuif). (See
primary and secondary antibodies' dilutions in Supporting Table 5).
All the photographs were taken with a Leica HMR microscope (Leica
Microsystems), inverted DMIRBE microscope (Leica) using a
20.times./0.4 N Plan objective (leica) and a CoolSnapES N&B
camera (Photometrics) driven by MetaVue software (Molecular
devices), SP5 LEICA DMI 6000 CS Microscope (Leica, Germany) and
treated with LAS AF 2.6.0 software, or ACT-1 Nikon software or
Cellomics ArrayScan VTI HCS Reader.
[0180] 3D cell culture immunostaining was performed as
described.sup.42. Briefly, cysts were fixed with 3%
paraformaldehyde for 20 min, permeabilized with 0.5% PBS-Triton
X-100 for 10 min, and quenched for 15 min with a glycine/PBS
solution (130 mM NaCl, 7 mM Na.sub.2HPO.sub.4, 3.5 mM
NaH.sub.2PO.sub.4, and 100 mM glycine). Cells were incubated in
primary block (10% FBS, 130 mM NaCl, 7 mM Na.sub.2HPO.sub.4, 3.5 mM
NaH.sub.2PO.sub.4, 7.7 mM NaN.sub.3, 0.1% BSA, 0.2% PBS-Triton
X-100, and 0.05% Tween-20) for 4 hours at room temperature. After
washes, cells were incubated overnight in primary block with
primary antibodies and DAPI. After washes, cells were incubated for
1 h with secondary antibodies, washed and mounted with Fluoromount
medium. 3D culture images of hESCs were taken with a Zeiss
LSM510-Meta/Axiovert 200M laser scanning confocal microscope (Carl
Zeiss, Iena, Germany) with an Achroplan 40.times., NA: 0.75
objective lens and scan zoom between 3.5 and 4). LSM ver4.2
software was used for acquisitions. Confocal images of hiPSCs and
HepaRG 3D cultures were taken with an SP5 LEICA DMI 6000 CS
Microscope (Leica, Germany). LAS AF 2.6.0 software was used for
acquisitions.
[0181] Transport of fluorescent dye and fluorescent bile salt:
Cysts were incubated in Leibovitz's no phenol red L-15 medium
(Gibco) containing 5 .mu.M cholyl-lysyl-fluorescein (CLF,
BD-451041) for 15 minutes at 37.degree. C. and 5% CO.sub.2. Cells
were then washed twice with L-15 medium and fluorescent bile salt
internalization was visualized with a SP5 LEICA DMI 6000 CS
Microscope (Leica, Germany) equipped with an incubator to maintain
the cells at 37.degree. C. during observation.
[0182] For rhodamine 123 transport test, cells were incubated in
fresh scrum-free medium containing 100 .mu.M rhodamine 123 (Sigma
R8004) for 5 min and washed with serum-free medium three times.
Cysts were incubated with 10 .mu.M verapamil (Sigma), for 30
minutes before adding rhodamine 123. Fluorescence internalization
was visualized with a confocal microscope and images were taken
after 30 minutes.
[0183] Flow cytometry: Cells were dissociated with cell
dissociation buffer, and suspended in 3% BSA-PBS. They were then
incubated with TRA-1-81-PE, SSEA4-PE and CD184-APC, EpCAM-FITC
conjugated antibodies or in control isotypes for 30 minutes at
4.degree. C. in the dark. Cells were then washed with PBS,
centrifuged and suspended in PBS-BSA 1% for analysis. Cells were
detected in FL2 and FL4 channels with an Accuri C6 flow cytometer
(BD biosciences). Dead cells were eliminated with 7AAD staining
(Beckman coulter A07704). For CFTR analysis, cells were fixed with
4% paraformaldehyde (10 min) and permeabilized with 0.1% PBS-Tween
for 20 min. Non-specific sites of interaction were blocked with 3%
PBS-BSA. Cells were incubated with anti-CFTR (CF-3) antibody
(Abeam) for 30 min at 4.degree. C. and then with the secondary
antibody Alexafluor 488 donkey anti-mouse (Molecular probes) for 30
min at room temperature. Cells were then washed twice, suspended in
0.1% PBS-Tween and analyzed in FL1 channel with an Accuri C6 flow
cytometer.
[0184] Cell cycle analysis: After 3 days of GH/EGF treatment, cells
were detached with trypsin, centrifuged and suspended in 3 ml of
70% ethanol and kept at -20.degree. C. On the day of analysis,
cells were centrifuged and suspended in 50 pg/ml propidium iodide
(Sigma P-4170) and 0.5 mg/ml RNase A (Sigma R6513), kept in the
dark at 4.degree. C. for 30 minutes and analyzed with an Accuri
flow cytometer.
[0185] Western blot analysis: Total proteins were extracted with
RIPA buffer. Briefly, 50 .mu.g of total protein was electrophoresed
on an 8% acrylamide-SDS gel and then transferred to a PVDF membrane
(GE healthcare RPN303F). The membrane was blocked with PBS
containing 5% (w/v) dry milk and 0.1% Tween-20, washed with PBS
containing 0.1% Tween-20 (PBST) and then incubated with primary
antibodies at 4.degree. C. overnight. After washing in PBST, the
membranes were incubated with horseradish peroxidase-conjugated
secondary antibodies at 37.degree. C. for 1 h. Beta-actin or HSC-70
was used as a control for protein amount.
[0186] Microarray analysis: mRNAs from biological replicates of H9
cells, hESC-HB, hESC-Chol, HepaRG-HB, HepaRG-Chol, HepaRG-cHep and
HepaRG-Hep were used. The purity and integrity of RNA were
evaluated on Agilent Bioanalyser (Agilent Technologies, Palo Alto,
Calif., USA). Genome-wide expression profiling was performed using
the low-input QuickAmp labeling kit and human SurePrint G3
8.times.60K pangenomic microarrays (Agilent Technologies, Santa
Clara, Calif., USA). Gene expression data were processed using
Feature Extraction and GeneSpring software (Agilent Technologies).
The data discussed in this publication have been deposited in
NCBI's Gene Expression Omnibus and are accessible through GEO
Series accession number GSE51791
(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE51791). To
determine genes significantly deregulated between hESC-HB and
hESC-Chol or HepaRG-HB and HepaRG-Chol, a t-test (Bonferroni FWER
correction) with a p-value <0.05 and a Fold Change (FC)>2 was
used.
[0187] Microarray meta-analysis: Home project Agilent chip
microarray data were combined with data from different GEO series
matrix (GEO data set) publicly available on the website. Public
data used are as follows: three samples of the cell line H69
untreated in the set GSE23991 (Illumina humanRef-8 v2.0 annotated
with platform GPL6104),.sup.29 and six samples of human normal
biliary epithelial cells in the set GSE32225 (WG-DASL Illumina
humanRef-8 v3.0 annotated with platform GPL8432)..sup.30 Individual
raw data from each GEO dataset and the house experimental set were
individually normalized on the standard deviation of the chip. Each
GEO experimental set was annotated with their respective GPL
platform. The cross experimental matrix was formed by SQL query.
The cross experimental mathematical adjustment was done with the
"batchadjust" function in the PAMR package.sup.43 of R software
available on the Bioconductor website..sup.44 The effect of
mathematical adjustment was verified by factorial projection by
principal component analysis before and after PAMR
correction..sup.45
[0188] Data Clustering: Global differentially expressed genes
during differentiation were identified by a one-way ANOVA test
(Bonferroni FWER correction) with a p-value <0.001 and an
absolute FC>3. We used Gene Cluster 3.0 software (Euclidean
metrics with complete distances) to cluster the set of genes
selected by ANOVA and TreeView 1.6 to visualize the clustering. The
accuracy of our genomic integration analysis was validated by
another analysis with the same number of genes (2920) randomly
selected. Heat maps of some gene expression were created by
importing data into the Java Treeview. When a gene was represented
by more than one probe-set on the array, a single probe-set was
chosen to represent gene expression in the heat map according to
the highest mean expression over all samples.
[0189] Ingenuity Pathway Analysis: Upstream regulators were
identified using upstream regulator analyses in the Ingenuity
Pathway Analysis Tool (IPA, Ingenuity Systems, Redwood City,
Calif.) based on the down-regulated or up-regulated gene lists. IPA
analysis was performed on the 126 up- and 71 down-regulated genes
(p<0.05; FC>2) for HepaRG differentiation. IPA was performed
on the 489 up- and 307 down-regulated genes (p>0.001; FC>7)
for hESC differentiation.
[0190] Transduction of cholangiocyte progenitors: Cholangiocyte
progenitors were transduced once at day 14 at MOI 30 with a
lentivector bearing a EF1.alpha.-GFP cassette and were analyzed 4
days later by flow cytometry.
[0191] Results
[0192] Differentiation of hESCs Into hepatoblasts: The present
strategy to induce differentiation of hESCs into hepatoblasts was
based on a previously published protocol.sup.11,12 with some
modifications (see Supporting Materials for details). The
differentiation protocol is illustrated in FIG. 1A. At day 0, hESCs
displayed typical colony morphology of stem cells (FIG. 1B, panel
i). The cells expressed pluripotency markers including OCT4, NANOG,
TRA-1-60 and SSEA4 and were negative for GATA4, confirming the
absence of endodermal differentiation. Flow cytometry analysis
showed that more than 70% of the cells expressed TRA-1-81. Five
days of endoderm induction treatment resulted in a homogeneous
monolayer of polygonal endodermal cells (FIG. 1B, panel ii).
Immunostaining revealed that most of the cells were positive for
the definitive endoderm markers GATA4, CXCR4, Hepatocyte nuclear
factor 3.beta. (HNF3.beta.) and SOX17. Flow cytometry analysis
confirmed that more than 90% of the cells expressed CXCR4.
Following the endodermal induction, cells were treated with the
hepatic commitment factors for 3 days, which gave rise to specified
hepatic endoderm/liver bud (FIG. 1B, panel iii). Immunostaining of
these cells confirmed that they expressed characteristic markers
HNF30, HNF4a Cytokeratin 19 (CK19), CK18, SOX9, SOX17, GATA4 and
Neural cell adhesion molecule (NCAM). In addition, these cells were
negative for .alpha.-fetoprotein (AFP) and CK7, the Cytokeratin
specifically expressed by cholangiocytes. Most of them (93%) were
also positive for Epithelial cell adhesion molecule (EpCAM) as
assessed by flow cytometry analysis. Finally, the medium was
supplemented with factors inducing differentiation of hepatic
endodermal cells into hepatoblasts with a polygonal morphology
(FIG. 1B, panel iv). Immunostaining of cells at day 10 showed that
they were positive for AFP and CK19. HESC-derived hepatoblasts
(hESC-HB) also expressed the transcription factors HNF3.beta.,
GATA4, HNF6 and HNF4.alpha.. Flow cytometry analysis revealed that
most of the cells (87%) remained positive for EpCAM
[0193] hESC-derived hepatoblasts exhibit the potential for
commitment toward cholangiocyte precursors: As NOTCH2 and SALL4,
Forkhead factor M1B (FOXM1B) is also reported to be critical for
differentiation of precursors toward biliary epithelial
cells..sup.15 To further define the biliary commitment potential of
our hepatoblasts, the inventors analyzed the expression of
different biliary markers. They found that hESC-HBs expressed
FOXM1B, NOTCH2 and SALL4.
[0194] The inventors then tested factors known to stimulate
cholangiocyte proliferation and differentiation in the prenatal
period: Epidermal growth factor (EGF), Growth hormone (GH), a
regulator of the insulin-like growth factor-1 pathway,.sup.16 and
Interleukin 6 (IL-6).sup.17 in diverse combinations. GH, a
regulator of the insulin-like growth factor-1 pathway, has not only
a mitogenic but also a developmental effect in liver. It stimulates
the synthesis of liver-specific transcription factors among which
HNF6, which enhances SOX9 expression..sup.18 Both GH and Sox9
negatively regulate CEBP/.alpha. which governs transcription of
mature hepatocyte marker genes..sup.19 Finally, IL-6 inhibits
GH-mediated gene expression in hepatocytes..sup.20
[0195] They then tested the effect of sodium taurocholate hydrate,
which not only stimulates proliferation and differentiation of
cholangiocytes but also displays anti-apoptotic activity on these
cells in rat..sup.21 The present final differentiation strategy is
illustrated in FIG. 2.
[0196] At the hepatoblast stage (Day 10), cells were passaged on
collagen I-coated plates and maintained for one day in William's
E/Ham's F12 medium as described in Materials and Methods. Cell
cycle analysis showed that after 3 days of GH/EGF the cells were
actively proliferating. The percentage of cells in S and G2/M phase
was 36% and 8% respectively. Differentiating cells remained in
proliferation after addition of IL-6 yielding up to 68%
Ki67-positive cycling cells This was confirmed by expression of
Cyclin A, Cyclin E, CDK1 and CDK2.
[0197] Gene expression of differentiating cells was analyzed after
addition of GH/EGF and IL-6 by qRT-PCR. Combination of these
factors increased the expression of cholangiocyte markers such as
CFTR, G protein-coupled bile acid receptor 1 (TGR5), SOX9 and
secretin receptor (SCTR).
[0198] In addition, one single transduction of cholangiocyte
progenitors yielded to GFP expression in 30% of the cell,
suggesting that, according to our experience, a second transduction
a few hours after the first one would result in 50-60% of GFP
positive cells.
[0199] hESC-derived hepatoblasts differentiate into functional
cholangiocyte-like cells: Treatment of hepatoblasts with GH/EGF
then IL-6 allowed the cells to reach confluency around day 17. This
population of proliferating biliary-committed cells was then
passaged on collagen I. The cells were treated with IL-6 for 3 days
and then with sodium taurocholate hydrate for 2 days. In the course
of differentiation the cell population progressively acquired a
cuboidal morphology (FIG. 2). QRT-PCR analysis revealed a
significant increase in gene expression level of cholangiocyte
markers such as CFTR, TGR5, Aquaporin-1 (AQP1), SOX9, SCTR and JAG1
in hESC-derived cholangiocytes (hESC-Chol) compared to hESC-HB. The
expression level of .gamma.-glutamyltransferase 1 (GGT1) was
equivalent to that of hESC-HB and control cells.
[0200] In addition, RT-PCR analysis on hESC-Chol revealed that
these cells expressed CK7, and also biliary markers including
Cl.sup.-/HCO.sub.3.sup.- Anion Exchanger 2 (AE2) and NCAM, a marker
of proliferating cholangiocytes..sup.22 The inventors observed that
SALL4, NOTCH2 and FOXM1B, were also expressed in hESC-Chol whereas
the stemness marker NANOG was extinguished.
[0201] The inventors also evaluated hepatocytic marker expression
by qRT-PCR in hESC-Chol. AFP and Albumin (ALB) expression level was
10.sup.4 and 10.sup.7 less than in fetal and adult hepatocytes
respectively. Aldolase B transcripts were not detectable.
[0202] Cholangiocyte differentiation was confirmed by
immunostaining analysis, showing that hESC-derived cholangiocytes
expressed CK7, CK19, CK18, Osteopontin (OPN), a downstream target
of SOX9 during normal development,.sup.23 SOX9, HNF6 and
HNF1.beta., whereas HNF4.alpha. expression was not detected, as
shown by co-staining experiments with CK7/HNF4.alpha.).
[0203] In addition, to further confirming the identity of these
cholangiocyte-like cells, they tested whether these cells expressed
specific biliary receptors. Immunostaining analysis revealed that
these cells expressed CFTR, SCTR, Apical Na.sup.+-dependent bile
acid transporter (ASBT), TGR5 and KDR (VEGFR-2). FACS analysis
revealed that 90% of the cells expressed CFTR. They also examined
expression of cholangiocyte transporters by Western blot analysis,
which confirmed expression of ASBT and TGR5 in hESC-derived
cholangiocytes.
[0204] By contrast to hepatocytes, cholangiocytes bear primary
cilia, which play an important role in modulating the secretory and
proliferative functions of fully differentiated cells..sup.24 The
presence of cilia (one cilium per cell) was detected using an
antibody to a ciliary marker, acetylated .alpha.-tubulin.
[0205] To assess the functionality of our hESC-Chol, they analyzed
their response to Adenosine triphosphate (ATP), acetylcholine and
Somatostatin, known to induce Ca.sup.2+ increase in biliary cells
via P2Y1 receptors,.sup.5 M3 muscarinic receptor (AChR M3),.sup.25
Somatostatin receptor type 2 (SSTR2).sup.26 respectively, as well
as type III Inositol 1,4,5-triphosphate (InsP3) receptor (InsP3R),
an intracellular Ca.sup.2+ channel, which stimulates release of
Ca.sup.2+ from intracellular stores into the cytosol..sup.27
[0206] RT-PCR on hESC-Chol revealed that the four different
receptors were specifically expressed in these cells compared to
human hepatocytes. ATP, acetylcholine or Somatostatin stimulation
resulted in Ca.sup.2+ increase. The percentage of responsive cells
was quantified to be 70%.+-.5, 40%.+-.4, and 31%.+-.9,
respectively.
[0207] HepaRG-progenitor cells can be driven to differentiate into
cholangiocytes: The inventors then assessed whether the present
protocol could be used to drive differentiation of HepaRG to a
cholangiocyte lineage. In the present conditions, addition of GH
did not improve proliferation and differentiation of
HepaRG-hepatoblasts (HepaRG-HB) (data not shown) since they
expressed HNF3.beta. and very low level of C/EBP.alpha...sup.13,14
However, two days of IL-6 followed by 2 days of sodium taurocholate
hydrate treatment was sufficient to drive commitment toward
cholangiocyte differentiation (FIG. 3). Then, to prevent
spontaneous differentiation of HepaRG-HB into hepatocytes, Na
Butyrate (NaBut) was added after 2 days of sodium taurocholate
hydrate treatment..sup.28
[0208] At the end of the differentiation protocol (day 10),
HepaRG-derived cholangiocytes (HepaRG-Chol) expressed high levels
of GGT1, CK19, JAGGED1 and TGR5 compared to hepatocyte-committed
HepaRG cells (HepaRG-cHep) and HepaRG-HB.
[0209] Immunostaining revealed that HepaRG-Chol cells expressed
OPN, CK19, SCTR and Tetraspanin-8 (CO-029), whereas they were not
expressed in our regular HepaRG culture conditions. Hepatocytic
markers such as ALB and HNF4a were repressed.
[0210] Immunostaining of acetylated .alpha.-tubulin revealed the
presence of primary cilia in HepaRG-Chol. In addition, HepaRG-Chol
expressed the receptors P2RY1, AChR M3, SSTR2 and InsP3R at levels
similar to that of hESC-Chol.
[0211] Finally, since cholangiocytes proliferate in response to
VEGF (Vascular endothelial growth factor), we stimulated
HepaRG-Chol with VEGF, which induced a significant two-fold
increase in the number of cells in G2/M phases.
[0212] Transcriptomic profiles of cholangiocytes derived from both
hESCs and HepaRG: To analyze gene expression profiling in
cholangiocytes derived from both hESCs and HepaRG in similar
conditions, the inventors first verified that addition of NaBut on
hESC-Chol did not significantly change expression of cholangiocyte
markers. Microarrays were conducted at day 23 and day 10 of
differentiation for hESC-Chol, and HepaRG-Chol respectively and
compared to hESC-HB and HepaRG-HB. Integrative genomics was first
used to determine the level of differentiation of the cells. The
2920 genes differentially expressed in cholangiocytes (ANOVA) were
integrated with gene expression profiles of normal human biliary
epithelial cells.sup.29 and of an immortalized human intrahepatic
biliary epithelial cell line (H69).sup.30. Hierarchical clustering
analysis revealed two main branches dividing the samples with
regard to the lineage: hESC/hepatoblasts/cholangiocytes in one
branch and hepatocytes in the other. Cluster one includes both
hESC-Chol and HepaRG-Chol as well as H69 and normal human biliary
epithelial cells. Cluster two included HepaRG-Hep and HepaRG-cHep.
Cluster one was divided into two branches driven by hESC- or
HepaRG-Chol differentiation. HepaRG-Chol clustered with both H69
cell line and human normal biliary epithelial cells. This
subdivision probably reflects the origin of the cells. HepaRG cells
derive from differentiated hepatocellular carcinoma, normal human
biliary epithelial cells were microdissected from six metastatic
liver tumors;.sup.30 and H69 cells are SV40-transformed
cells..sup.29
[0213] During hESC-HB differentiation, they found that up to 3429
genes were deregulated (p<0.05; FC>2). In HepaRG-Chol only
197 genes were significantly deregulated (p<0.05; FC>2).
Interestingly Ingenuity Pathway Analysis (IPA) revealed that the
489 strongest up-regulated genes (p<0.001; FC>7) in hESC-Chol
and the 126 up-regulated genes in HepaRG-Chol were involved in
common pathways including Farnesoid X receptor (FXR)/RXR activation
pathway. In addition, Vitamin D receptor/Retinoid X receptor
(VDR/RXR) was strongly upregulated in hESC-Chol. Two of the main
upstream regulators of up-regulated genes were Transforming growth
factor beta 1 (TGF.beta.1) and Estrogen receptor 1 (ESR1), known to
play a role in modulating rat cholangiocyte
proliferation..sup.31
[0214] Comparison of both expression profiles of hESC-Chol and
HepaRG-Chol highlighted up-regulation of common and distinct genes.
For instance, OPN, E-cadherin (CDH1), and KCNN4, a Potassium
intermediate-conductance Ca.sup.2+-activated K.sup.+ channel were
upregulated in both cell types. By contrast, Peroxysome
proliferator activated receptor gamma (PPAR.gamma.) or the ABCB8
transporter were specifically upregulated only in hESC-Chol. Other
transporters such as ABCA3 and ABCB4 were induced specifically in
HepaRG-Chol. Expression of these genes was confirmed by
qRT-PCR.
[0215] Generation of cholangiocytes from hiPSCs: Finally, the
inventors investigated whether the culture conditions developed to
differentiate hESCs into cholangiocytes could also efficiently
drive differentiation of hiPSCs. To this end, they used iPS P2 cell
line which was reprogrammed using a polycistronic retroviral vector
encoding OCT4, SOX2, KLF4 and C-Myc..sup.32 iPSCs were
characterized for pluripotency markers such as OCT4, NANOG,
TRA-1-60 and SSEA4 by immunostaining and flow cytometry. IPS P2
cells were differentiated into hepatoblasts as described for hESCs.
Immunostaining confirmed that hiPSC-derived hepatoblasts (hiPSC-HB)
expressed hepatic progenitor markers AFP, HNF4.alpha., CK19, HNF6
and HNF30, whereas they were negative for biliary marker CK7.
hiPSC-HB were then differentiated into cholangiocytes (hiPSC-Chol).
Analysis of biliary markers expressed in hiPSC-Chol by qRT-PCR
revealed a significant increase in CFTR, TGR5 and AQP1 expression
compared to that of hiPSC-HB. In addition, hiPSC-Chol expressed
biliary markers CK7, CK19, HNF6, SOX9 and CFTR, whereas expression
of HNF4a was not detected.
[0216] As in hESC-Chol, AFP, ALB and Aldolase B expression level
was 10.sup.4, 10.sup.6 and 10.sup.6 less than in fetal and adult
hepatocytes respectively. Expression of cholangiocyte-specific
calcium signaling receptors SSTR2, P2RY1, InsP3R type III and AChR
M3 was confirmed in hiPSC-Chol. Finally, the presence of primary
cilia was visualized by immunostaining. Taken together, these data
suggest that the present approach, developed initially for hESCs,
can be used to generate cholangiocyte-like cells from hiPSCs as
well as from HepaRG cells.
[0217] Functionality of cholangiocyte-like cells generated from
pluripotent stem cells and HepaRG: To assess the potential of
cholangiocytes derived from hESC/hiPSC and HepaRG to form cysts and
tubules, the inventors used a three-dimensional culture system.
After 7 days of culture, cholangiocytes formed round cysts with
luminal space. Moreover, when the cysts were kept in culture for
one more week, they were able to bud, and formed branching tubular
structures. Of note, HepaRG-cHep cultured in 3D did not form cysts
and tubules. Cyst polarity was verified by basolateral and apical
localization of .beta.-catenin and F-actin, respectively. A
physiological function of cholangiocytes is the secretion of small
substances by various transmembrane channel proteins such as
multidrug resistance protein 1 (MDR1). After incubation with
rhodamine 123, MDR1 substrate, fluorescence accumulation was
detected inside the central lumen of hESC/hiPSC/HepaRG-cysts.
Furthermore, when cysts were incubated in verapamil, an MDR1
inhibitor, rhodamine 123 transport was abolished. Since MDR1 is
also expressed by progenitor cells.sup.33 we further investigated
the transport of fluorescent bile salt, a property of functional
cholangiocytes. When cysts were incubated with
cholyl-lysyl-fluorescein (CLF), fluorescence accumulation was
detected inside the central lumen.
[0218] Discussion:
[0219] This present study provides the first evidence of the
differentiation of functional cholangiocyte-like cells from hepatic
progenitors generated from human pluripotent stem cells and HepaRG
cells. The present approach is based on fully defined culture
conditions devoid of serum and of feeder cells.
[0220] In vivo maturation of hepatoblasts to cholangiocytes is
regulated by several factors secreted in the microenvironment
around the portal vein, which consists of mesenchymal cells, other
cells and extracellular matrix. After liver specification, HNF3p,
HNF6 and HNF1.beta. are required for normal bile duct
development..sup.34 HNF1.beta. is a direct target of HNF6 and is
also required for duct morphogenesis. The inventors found that
these transcription factors were expressed in hepatoblasts and in
differentiating cholangiocytes.
[0221] As hepatoblasts forming primitive ductal structures respond
to periportal mesenchymal cues such as Bone morphogenetic protein 4
(BMP4), TGF.beta.1 and VEGF, they first analyzed the effect of
these factors on hepatic progenitors. They found that addition of
these factors did not induce significant cell commitment toward
cholangiocytes in our conditions (data not shown).
[0222] Therefore, since it seemed difficult to find a signal
pathway truly specific for cholangiocytes, they reasoned that
addition of GH on hepatoblasts, followed by IL-6 should efficiently
trigger these cells toward the cholangiocyte lineage via HNF6 and
HNF3.beta. which are restricted to biliary lineage throughout
development and are expressed in their hepatoblasts. Interestingly,
transcriptome analysis revealed that factors important for ductal
plate commitment and/or their downstream targets were activated in
both hESC/HepaRG-cholangiocytes. For instance, downstream target
genes of TGF.beta., including those coding for matrix proteins such
as Fibronectin 1, Integrin .alpha.6 and Laminin 3, were expressed
by both hESC/HepaRG-Chol. Of note, they confirmed by immunostaining
that the cells also expressed OPN and VEGFR-2. NOTCH2 and
downstream effectors of Transcription factor HES1, a target of
NOTCH signaling, were also up-regulated. Altogether, these data
suggest that the present differentiation protocol allows activation
of appropriate signaling pathways known to be involved in biliary
differentiation.
[0223] Gene expression profile analysis allowed the inventors to
identify a panel of genes and pathways characteristic of
cholangiocytes. It is known that cholangiocytes express a subset of
nuclear receptors including VDR, FXR, PPAR delta/gamma, ESR1 and
ABCA..sup.35,36 The present data show that most of them were
induced in hESC-Chol and most of their downstream targets were
activated in both hESC/HepaRG-Chol. Human cholangiocytes also
express a variety of Toll-Like-Receptors (TLRs) which mediate (via
TLR2 and 4) host epithelial defense responses to microbial
infection..sup.37 Interestingly. TLR4 was significantly induced in
hESC-Chol.
[0224] Genomic integration of their dataset with gene expression
profiles of normal human biliary epithelial cells and H69 cell line
confirmed the commitment of progenitors into cholangiocytes.
Importantly, both hESC/HepaRG-Chol as well as H69 and normal human
biliary epithelial cells clustered in the same branch.
[0225] Morphogenesis of the biliary tree is tightly linked to the
differentiation of the cholangiocytes that line the lumen of the
biliary tree. According to the position of the cholangiocytes along
the intrahepatic biliary tree, their size, morphology,
proliferation activity and function differ..sup.38 Small
cholangiocytes are thought to be committed biliary progenitors
lining the interlobular bile ducts, ductules, and the canal of
Hering expressing CK7, CK19, NCAM, CFTR and AQP1, while large
cholangiocytes line large ducts and express SCTR, GGT1, TGR5 and
AE2..sup.39 Cholangiocytes also express ASBT, providing a mechanism
to mediate bile acid uptake.sup.21 and TGR5 that may function as a
bile sensor coupling biliary bile acid concentration to ductular
bile formation and bile flow..sup.40 hESC/hiPSC-Chol and
HepaRG-Chol expressed various levels of these markers, suggesting a
mixed population containing both types of cholangiocytes. Of note
CFTR, ASBT and EpCAM are mutated in HepaRG (Dubois-Pot-Schneider,
personal communication).
[0226] The present data show that differentiated cholangiocytes
express SSTR2, P2RY1, AChR M3 and InsP3R type Ill receptors, known
to be restricted to bile ducts in human liver,.sup.5,25-27 and that
their stimulation resulted in an increase of intracellular
Ca.sup.2+. Interestingly, hESC-, HepaRG- and hiPSC-Chol have
primary cilia, the sensory organelle present on cholangiocyte
apical surface, and form polarized cysts able to transport a
fluorescent bile acid. Altogether the present data reveal the
functionality of the cholangiocyte-like cells thus obtained and
highlight the importance of the in vitro model.
[0227] Pathogenic aspects of the most important primary
cholangiopathies (polycystic and fibropolycystic liver diseases,
Alagille syndrome) are related to altered biliary development,
which in some cases (polycystic disease notably), can be due to
defects in cilia. Thus cholangiocytes generated by the present
approach represent a useful model for studying not only the
molecular mechanisms of bile duct development but also the
pathogenic mechanisms leading to liver fibrosis or ciliopathies. It
may contribute to the development of therapeutic strategies,
including those with bioengineered livers.
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