CN115216436A - Method for establishing hepatic stem cell line with bidirectional differentiation potential by using pluripotent stem cells and application thereof - Google Patents

Abstract

The invention provides a method for establishing a liver stem cell line HepSC with bidirectional differentiation potential by utilizing pluripotent stem cells and application thereof, in particular to a method for inducing human pluripotent stem cells to be differentiated into HepSC, which comprises the following steps: culturing the pluripotent stem cells in a culture system to obtain functional HepSC, and continuing to culture the HepSC to obtain functional parenchymal hepatocytes or a population of parenchymal hepatocytes; and/or a biliary epithelial cell population. A functional HepSC, hepatocyte, or population of hepatocyte cells of the invention; and/or the cholangiocytes or cholangiocytes epithelial cell population has a very high differentiation rate and purity, and the HepSC, the parenchymal hepatocytes or the parenchymal hepatocyte population of the present invention; and/or bile duct epithelial cells or bile duct epithelial cell populations may be used as in vitro models for drug screening.

Description

Method for establishing hepatic stem cell line with bidirectional differentiation potential by using pluripotent stem cells and application thereof

Technical Field

The present invention relates to the fields of biotechnology and cell therapy. Specifically, the invention relates to a method for establishing a hepatic stem cell line with bidirectional differentiation potential by using pluripotent stem cells and application thereof.

Background

Liver disease has a high incidence and mortality worldwide. In China, the influence of liver diseases is far beyond that of other countries, the number of patients with viral hepatitis, drug-induced hepatitis, liver cancer, hereditary liver diseases and the like reaches more than 3 hundred million at present, and about 30 to 40 million people die of liver diseases each year, so that the influence on the health of Chinese people and social economy is huge.

End-stage liver disease and bile duct disease are exemplified.

For end-stage liver disease, the best treatment is liver transplantation. However, donor liver sources are very scarce, and thus a liver cell transplantation therapy has been developed, which refers to a therapeutic approach for achieving substitute liver functions by transplanting liver cells. Compared with liver transplantation, cell transplantation has the following advantages: 1) Primary hepatocytes of one donor origin can be used in the treatment of multiple patients; 2) Compared with complex surgery, the operation of cell transplantation is simpler; 3) Primary hepatocytes can be cryopreserved, which also enables cell transplantation to be often applied in emergency situations; 4) Repeated transplantation treatments can be carried out for a plurality of times; 5) Cell transplantation costs far less than liver transplantation. However, the source of mature parenchymal hepatocytes available for transplantation has been a limitation of this treatment.

Aiming at bile duct diseases. Bile duct diseases are mainly divided into two categories: PSC and PBC (Banales et al, 2019). PSC and PBC are rare but are important causes of chronic liver disease. Both of these can lead to inflammation of the bile duct, thereby inducing the processes of biliary cirrhosis, portal hypertension and liver failure. Bile duct diseases are often important causes of liver disease, such as Cystic Fibrosis (CF) and Alagille syndrome. More complex biliary diseases, such as primary sclerosing cholangitis and biliary atresia, have very few therapeutic drugs available due to the lack of an understanding of their pathophysiology and an adequate model for screening pharmaceutical formulations.

The problem common to both classes of diseases is the lack of donor sources of cells.

ES (endothelial stem cell) cells and iPS (induced pluripotent stem cell) cells have the ability to proliferate indefinitely in vitro, and thus can theoretically obtain sufficient quantities of parenchymal hepatocytes and biliary epithelial cells). And the iPS cells are used as the starting points for hepatic cell differentiation, so that various ethical problems can be avoided on one hand, and on the other hand, the patient specific iPS cells can be easily obtained for differentiation, so that the problem of immunological rejection is solved. However, cells obtained by differentiation of ES or iPS cells often contain ES or iPS cells mixed therein, and when transplanted into the body, teratomas are easily caused.

Therefore, there is an urgent need in the art to develop a novel method capable of establishing a hepatic stem cell line having bidirectional differentiation potential.

Disclosure of Invention

The invention discloses a novel method capable of establishing a hepatic stem cell line with bidirectional differentiation potential.

In a first aspect of the present invention, there is provided a method of inducing differentiation of pluripotent stem cells into hepatic stem cell lines, comprising the steps of:

(a) Culturing pluripotent stem cells in a culture system under first culture conditions, thereby obtaining a hepatic stem cell line (HepSC); wherein the culture system comprises: l-glutamine, ascorbic Acid, MTG, bFGF, EGF, VEGF, BMP4, A8301, CHIR99021.

In another preferred embodiment, in step (a), comprising step (b 1), the pluripotent stem cells are cultured in a culture system under conditions suitable for culture, thereby obtaining definitive endoderm cells.

In another preferred example, in step (a), comprising step (b 2), culturing the definitive endoderm cells obtained in step (b 1) in a culture system under first culture conditions, thereby obtaining a hepatic stem cell line (HepSC), wherein the culture system comprises: l-glutamine, ascorbic Acid, MTG, bFGF, EGF, VEGF, BMP4, A8301, CHIR99021.

In another preferred embodiment, the first culture condition comprises a first medium.

In another preferred embodiment, the first medium is selected from the group consisting of: RPMI medium 1640, SFD, ham's F12, or a combination thereof.

In another preferred example, in the step (b 1), the culture system is a culture solution containing a second medium and additives, and the additives include L-glutamine, MTG, ascorbic Acid, bFGF, VEGF, activin A, CHIR99021 and BMP4.

In another preferred embodiment, the second medium is selected from the group consisting of: RPMI medium 1640, SFD, ham's F12, or a combination thereof.

In another preferred example, the second medium includes a second a medium, a second B medium, and a second C medium.

In another preferred example, the second A medium is DE (Definitive endoderm) differentiation first stage medium.

In another preferred example, the second B medium is a DE differentiation second stage medium.

In another preferred embodiment, the second C medium is a DE differentiation third stage medium.

In another preferred example, the second a medium comprises RPMI medium 1640.

In another preferred embodiment, the second a medium contains one or more additives selected from the group consisting of: l-glutamine, MTG, activin A, and CHIR99021.

In another preferred example, the second B medium comprises RPMI medium 1640.

In another preferred embodiment, the second B medium contains one or more additives selected from the group consisting of: l-glutamine, ascorbic Acid, MTG, bFGF, VEGF, activin A and BMP4.

In another preferred embodiment, the second C medium comprises Serum free medium (SFD).

In another preferred embodiment, the second C medium contains one or more additives selected from the group consisting of: l-glutamine, ascorbic Acid, MTG, bFGF, VEGF, activin A, and BMP4.

In another preferred embodiment, the pluripotent stem cells are derived from human embryonic stem cells or human induced pluripotent stem cells.

In another preferred embodiment, the pluripotent stem cells are selected from the group consisting of: human embryonic stem cell lines (e.g., H9-ES, HES2-ES, 9-3-ES), human induced pluripotent stem cell lines (e.g., iPS17, WT 6-iPS), or combinations thereof.

In another preferred embodiment, the pluripotent stem cells are selected from the group consisting of: H9-ES, 9-3-ES, iPS17, or a combination thereof.

In another preferred example, in step (a), the pluripotent stem cells are cultured under the first culture conditions for 2 to 15 days, preferably 3 to 10 days, more preferably 6 to 7 days.

In another preferred example, in step (b 1), the pluripotent stem cells are cultured in the second medium for 5 to 6 days.

In another preferred embodiment, in step (b 2), definitive endoderm cells are cultured under the first culture conditions for 4 to 8 days, preferably 8 to 12 days, more preferably 12 to 20 days.

In another preferred embodiment, the method further comprises the step of (c) culturing the HepSC obtained in step (a) in a culture system under second culture conditions, thereby obtaining hepatocytes; wherein the culture system comprises: bFGF, BMP4, IWP2 and A8301.

In another preferred embodiment, the parenchymal hepatic cells are mature parenchymal hepatic cells.

In another preferred embodiment, the second culture condition comprises a third medium.

In another preferred embodiment, the third medium is selected from the group consisting of: SFD, HCM-cAMP, ham's F12, or a combination thereof.

In another preferred example, in the step (c), comprising the step (c 1), the HepSC obtained in the step (a) is cultured in a culture system under the second culture condition, thereby obtaining the hepatoblasts, wherein the culture system comprises: bFGF, BMP4, IWP2 and A8301.

In another preferred embodiment, in step (c 1), the culture system further comprises one or more additives selected from the group consisting of: l-glutamine, ascorbic Acid, MTG, EGF, VEGF, ROCKi and CHIR99021.

In another preferred example, in the step (c), comprising the step (c 2), the hepatoblasts obtained in the step (c 1) are cultured in a culture system under conditions suitable for the culture, thereby obtaining immature hepatocytes.

In another preferred example, in the step (c), comprising the step (c 3), the immature parenchymal cells obtained in the step (c 2) are cultured in a culture system under conditions suitable for culture, thereby obtaining mature parenchymal cells.

In another preferred example, in the step (c 2), the culture system is a culture solution containing a fourth medium and additives, and the additives include one or more selected from the group consisting of: l-glutamine, ascorbic Acid, MTG, HGF, OSM, DEXAMETHASONE, vitamin K1, CE, A8301, and EGFi.

In another preferred embodiment, the fourth medium is selected from the group consisting of: SFD, HCM-cAMP, ham's F12, or a combination thereof.

In another preferred example, in the step (c 3), the culture system is a culture solution containing a fifth medium and additives, and the additives include one or more selected from the group consisting of: l-glutamine, ascorbic Acid, MTG, HGF, OSM, DEXAMETHASONE, vitamin K1, CE, A8301, and EGFi.

In another preferred embodiment, the fifth medium is selected from the group consisting of: SFD, HCM-cAMP, ham's F12, or a combination thereof.

In another preferred example, in step (c), the HepSC is cultured under the second culturing condition for 10 to 40 days, preferably 12 to 30 days, more preferably 15 to 25 days.

In another preferred example, in step (c 1), hepSC is cultured under the second culturing conditions for 3 to 15 days, preferably, 4 to 12 days, more preferably, 5 to 10 days.

In another preferred example, in step (c 2), the hepatoblasts are cultured in the fourth medium for 3 to 15 days, preferably for 4 to 12 days, more preferably for 5 to 10 days.

In another preferred example, in step (c 3), the immature parenchymal liver cells are cultured in the fifth medium for 3 to 15 days, preferably 4 to 12 days, more preferably 5 to 10 days.

In another preferred example, the method further comprises the step (d) of culturing the HepSC obtained in the step (a) in a culture system under third culture conditions, thereby obtaining cholangiocytes; wherein the culture system comprises: bFGF, CHIR99021, noggin and TGF beta 2.

In another preferred embodiment, said third culture condition comprises a sixth culture medium.

In another preferred embodiment, the sixth medium is selected from the group consisting of: SFD, ham's F12, or a combination thereof.

In another preferred example, in the step (d), comprising the step (d 1), the HepSC obtained in the step (a) is cultured in a culture system under a third culture condition, so as to obtain bile duct precursor cells, wherein the culture system comprises: bFGF, CHIR99021, noggin and TGF beta 2.

In another preferred example, in the step (d), comprising the step (d 2), the bile duct precursor cells obtained in the step (d 1) are cultured under conditions suitable for culture, thereby obtaining bile duct epithelial cells.

In another preferred example, the bile duct cells are mature bile duct epithelial cells.

In another preferred embodiment, in step (d 1), the culture system further comprises one or more additives selected from the group consisting of: l-glutamine, ascorbic Acid, MTG, HGF, EGF and TGF beta 2.

In another preferred example, in the step (d 2), the culture system is a culture solution containing a seventh medium and additives, and the additives include one or more selected from the group consisting of: l-glutamine, ascorbic Acid, MTG, HGF, EGF and TGF beta 2.

In another preferred embodiment, the seventh medium is selected from the group consisting of: SFD, ham's F12, or a combination thereof.

In another preferred example, in step (d), hepSC is cultured under the third culture condition for 10-30 days, preferably, 11-20 days, more preferably, 12-19 days.

In another preferred example, in step (d 1), the HepSC is cultured under the third culturing condition for 3 to 15 days, preferably, 4 to 12 days, more preferably, 5 to 10 days.

In another preferred example, in step (d 2), the bile duct precursor cells are cultured in the seventh medium for 3 to 15 days, preferably 4 to 12 days, more preferably 5 to 10 days.

In another preferred embodiment, the method has one or more characteristics selected from the group consisting of:

(i) High HepSC differentiation rate, said differentiation rate being 50-80%, preferably 70-80%;

(ii) High hepatic parenchymal cell differentiation rate, wherein the differentiation rate is 50-80%, preferably 70-80%;

(iii) High bile duct cell differentiation rate, wherein the differentiation rate is 70-90%, preferably 80-90%;

(iv) During the culture, 2X 10 cells were inoculated per 1ml of the culture ⁵ Multiple pluripotent stem cells capable of producing 1.8 × 10 ⁶ A HepSC;

(v) During the culture, 2X 10 cells were inoculated per 1ml of the culture ⁵ Multiple pluripotent stem cells capable of producing 2 × 10 ⁶ Individual hepatic parenchymal cells;

(vi) During the culture, 2X 10 cells were inoculated per 1ml of the culture ⁵ Multiple pluripotent stem cells capable of producing 2 × 10 ⁶ Bile duct cells;

(vii) The HepSC has partial specific expression genes (such as HNF4A, AFP, TBX3, CEBPA, SOX9, HNF1 beta and CK 19) of liver parenchymal cells and bile duct epithelial cells, maintains the unlimited proliferation capacity, and can be directionally induced and differentiated into liver parenchymal cells and bile duct epithelial cells in vitro;

(viii) The parenchymal hepatic cells are closed single-layer cell balloons with hollow structures, wherein the diameter of the balloon of the parenchymal hepatic cells is 300-500 mu m, the size of the parenchymal hepatic cells on the surface of the balloon is 80-100 mu m, and the thickness of the cell layer is 8-15 mu m;

(ix) Bile duct epithelial cells are in vacuole-like forms and are connected with each other to gradually form a pipe network structure. Wherein the diameter of the bile duct network is 20-40 μm, the thickness of the bile duct network is different, and the thickness of the cell layer is 8-12 μm.

In another preferred embodiment, in steps (a) to (d), the culture system further comprises other substances for promoting differentiation, selected from the group consisting of: matrigel (Matrigel), laminin (Laminin), basement Membrane Extracts (base Membrane Extracts), or combinations thereof.

In another preferred embodiment, the method comprises therapeutic and non-therapeutic.

In another preferred embodiment, the culture system has a density of pluripotent stem cells of 1X10 ⁵ －5×10 ⁵ Cells/ml, preferably, 2X 10 ⁵ －4×10 ⁵ Cells/ml.

In another preferred embodiment, the culture system has a volume of 50-125ml, preferably 80-125ml, and most preferably 80-100ml.

In another preferred embodiment, the ratio M2/M1 of the number M2 of obtained HepSCs to the number M1 of pluripotent stem cells is 1 to 3, preferably 1 to 2, more preferably 1 to 1.5.

In another preferred embodiment, the ratio M3/M2 of the number M3 of obtained liver parenchymal cells to the number M2 of the HepSC is 1 to 3, preferably 1 to 2, more preferably 1 to 1.5.

In another preferred embodiment, the ratio M4/M2 of the number M4 of bile duct epithelial cells obtained to the number M2 of the HepSC is 1 to 3, preferably 1 to 2, more preferably 1 to 1.5.

In another preferred embodiment, the HepSC is a functional hepatic stem cell line.

In another preferred embodiment, the liver parenchymal cells are functional liver parenchymal cells.

In another preferred example, the bile duct epithelial cells are functional bile duct epithelial cells.

In another preferred embodiment, in steps (a) to (d), the culture is a suspension culture.

In another preferred example, the method further comprises:

(e) The phenotype of the cells was examined for the HepSC formed.

In a second aspect, the invention provides a Hepatic Stem Cell line (HepSC, a liver Stem Cell), wherein the HepSC is formed by differentiation of pluripotent Stem cells and has a phenotype of HNF4A +, SOX9+, AFP + and ALB-.

In another preferred example, the hepatic stem cell line has a phenotype of HNF4A +, TBX3+, SOX9+, AFP +, ALB-, CYP3A 7-.

In another preferred embodiment, the HepSC has one or more of the following characteristics:

the cell has the differentiation potential of liver parenchymal cells and bile duct epithelial cells in vivo and in vitro;

expressing the hepatic specific transcription factor HNF4A, hepatic parenchymal cell specific protein AFP;

expressing bile duct specific transcription factors SOX9 and HNF1 beta;

maintain the unlimited proliferation capacity.

In another preferred embodiment, the hepatic stem cell line is a human hepatic stem cell line.

In another preferred embodiment, the HepSC has one or more characteristics selected from the group consisting of:

(i) More than 99% of the cells have the surface antigen CD99;

(ii) The purity is high and is more than or equal to 95-99 percent;

(iii) The animal source is low, the matrigel dependence is low, and the method is independent of mouse fibroblasts;

(iv) Performing in vitro infinite amplification;

(v) No tumor is formed in the body;

(vi) The culture cost is low;

(vii) Differentiating into functional liver parenchymal cells and bile duct epithelial cells in vitro with high efficiency;

(viii) In the process of forming bile duct epithelial cells in vitro, the bile duct epithelial cells can be self-assembled into a pipe network structure through three-dimensional culture.

In another preferred embodiment, the HepSC has the ability to differentiate into hepatocytes.

In another preferred embodiment, the HepSC has the ability to differentiate into biliary epithelial cells.

The invention provides a hepatic parenchymal cell and/or a hepatic parenchymal cell population, wherein the hepatic parenchymal cell and/or the hepatic parenchymal cell population is a closed single-layer cell balloon with a hollow structure, the diameter of the balloon of the hepatic parenchymal cell is 300-500 μm, the size of the hepatic parenchymal cell on the surface of the balloon is 80-100 μm, and the thickness of a cell layer is 8-15 μm.

In another preferred embodiment, the liver parenchymal cells and/or liver parenchymal cell population has one or more characteristics selected from the group consisting of:

(a) 60% of the cells were positive for ALBUMIN expression (ALBUMIN +);

(b) 97% of the cells were positive for AFP expression (AFP +);

(c) 99% of the cells were positive for HNF4A expression (HNF 4A +).

In another preferred embodiment, the parenchymal hepatocytes and/or the parenchymal hepatic cells are prepared by the method of claim 1.

In another preferred embodiment, the liver parenchymal cells and/or the liver parenchymal cell population is obtained by induction of HepSC.

The invention provides a bile duct epithelial cell or bile duct epithelial cell population, wherein the bile duct epithelial cell or bile duct epithelial cell population is in a vacuole-like form, and is connected with each other to gradually form a pipe network structure. Wherein the diameter of the bile duct network is 20-40 μm, the thickness of the bile duct network is different, and the thickness of the cell layer is 8-12 μm.

In another preferred example, the bile duct epithelial cell or bile duct epithelial cell population has one or more characteristics selected from the group consisting of:

(a) 99% of the cells were positive for CK19 expression (CK 19 +);

(b) 90% of the cells were positive for CK7 expression (CK 7 +);

(c) 80% of the cells are positive for HNF1 BETA expression (HNF 1 BETA +);

(d) 99% of the cells were down-regulated for hepatic specific gene expression.

In another preferred embodiment, the hepatic-specific gene is selected from the group consisting of: HNF4A, AFP, ALB, CYP3A7, CYP3A4, or a combination thereof.

In another preferred embodiment, the cholangiocytes or cholangiocytes epithelial cell population is prepared by the method of claim 1.

In another preferred example, the bile duct epithelial cells or bile duct epithelial cell population is obtained by HepSC induction.

In a fifth aspect, the invention provides the use of a HepSC according to the second aspect of the invention or a population of parenchymal hepatocytes and/or hepatocytes according to the third aspect of the invention or a population of biliary epithelial cells or biliary epithelial cells according to the fourth aspect of the invention for (i) screening for a drug that promotes differentiation of the HepSC, the hepatocytes and/or the population of parenchymal hepatocytes, or the biliary epithelial cells or the population of biliary epithelial cells; and/or (ii) the prevention and/or treatment of liver-related diseases.

In another preferred embodiment, the liver-related disease is selected from the group consisting of: biliary related diseases, chronic liver failure, or a combination thereof.

In another preferred example, the bile duct related disease includes a bile duct injury disease.

In another preferred example, the bile duct related disease comprises a DDC (3, 5-diethoxycarbonyl-1, 4-dihydro-2, 4, 6-trimethylpyridine) induced bile duct injury disease.

In a sixth aspect, the present invention provides a pharmaceutical composition for preventing and/or treating liver-related diseases, wherein the pharmaceutical composition comprises: an effective amount of HepSC according to the second aspect of the invention, parenchymal hepatocytes and/or a population of parenchymal hepatocytes according to the third aspect of the invention or biliary epithelial cells or a population of biliary epithelial cells according to the fourth aspect of the invention, and a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

In another preferred embodiment, the pharmaceutical composition is a cell preparation.

In another preferred embodiment, the pharmaceutical composition is an intravenous agent.

In another preferred embodiment, the pharmaceutically acceptable carrier includes (but is not limited to): saline, buffer, glucose, and combinations thereof.

In another preferred embodiment, the concentration of the HepSC, the hepatic parenchymal cells and/or the hepatic parenchymal cell population or the biliary epithelial cell population is 1 × 10 ³ 1X10 pieces/ml ⁷ One/ml, preferably 1X10 ⁴ -1×10 ⁶ One/ml, more preferably 1X10 ⁵ Per ml-9.9X 10 ⁵ Each/ml.

In another preferred embodiment, the liver disease is selected from the group consisting of: bile duct related diseases, chronic liver failure, or a combination thereof.

The seventh aspect of the invention provides an induction medium, which contains a basic medium and additives; wherein the basal medium is selected from the group consisting of: RPMI medium 1640, SFD, HCM-cAMP, ham's F12, or a combination thereof; and, the supplement includes BMP4, A8301 and CHIR99021.

In another preferred embodiment, the additive further comprises bFGF, EGF and VEGF.

In another preferred example, the additive also comprises L-glutamine, ascorbic Acid and MTG.

In an eighth aspect, the invention provides an inducing composition comprising:

(a) A first inducing factor comprising L-glutamine, ascorbic Acid, MTG, bFGF, EGF, VEGF, BMP4, A8301, CHIR99021;

(b) Optionally a second induction factor comprising bFGF, BMP4, IWP2, a8301;

(c) Optionally a third inducing factor comprising bFGF, CHIR99021, noggin, TGF beta 2; and

(d) (ii) other substances that promote differentiation selected from the group consisting of: matrigel (Matrigel), laminin (lamin), basement Membrane Extracts (base Membrane Extracts), or combinations thereof.

In a ninth aspect, the present invention provides a use of the inducing composition according to the eighth aspect of the present invention for inducing differentiation of pluripotent stem cells into hepscs according to the second aspect of the present invention; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells according to the third aspect of the invention; and/or a cholangiocyte or cholangiocyte population according to the fourth aspect of the present invention.

The tenth aspect of the present invention provides a method for screening or determining the promotion of differentiation of pluripotent stem cells into HepSC; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or potential material of biliary epithelial cells or bile duct epithelial cell populations, comprising the steps of:

(a) Culturing pluripotent stem cells in a culture system for a period of time T1 in the presence of a test compound in a test group, and detecting the number S1 of HepSCs in the culture system of the test group; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or the number of biliary epithelial cells or bile duct epithelial cell populations S2;

and detecting the amount S3 of hepscs in said culture system in a control group in the absence of said test compound and otherwise identical conditions; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or the number of biliary epithelial cells or bile duct epithelial cell populations S4;

(b) Comparing S1 and S3 detected in the previous step, and S2 and S4, thereby determining whether the test compound is one that promotes differentiation of pluripotent stem cells into hepscs; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or potential material of biliary epithelial cells or biliary epithelial cell populations;

wherein if S1 is significantly higher than S3, and/or S2 is significantly higher than S4, it is indicative that the test compound promotes differentiation of pluripotent stem cells into hepscs; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or potential material of biliary epithelial cells or populations of biliary epithelial cells.

In another preferred example, the expression "significantly higher" means that S1/S3 is ≧ 2, preferably ≧ 3, more preferably ≧ 4.

In another preferred embodiment, the expression "significantly higher than" means that S2/S4 is not less than 2, preferably not less than 3, more preferably not less than 4.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In another preferred embodiment, the pluripotent stem cells are cells cultured in vitro.

In another preferred embodiment, the method is an in vitro method.

In an eleventh aspect, the present invention provides a method of treating liver disease comprising the steps of:

administering to a subject in need thereof a HepSC according to the second aspect of the invention, a hepatocyte or a population of hepatocyte according to the third aspect of the invention; or a cholangiocytes or cholangiocytes cell population according to the fourth aspect of the invention, or a mixture comprising hepscs according to the second aspect of the invention, or hepatocytes or a liver parenchymal cell population according to the third aspect of the invention; or a biliary epithelial cell population according to a fourth aspect of the present invention.

In another preferred embodiment, the subject is a human or non-human mammal, preferably a human.

In another preferred embodiment, the site of administration is within the hepatic portal vein of the subject.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the establishment of a novel human hepatic stem cell line; wherein the content of the first and second substances,

(A) Schematic representation of HepSC in establishment and maintenance.

(B) Morphograms of HepSC at passage 15. Scale bar: 200um.

(C) And detecting the characteristic gene expression of the HepSC by using qRT-PCR. The data are relative values to the expression level of the housekeeping gene TBP. HepSC down-regulates definitive endoderm characteristic genes EOMEs and SOX17, up-regulates hepatic characteristic genes HNF4A, TBX3 and AFP, and does not express characteristic gene ALB of mature liver; bile duct epithelial cell characteristic genes HNF1 beta, SOX9 and CK19 are up-regulated, but a mature bile duct epithelial cell characteristic gene CK7 is not expressed.

(D) And (3) detecting the expression of the HepSC characteristic gene at the protein level by using a cell flow method. HepSC is positive for a group of CK19, SOX9, HNF4A, negative for ALB, and AFP expression has some heterogeneity in HepSC. The cell is shown to have characteristic genes of liver parenchymal cells and bile duct epithelial cells at the same time, and has certain heterogeneity.

(E) And (3) detecting the expression of the characteristic gene of the HepSC at the protein level by using cellular immunofluorescence. Hepscs were a population FOXA1, FOXA2, CK19, SOX9, HNF4A positive, SOX17 and ALB negative. The cell is shown to have the characteristic genes of the liver parenchymal cell and the bile duct epithelial cell at the same time, and does not express the characteristic gene of mature liver parenchymal cell and the characteristic gene of definitive endoderm. And (C) graph qPCR data.

(F) Karyotype identification map of HepSC thirty th generation cells. Indicating that the cells still maintain diploid karyotype stability.

(G) Proliferation curves of HepSC from first generation to thirtieth generation.

(H) And (5) verifying a HepSC tumorigenicity experiment. HepSC were transplanted into neck skin or medial thigh muscle of immunodeficient mice Scid Beige, and the data indicated that HepSC did not have tumorigenicity compared to the control group.

(I) And (5) verifying the self-differentiation potential of the HepSC. (H) The transplanted cells were removed at about 4 weeks, cryosectioned, and immunofluorescent-stained. The data show that the cell mass retains the bile duct characteristic gene SOX9 at the transplantation site and expresses the characteristic gene CFTR of the mature bile duct; the characteristic gene HNF4A of the liver parenchymal cells is lost. It was shown that HepSC self-differentiated in vivo to form biliary epithelial cells.

FIG. 2 shows the efficient in vitro directed induced differentiation of a novel human hepatic stem cell line into functional liver parenchymal cells, wherein,

(A) Schematic representation of HepSC liver differentiation towards directed induction.

(B) HepSC induced the formation of a morphogram of hepatocytes. Scale bar: 200um.

(C) The qRT-PCR was used to detect the characteristic gene expression of HepSC induced hepatocyte formation. The data are relative values to the expression level of the housekeeping gene TBP. When HepSC induces the formation of hepatoblasts, the hepatic parenchymal cell characteristic genes AFP, ALB and HNF4A are up-regulated; characteristic genes HNF1 beta, SOX9 and the like in the bile duct direction are down-regulated. When the mature hepatic parenchymal cells are induced to form, the immature hepatic characteristic gene AFP is regulated down, and characteristic genes of the mature hepatic parenchymal cells, such as ALB, HNF4A, CYP3A7 and the like, are maintained or improved.

(D) And (3) performing cell flow detection on the expression of the characteristic genes of the liver parenchymal cells induced by HepSC at the protein level. HepSC-derived hepatic parenchymal cells were shown to be AFP + ALB + (efficiency at 60-70%).

(E) Cell immunofluorescence detects the expression of HepSC induced liver parenchymal cell characteristic genes at the protein level. The data show that the induction of the formed parenchymal hepatic cells expresses characteristic genes HNF4A, AFP and ALB of the parenchymal hepatic cells, and the expression of the polar protein E-cadherin also indicates that the three-dimensional hepatocyte spheroids have certain polarity. Scale bar: 100um.

FIG. 3 shows the in vitro high-efficiency directional induced differentiation of a novel human hepatic stem cell line into functional bile duct epithelial cells, wherein,

(A) Schematic diagram of directional induction of differentiation of HepSC biliary epithelial cells.

(B) HepSC induced the formation of a morphology map of biliary epithelial cells. The images show that the cells are gradually connected into a pipe network structure in the matrigel, and form a shape similar to the intrahepatic bile duct network. Scale bar: 200um.

(C) And detecting the characteristic gene expression of the bile duct epithelial cells induced by the HepSC by using qRT-PCR. The data are relative values to the expression level of the housekeeping gene TBP. When HepSC induces and forms bile duct precursor cells, hepatic characteristic genes HNF4A and AFP are down-regulated, bile duct characteristic genes SOX9 and CK19 and the like are up-regulated, but bile duct mature genes are still not expressed; when bile duct epithelial cells are further induced to form, the cells further down-regulate characteristic genes HNF4A, AFP and ALB in the hepatic direction, and maintain genes such as characteristic genes SOX9, HNF1 beta and CK19 in the bile duct direction. And relevant genes HES1 and NOTCH2 expressing a NOTCH signal pathway which is important in bile duct development are up-regulated. Indicating that HepSC indeed has cholangiogenic differentiation potential in vitro.

(D) And detecting the expression of characteristic genes of bile duct epithelial cells induced by HepSC at the protein level by using a cell flow method. HepSC-derived liver parenchymal cells were shown to be SOX9+ CK7+ (efficiency at 80-90%).

(E) And detecting the expression of characteristic genes of bile duct epithelial cells induced by HepSC at the protein level by using cellular immunofluorescence. The data show that the characteristic genes SOX9 and CK7 of the primary bile duct epithelial cells are expressed by inducing the formation of a bile duct ductal network structure. Scale bar: 100um

FIG. 4 shows the integration of a novel human hepatic stem cell line transplanted into the liver of a bile duct-injured mouse, wherein,

(A) Schematic diagram of mouse bile duct injury model construction and cell transplantation.

(B) And (4) performing fluorescence imaging on liver tissues. The pictures show that HepSC is well integrated in mouse liver tissue.

(C) Mouse liver tissue frozen section immunofluorescence staining map. The panels show that integrated HepSC formed a good luminal structure in the mouse liver and that the integrated cells did not express the hepatic signature gene HNF4A. Scale bar: 200um.

Detailed Description

The present inventors have conducted extensive and intensive studies and, for the first time, have unexpectedly found that, by culturing pluripotent stem cells in a culture system under the first culture conditions of the present invention, hepscs having a hepatic and cholangiogenic bidirectional differentiation potential with a specific cell phenotype can be obtained, and that hepscs can be directionally induced to differentiate in vitro by a combination of small molecules and cytokines to form mature functional hepatocytes and cholangiocytes with specific structures. The liver parenchymal cells can be used for specific treatment of liver-related diseases and the like or screening of drugs for treating the liver-related diseases. The bile duct epithelial cells of the invention can be used for the construction of tissue engineering bile duct networks and the cell therapy of bile duct diseases.

In addition, the invention also unexpectedly discovers that the differentiation rate of the HepSC is more than 50%, the differentiation rate of mature parenchymal hepatocytes is more than 60%, and the differentiation rate of biliary epithelial cells is more than 86%. On the basis of this, the present inventors have completed the present invention.

Specifically, starting from human ES or iPS cells, differentiation is carried out to form definitive endoderm, and then the definitive endoderm cells can be directionally induced to differentiate under the culture conditions of bFGF, EGF, VEGF, BMP4, A8301 and CHIR99021, and can be maintained in a certain state to establish a cell lineage.

Taking the HepSC as a starting point, a three-dimensional suspension differentiation system can be used for directionally inducing and differentiating in vitro to form mature hepatic parenchymal cells. And can utilize a three-dimensional 'glue dripping' differentiation system to directionally induce and differentiate to form bile duct epithelial cells. The HepSC has important significance for liver tissue engineering and cell therapy of liver diseases.

Term(s) for

Hepatoblasts

In the invention, hepSC forms bipotent hepatic progenitors which generate cholangiocytes and hepatic parenchymal cells under specific factors of the hepatic aspect.

The hepatoblasts are closed monolayer cell balloons with hollow structures, wherein the diameter of the balloon of the hepatoblasts is 300-500 mu m, the size of the hepatoblasts on the surface of the balloon is 60-80 mu m, and the thickness of the cell layer is 6-12 mu m.

Parenchymal hepatic cells

As used herein, the terms "hepatocyte", "hepatocyte population", and "hepatocyte" are used interchangeably, as not to refer to a single cell.

In the present invention, hepatoblasts form parenchymal cells having liver functions under the action of hepatocyte maturation-inducing factors.

The parenchymal hepatic cell is a closed monolayer cell balloon with a hollow structure, wherein the diameter of the balloon of the parenchymal hepatic cell is 300-500 mu m, the size of the parenchymal hepatic cell on the surface of the balloon is 80-100 mu m, and the thickness of the cell layer is 8-15 mu m.

Bile duct precursor cell

In the invention, under the induction of specific factors in bile duct, hepSC forms progenitor cells with the potential of generating bile duct epithelial cells.

The bile duct precursor cells of the invention are in vacuole-like forms and are connected with each other to gradually form a pipe network structure. Wherein the diameter of the bile duct network is 20-40 μm, the thickness of the bile duct network is different, and the thickness of the cell layer is 8-12 μm.

Bile duct epithelial cells

As used herein, the terms "biliary epithelial cell", "biliary epithelial cell population", and "bile duct epithelial cell population", are used interchangeably, as non-specific to a single cell.

In the invention, bile duct precursor cells form a bile duct network with functions and molecular characteristics of mature primary bile ducts under the action of bile duct cell maturation induction factors.

The bile duct precursor cells are in vacuole-like forms and are connected with each other to gradually form a pipe network structure. Wherein the diameter of the bile duct network is 20-40 μm, the thickness of the bile duct network is different, and the thickness of the cell layer is 8-12 μm.

As used herein, the term "A8301" has the formula C25H19N5S with the CAS number 909910-43-6."CE" has the formula C27H24F2N4O3, CAS number 209986-17-4. The "EGFi" has the formula C22H23N3O4.HCl, CAS No. 183319-69-9."CHIR99021" has the chemical formula of C22H18Cl2N8 and CAS number of 252917-06-9."IWP2" has the formula C22H18N4O2S3 and CAS number 686770-61-6. The "ROCKI" formula is C14H21N3O, CAS number 146986-50-7.

Pluripotent stem cells

Starting seed cells established by HepSC as described in the present invention. Pluripotent Stem Cells (Pluripotent Stem Cells) are a class of Pluripotent Cells that have the ability to self-renew and self-replicate. Under certain conditions, pluripotent stem cells (HSCs) have the potential to differentiate into a variety of cellular tissues. Cells from two sources, the inner cell mass at the blastocyst stage of the human embryo; one is induced pluripotent stem cells obtained by reprogramming adult cells.

Induction culture method of hepatic stem cell line, hepatic parenchymal cell and bile duct epithelial cell

The starting cells of the hepatic stem cell line are human pluripotent stem cells, and the hepatic stem cell line is obtained by culturing the starting cells under a first culture condition (such as containing a first culture medium), wherein a culture system under the first culture condition comprises L-glutamine, ascorbic Acid, MTG, bFGF, EGF, VEGF, BMP4, A8301 and CHIR99021.

Then, further culturing the HepSC under conditions suitable for culturing (e.g., in the presence of a third medium and additives including bFGF, BMP4, IWP2, a 8301) to obtain hepatocytes; or

The HepSC is cultured under conditions suitable for culture (e.g., in the presence of a sixth medium and additives including bFGF, CHIR99021, noggin, TGF β 2) to obtain biliary epithelial cells.

In a preferred embodiment, the culture system of the present invention further comprises an additional substance for promoting differentiation selected from the group consisting of: matrigel (Matrigel), laminin (Laminin), basement Membrane Extracts (base Membrane Extracts), or combinations thereof.

In a preferred embodiment, the method of the present invention for inducing differentiation of human pluripotent stem cells into HepSC lineage is described in general materials method 8.

In a preferred embodiment, the RPMI medium 1640 medium is commercially available (available from GIBCO, inc.).

Ham's F12 medium is commercially available (from Cellgro).

Screening or determining the lines that promote the differentiation of pluripotent stem cells into hepatic stem cells; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or potential material of biliary epithelial cells or bile duct epithelial cell populations

In the present invention, a screen or assay is provided that promotes differentiation of pluripotent stem cells into hepscs; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or potential material of biliary epithelial cells or bile duct epithelial cell populations, comprising the steps of:

(a) Culturing pluripotent stem cells in a culture system for a period of time T1 in the presence of a test compound in a test group, and detecting the number S1 of HepSCs in the culture system of the test group; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or the number of biliary epithelial cells or bile duct epithelial cell populations S2;

and detecting the number S3 of hepscs in said culture system in a control group in the absence of said test compound and otherwise identical conditions; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or the number of biliary epithelial cells or bile duct epithelial cell populations S4;

(b) Comparing S1 and S3 detected in the previous step, and S2 and S4, to determine whether the combination of test compounds promotes differentiation of pluripotent stem cells into hepscs; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or a cholangiocyte or cholangiocyte population;

wherein if S1 is significantly higher than S3 and S2 is significantly higher than S4, it is indicative that the test compound promotes differentiation of pluripotent stem cells into hepscs; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or a cholangiocyte or cholangiocyte population.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

The main advantages of the invention include:

(1) The invention discovers for the first time that functional HepSC with extremely high differentiation rate (as high as 80-90%) can be obtained by culturing the pluripotent stem cells in a culture system; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or biliary epithelial cells or populations of biliary epithelial cells, and the hepscs of the invention have a particular cellular phenotype. The parenchymal hepatic cells and/or the parenchymal hepatic cell population are closed single-layer cell balloons with hollow structures, wherein the diameter of the balloon of the parenchymal hepatic cells is 300-500 mu m, the size of the parenchymal hepatic cells on the surface of the balloon is 80-100 mu m, and the thickness of the cell layer is 8-15 mu m. Bile duct epithelial cells or bile duct epithelial cell populations are in vacuole-like forms and are connected with one another to gradually form a pipe network structure. Wherein the diameter of the bile duct network is 20-40 μm, the thickness of the bile duct network is different, and the thickness of the cell layer is 8-12 μm.

And a functional HepSC of the invention; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or the purity of the bile duct epithelial cells or bile duct epithelial cell populations is also very high.

(2) The invention firstly establishes a HepSC line in vitro, the stem cell line reserves the differentiation potential of parenchymal hepatocytes and bile duct epithelial cells in vitro, has unlimited proliferation capacity, and keeps the chromosome diploid karyotype stable after being cultured for 30 generations. The method is independent of mouse-derived fibroblasts, has low culture cost and high safety, is closer to parenchymal hepatocytes and bile duct epithelial cells in a development path, only has the differentiation potential of the parenchymal hepatocytes and the bile duct epithelial cells, and is a good differentiation starting point of the parenchymal hepatocytes and the bile duct epithelial cells.

(3) The invention firstly induces and differentiates the novel HepSC to obtain mature liver parenchymal cells. Provides a good cell source for cell therapy and drug screening of liver tissue engineering and liver diseases.

(4) The invention firstly obtains mature bile duct epithelial cells from the novel HepSC by induced differentiation, and firstly constructs a continuous bile duct network in vitro. Provides a good cell source for cell therapy and drug screening of liver tissue engineering and bile duct diseases.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Unless otherwise indicated, reagents and materials used in the examples of the present invention were all commercially available products.

Versatile materials and methods

1. Laboratory animal

All animals involved in this chapter were housed in SPF animal houses. SCID-Beige mice were purchased from Shanghai Ling. All animal experimental procedures were performed strictly in accordance with the regulations of the animal care committee of the biochemical cell institute.

2. Mouse bile duct injury model construction and cell transplantation

RG mice at 6-8 weeks were fed with DDC drug-containing feed starting four days before cell transplantation and maintained on DDC feed at a later stage. For cell transplantation, spleen transplantation was performed, and cells (transplanted cells are HepSC with green fluorescent protein EGFP) were resuspended in HepSC cell culture medium and 1uM ROCki was added, and the cell concentration was controlled to 1-2x 10 ⁶ Cells/100 ul, 1-2x 10 per mouse injection ⁶ A cell. And mice were sacrificed around week 4 and liver tissue was taken for analysis.

In the case of cell transplantation, after the mice were anesthetized with isoflurane gas, their livers were exposed after the abdomen was opened, the spleen was exposed by pulling fat attached to the spleen, the front end of the spleen was ligated with silk thread, and the cell suspension was injected from the spleen with an insulin needle. After completion of the injection, the spleen and the like were returned to their original positions, and the abdomen was sutured.

3. Frozen tissue section

Fresh liver was fixed with 4% PFA overnight at 4 deg.C, then washed with DPBS for 30 minutes on a shaker, then transferred to 30% sucrose solution (diluted with DPBS) for dehydration overnight at 4 deg.C, then washed with DPBS on a shaker for 30 minutes, embedded with OCT in a tissue embedding frame, and the tissue frozen at-80 deg.C. Then, the film is sliced by an ice cutter to be 8-10um thick, stuck on a glass slide and stored at minus 80 ℃. For staining, the slides were removed, baked at 55 ℃ for 15 minutes, and then washed with PBST (0.1% Triton-X-100 inPBS) at room temperature for 20 minutes. Blocking was performed with blocking solution 2% BSA (PBST dilution) at room temperature for 1 hour. Subsequent staining was identical to immunofluorescence staining.

4.QPCR detection

All cell samples or tissue samples were subjected to qPCR (Rhoche) by SYBR method using a microtool (purchased from Tiangen).

5. Flow cytometric analysis

Staining cell surface protein, digesting cell sample into single cell with pancreatin, re-suspending 5 × 104-1 × 105 cells with corresponding antibody work liquid, incubating on ice for 30min, washing with FACS buffer solution for 3 times, re-suspending with 300 μ l FACS buffer solution, protecting from light on ice, and loading onto machine as soon as possible. Most of the existing surface protein antibodies in a laboratory are directly marked by fluorescein, if secondary antibody is needed to be marked, the primary antibody is incubated and washed, then secondary antibody working solution is continuously incubated, and the subsequent operation is consistent.

Cell flow antibody usage ratio: the CK7 antibody was used at a concentration of 1, the ck19 antibody was used at a concentration of 1, the sox9 antibody was used at a concentration of 1. The antibody dilution was 0.1% BSA in DPBS.

6. Intracellular protein staining

After the cell sample was trypsinized into single cells, 1X 106 cells were fixed with 1.6% PFA 37 ℃ for 30min, washed 3 times with FACS buffer, and 500. Mu.l FACS buffer was resuspended and stored at 4 ℃ (the fixed cell sample can be stored for up to 1 month). During the staining procedure 1 × Saponin buffer was used, all antibodies were diluted with 1 × Saponin buffer, primary antibody was incubated for 30 minutes at room temperature, 20 × Saponin buffer was washed 3 times, secondary antibody was incubated for 30 minutes in the dark, 1 × Saponin buffer was washed 3 times, 300 μ l FACS buffer was resuspended, protected from light on ice, and loaded as soon as possible. All intracellular proteins were stained with isotype controls. Flow cytometry was purchased from BD and model LSR ii. Flowjo software is used for streaming data.

7. Immunofluorescence staining

The cells or tissues were first fixed with 4% PFA 37 ℃ for 1 hour, and then incubated for permeabilization at room temperature for 10 minutes with PBS buffer containing 0.5% TritonX-100 (purchased from Sigma) 0.05% BSA. 30 then washed 3 times with PBS and blocked with 3% BSA solution at room temperature for 1 hour. After incubation of the primary antibody at 4 ℃ overnight or at room temperature for 2 to 3 hours, it was washed three times with PBS and incubated for 1 hour with the secondary antibody. After 3 washes with PBS, they were stained with ready-to-stain DAPI for 5 minutes. Photographs were taken using a Leica TCS SP5 laser confocal microscope.

Immunofluorescence antibody use ratio:

the FOXA1 antibody was used at a concentration of 1, the FOXA2 antibody was used at a concentration of 1, the soxh17 antibody was used at a concentration of 1. The antibody dilution was 0.1% BSA in DPBS.

Establishment of HepSC

The starting cells are human pluripotent stem cell lines. Cells were first plated in six-well plates using Matrigel 1. The human pluripotent stem cells that grew out of the six-well plate were passaged according to the ratio of 1. 2-3days were cultured after passage, and ESC/iPSC clones grown to 80-90% density were used for DE differentiation, with the following specific culture conditions:

day 1 to day 2, second A medium (such as RPMI 1640 medium) was supplemented with L-glutamine (1X, cellgro), MTG (4.5X 10-4M), activin A (10 ng/ml), CHIR99021 (3 uM).

Day 2 to day 4, second B medium (such as RPMI 1640 medium) was supplemented with L-glutamine (1X, cellgro), ascorbic Acid (50. Mu.g/ml, wako, cat # 013-19641), MTG (4.5X 10) ^-4 M)，bFGF(5ng/ml)，hVEGF(10ng/ml)，Activin A(10ng/ml)，hBMP4(0.25ng/ml)。

From day 4 to day 6, the second C medium (such as SFD medium) was supplemented with L-glutamine (1X, cellgro), ascorbic Acid (50. Mu.g/ml, wako, cat # 013-19641), MTG (4.5X 10) ^-4 M)，bFGF(5ng/ml)，hVEGF(10ng/ml)，Activin A(10ng/ml)，hBMP4(0.25ng/ml)。

DE differentiation yielded definitive endoderm cells. HepSC can be established under specific culture conditions starting from definitive endoderm cells, as follows:

definitive endoderm cells were first plated at 2 × 10 using Matrigel seeded six-well plates of 1 ⁵ Cell density per well was seeded into culture plates and passed into six well plates as described above. The medium was SFD medium supplemented with L-glutamine (1X, cellgro), ascorbic Acid (50. Mu.g/ml, wako, cat # 013-19641), MTG (4.5X 10) ^-4 M), bFGF (10 ng/ml), hVEGF (10 ng/ml), hEGF (10 ng/ml), hBMP4 (50 ng/ml), A8301 (5 uM), CHIR99021 (3 uM). After 3-4 days of culture at 37 ℃, the clones were passaged when they grew to 80-90% density. At the time of cell passage, trypsin was digested at 0.25% at 37 ℃ for 3-5 minutes, digestion was terminated with FBS of 1/2 digestive enzyme volume, after blowing to single cells, centrifuged at 1300rpm for 3 minutes, after removing the supernatant, resuspended with fresh above medium, inoculated into the above described plate, and passage was completed. After passage 3-5, hepSC was stably established.

9. Directional induction and differentiation of liver parenchymal cells

Novel HepSC (1.2-2X 10) ⁶ Individual cells/plate) were cultured on matrix (Corning, cat # 354230) coated cell culture plates with HepSC medium. After the HepSC confluent cell culture plates, they were digested with 0.25% trypsin at 37 ℃ for 5-7 minutes, and then single cells were blown out by mechanical force and plated in six well plates (Corning, cat # 3471) with 1X10 cells per well ⁶ Cells/ml, 3ml of medium per well, 10ul/ml Matrigel was added to the medium. The cell plate was placed at 150rpmCulturing on a shaker in a hypoxic incubator. The solution was changed every 2 days.

From day 1 to day 6, SFD medium was supplemented with L-glutamine (1X, cellgro), ascorbic Acid (50. Mu.g/ml, wako, cat # 013-19641), MTG (4.5X 10) ^-4 M), hBMP4 (50 ng/ml), bFGF (10 ng/ml), IWP2 ((0.5 mM), A8301 (5 uM), ROCKI (1 uM). Directed differentiation into hepatoblasts. The hepatoblasts are closed monolayer cell balloons with hollow structures, wherein the diameter of the balloon of the hepatoblasts is 400 mu m, the size of the hepatoblasts on the surface of the balloon is 70 mu m, and the thickness of the cell layer is 8 mu m.

Day 6 to day 12, the SFD medium was supplemented with L-glutamine (1X, cellgro), ascorbic Acid (50. Mu.g/ml, wako, cat # 013-19641), MTG (4.5X 10) ^-4 M), HGF (25 ng/ml), OSM (20 ng/ml), dexamethasone (40 ng/ml), vitamin K1 (6 g/ml, sangon), C-E (0.1 uM), A83-01 (0.5 uM), EGFi (2 uM) differentiated into immature hepatocytes.

From day 12 to day 18, HCM medium (Lonza) was supplemented with L-glutamine (1X, cellgro), ascorbic Acid (50. Mu.g/ml, wako, cat # 013-19641), MTG (4.5X 10) ^-4 M), HGF (25 ng/ml), OSM (20 ng/ml), dexamethasone (40 ng/ml), vitamin K1 (6 g/ml, sangon), C-E (0.1 uM), A83-01 (0.5 uM), EGFi (2 uM), differentiation into hepatocyte cells to give 6X 10 ⁶ The liver parenchyma cells are closed single-layer cell balloons with hollow structures, wherein the balloon diameter of the liver parenchyma cells is 400 microns, the balloon surface liver parenchyma cells are 90 microns, and the cell layer thickness is 10 microns.

All related small molecule compounds are purchased from seleck, and the small molecule compounds are prepared according to the instruction. Cytokines were purchased from RD and the lysis method was according to the instructions.

In the present invention, the basic medium is SFD and HCM (platelet culture medium). SFD (Serumfree differentiation medium) comprises the following components: 75% Iscove's Modified Dulbecco's Medium (IMDM) (Cellgro), supplemented with 1% N2 and B27 supplements (Gibco-BRL), 1% penicillin/streptomycin, 0.05% bovine serum albumin in 25% ham's F12 medium (Cellgro).

HCM medium is commercially available (Commercial medium: lonza:15 CC-4182) (Ogawa, S., surapitchat, J., virtanen, C., ogawa, M., niapour, M., sugamori, K.S., zhao, B. (2013), three-dimensional culture and signaling promoter the reaction of human multiple step cell-derived procedure. Development,140 (15), 3285-3296.).

10. Directional induced differentiation of bile duct epithelial cells

Novel HepSC (1.2-2X 10) ⁶ Individual cells/plate) were cultured on Matrigel (Corning, cat # 354230) coated cell culture plates with HepSC medium. After HepSC overgrows the cell culture plate, it is digested with 0.25% trypsin at 37 ℃ for 5-7 minutes, then mechanically blown into single cells, resuspended in Matrigel at a density of 15X106 cells per ml, controlled at 20. Mu.l per drop, and after coagulation at 37 ℃, cultured in a hypoxic incubator. The solution was changed every 2 days.

Day 1 to day 2, SFD medium was supplemented with L-glutamine (1X, cellgro), ascorbic Acid (50. Mu.g/ml, wako, cat # 013-19641), MTG (4.5X 10) ^-4 M)，hBMP4(50ng/ml)，bFGF(10ng/ml)，hVEGF(10ng/ml)，hEGF(10ng/ml)，A8301(5uM)，CHIR99021(3uM)，ROCKi(1uM)。

Day 2 to day 8, the SFD medium was supplemented with L-glutamine (1X, cellgro), ascorbic Acid (50. Mu.g/ml, wako, cat # 013-19641), MTG (4.5X 10) ^-4 M), bFGF (10 ng/ml), noggin (100 ng/ml), CHIR99021 (3 uM), TGF beta 2 (0.1 ng/ml). Bile duct precursor cells are obtained.

Day 8 to day 16, the SFD medium was supplemented with L-glutamine (1X, cellgro), ascorbic Acid (50. Mu.g/ml, wako, cat # 013-19641), MTG (4.5X 10) ^-4 M), HGF (25 ng/ml), EGF (50 ng/ml), TGF beta 2 (0.1 ng/ml). And (4) obtaining bile duct epithelial cells, wherein the cells form vacuole-like forms and are connected with each other to gradually form a pipe network structure. Wherein the diameter of the bile duct network is 20-40 μm, the thickness of the bile duct network is different, and the thickness of the cell layer is 8-12 μm.

All related small molecule compounds are purchased from seleck, and the small molecule compounds are prepared according to the instruction. Cytokines were purchased from RD and the lysis method was according to the instructions.

Example 1 establishment and characterization of HepSC cell lines

The HepSC cell line was obtained by directed differentiation of human pluripotent stem cells (fig. 1. A-B); the combination of special small molecule CHIR99021 etc. with cytokine bFGF etc. can maintain HepSC at a developmental stage similar to hepatoblasts, in culture conditions not dependent on any feeder cells and Matrigel (FIG. 1. C-E); hepSC expresses key hepatic transcription factors HNF4A and TBX3, hepatoblast marker AFP, biliary transcription factors CK19, SOX9 and HNF1B, but does not express mature hepatic parenchyma or cholangiocellular marker ALB or P450 family protein, nor express mature cholangiocellular marker genes ASBT or GGT1, etc. (fig. 1. C-E). HepSC can be propagated indefinitely (> P70) in vitro and maintain phenotypic and genomic stability for long periods (fig. 1. F-G), and subclone lines can be created from a single HepSC cell, a truly significant stem cell. HepSC has no in vivo neoplasia (fig. 1. H), and in immune-deficient mice, can self-differentiate to form luminal structures which do not express the hepatic specific transcription factor HNF4A, but up-regulate the expression amount of bile duct epithelial cell specific expression genes CFTR, ASBT, AQP1 and the like on the basis of retaining bile duct epithelial cell transcription factor SOX 9. (FIG. 1. I)

Example 2 in vitro establishment of hepatic differentiation System of HepSC

At present, the applicant has established an in vitro hepatic differentiation system of HepSC, which can efficiently prepare liver parenchyma (fig. 2-3).

First, we established a three-dimensional suspension differentiation system of HepSC hepatocytes (fig. 2. A-B), the differentiation process including two major stages of hepatoblasts and hepatocytes. As differentiation progresses, hepSC gradually up-regulates the hepatic parenchymal cell characteristic genes AFP, ALB and CYP3A7, while down-regulating the biliary transcription factors HNF1B and SOX9, on the basis of maintaining the hepatic key transcription factors HNF4A; the hepatoblast characteristic factor AFP was significantly down-regulated in mature hepatic parenchymal cells (fig. 2. C). In the final stage, hepatic parenchymal cells with AFP + ALB + accounted for 60% of the total number, and mature hepatic parenchymal cells without AFP-ALB +, indicating that this system is in need of improvement in terms of efficiency in obtaining mature hepatic parenchymal cells (fig. 2. D-E).

Secondly, we established the bile duct-directed differentiation system of HepSC in vitro (fig. 3. A), using Matrigel encapsulated cell culture (fig. 3. A), and HepSC spontaneously formed dense bile duct network structures during differentiation (fig. 3. B). HepSC-derived biliary epithelial cells significantly down-regulated the hepatic marker genes HNF4A, AFP and ALB, up-regulated or maintained the biliary epithelial marker genes SOX9, HNF1B, CK19, CK7 and GGT1 and AE2 (fig. 3. C-E).

Example 3 integration of HepSC in DDC-induced bile duct injury mouse model

Prophase data indicate that HepSC possess the differentiation potential of hepatic parenchymal and biliary epithelial cells in vitro and auto-differentiate into biliary epithelium-like cells in vivo, either subcutaneously or intramuscularly in immunodeficient mice (figure 1. I). We further evaluated the integration and differentiation capacity after HepSC transplantation using DDC-induced chronic bile duct injury mouse model (fig. 4. A). The rat liver one month after transplantation was observed under a fluorescence microscope and it was found that HepSC was efficiently integrated into the host and formed bile duct-like structures (fig. 4. B). Integrated GFP + cells did not express the hepatic critical transcription factor HNF4A (fig. 4. C). These data preliminarily indicate that the HepSC has integration and differentiation capacity after being transplanted into the body of the mouse with the chronic bile duct injury, and therefore has the potential of being directly used for the replacement therapy of the liver disease cells.

Discussion of the related Art

Compared with the existing endoderm cell line maintained in vitro, the new HepSC is established and maintained in vitro for the first time, has a plurality of advantages, such as independence of stroma and helper cells of many animal sources, no tumorigenicity, closer development path to adult parenchymal cells and bile duct epithelial cells, and shorter time period for obtaining the adult cells in the final stage. Therefore, the cell line has great value and significance in the fields of tissue engineering and cell therapy.

The data of the invention show that the HepSC also retains the characteristic genes or transcription factors SOX9, HNF1 beta, CK19 and the like of biliary duct epithelial cells besides the characteristic gene or transcription factor HNF4A of the hepatic parenchymal cells. But do not express functional genes characteristic of the mature liver parenchyma, such as ALB and CYP enzyme series genes. At the same time, ki67 was almost all positive, indicating that the cells were in the cell cycle and were proliferating. This also corresponds to the molecular characteristics of its HepSC. Further, it is desirable to know what state the cell line is in at the single cell level, whether a cell population in such a cell state is also present in fetal liver or adult liver. And what the maintenance mechanism is different from that of the existing endoderm cell lines maintained in vitro.

The potential of this novel HepSC for hepatic parenchymal differentiation also has great clinical value. Liver transplantation is currently the most effective means for the treatment of severe liver diseases, and liver parenchymal cell transplantation has also shown positive efficacy as an alternative means for the treatment of various liver diseases. Human primary hepatocytes are the only cell type currently used for cell replacement therapy of liver diseases. However, the development of primary liver parenchymal cell transplantation is severely hampered by donor liver quality and quantity problems. Therefore, how to obtain large-scale hepatic parenchymal cells in vitro still remains an urgent problem to be solved. Even though the final-stage parenchymal hepatic cells obtained by differentiation of the HepSC and the primary liver have a certain distance, the unlimited capacity of the novel HepSC and the characteristic of low maintenance cost of the novel HepSC also provide a good idea for solving the problem in the future.

The research field of bile ducts is at a newer stage at present, and the main research work focuses on two aspects, namely the problem of cell source of bile duct epithelial cells, which relates to the establishment of bile duct disease models in vitro for drug screening or cell therapy; one is how to realize hepatocholangization when constructing the engineered tissue liver, which also relates to the tissue engineering of bile duct. The HepSC established by the invention can well meet the two problems.

Firstly, the HepSC is derived from human pluripotent stem cells, so that the ethical problem can be avoided; secondly, the pluripotent stem cells of the origin or the HepSC have unlimited proliferation capacity, and the cell line has low in-vitro culture cost, so that the problem of large-scale cell culture can be well solved; the cells have good bile duct differentiation potential in vitro, and can well solve the problem of donor bile duct cell source by combining the convenience of large-scale amplification, thus being beneficial to constructing bile duct related disease models in vitro and being used for drug screening.

Our data show that the HepSC has good differentiation potential and integration capability of bile duct epithelial cells in an in vivo mouse environment, and this also provides a thought for the direct cell therapy of bile duct diseases by using the stem cells. We currently expect two bile duct diseases that the HepSC can treat. One is drug-induced bile duct cell damage; one is genetic diseases, such as natural biliary atresia syndrome. The former has damage to bile duct epithelial cells, damage to the bile duct system, and incapability of bile excretion, resulting in liver damage; the latter is deficient in the native biliary system and fails to excrete bile. Both require bile duct epithelial cell repair and even remodeling of the biliary system that can integrate. The HepSC is a good source of cells.

The data of the invention show that the cell line has good capacity of self-assembling into a pipe network structure in vitro, which is the purpose that people want to realize the in vitro construction of the complex bile duct network by means of a tissue chip or a micro-fluidic or 3D printing technology. At present, liver tissue engineering focuses more on the construction of liver parenchymal cells, and rarely relates to a bile duct system, but an intrahepatic bile duct plays an important role in assisting proliferation and differentiation and excreting bile in development and physiological processes. Therefore, the integration of the bile duct system into the tissue engineering liver is of great significance.

At present, the HepSC has certain defects in vitro differentiation of bile ducts, namely, the maturity of the obtained terminal bile duct epithelial cells and primary bile duct epithelial cells have certain distances, and further optimization of differentiation conditions is needed. But the differentiation potential in vivo and the integration capability in vivo of the cell line allow the cell line to have very high clinical use potential.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (10)

1.A method of inducing differentiation of pluripotent stem cells into hepatic stem cell lines comprising the steps of:

(a) Culturing pluripotent stem cells in a culture system under first culture conditions, thereby obtaining a hepatic stem cell line (HepSC); wherein, the culture system comprises: l-glutamine, ascorbic Acid, MTG, bFGF, EGF, VEGF, BMP4, A8301, CHIR99021.

2.A liver Stem Cell line (HepSC), wherein the HepSC is formed by differentiation of pluripotent Stem cells and has the phenotype of HNF4A +, SOX9+, AFP + and ALB-.

3. The parenchymal liver cells and/or the parenchymal liver cell population are closed single-layer cell balloons with hollow structures, wherein the diameter of the balloons of the parenchymal liver cells is 300-500 mu m, the size of the parenchymal liver cells on the surfaces of the balloons is 80-100 mu m, and the thickness of the cell layer is 8-15 mu m.

4.A bile duct epithelial cell or bile duct epithelial cell population, characterized in that the bile duct epithelial cell or bile duct epithelial cell population is vacuole-like and connected with each other to gradually form a pipe network structure. Wherein the diameter of the bile duct network is 20-40 μm, the thickness of the bile duct network is different, and the thickness of the cell layer is 8-12 μm.

5. Use of the HepSC of claim 2 or the population of hepatocytes and/or hepatocytes of claim 3 or the population of cholangiocytes or cholangiocytes of claim 4 for (i) screening for a drug that promotes differentiation of the HepSC, the hepatocytes and/or the population of hepatocytes or cholangiocytes or the population of cholangiocytes; and/or (ii) the prevention and/or treatment of liver-related diseases.

6. A pharmaceutical composition for preventing and/or treating liver-related diseases, comprising: an effective amount of the HepSC of claim 2, the parenchymal liver cells and/or cell populations of claim 3 or the cholangiocytes or cell populations of bile duct epithelium of claim 4, and a pharmaceutically acceptable carrier.

7. An induction medium comprising a basal medium and an additive; wherein the basal medium is selected from the group consisting of: RPMI medium 1640, SFD, HCM-cAMP, ham's F12, or a combination thereof; and, the supplement includes BMP4, a8301, and CHIR99021.

8. An inducing composition, comprising:

(a) A first inducing factor comprising L-glutamine, ascorbyl Acid, MTG, bFGF, EGF, VEGF, BMP4, A8301, CHIR99021;

(b) Optionally a second induction factor comprising bFGF, BMP4, IWP2, a8301;

(c) Optionally a third inducing factor comprising bFGF, CHIR99021, noggin, TGF beta 2; and

(d) (ii) other substances that promote differentiation selected from the group consisting of: matrigel (Matrigel), laminin (lamin), basement Membrane Extracts (base Membrane Extracts), or combinations thereof.

9. Use of the inducing composition of claim 8 for inducing differentiation of pluripotent stem cells into hepscs of claim 2; and/or the parenchymal hepatic cell or a population of parenchymal hepatic cells of claim 3; and/or the biliary epithelial cell or biliary epithelial cell population of claim 4.

10. A screening or determination to promote differentiation of pluripotent stem cells into hepscs; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or potential material of biliary epithelial cells or biliary epithelial cell populations, comprising the steps of:

(a) Culturing pluripotent stem cells in a culture system for a period of time T1 in the presence of a test compound in a test group, and detecting the number S1 of HepSCs in the culture system of the test group; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or the number of biliary epithelial cells or bile duct epithelial cell populations S2;

and detecting the amount S3 of hepscs in said culture system in a control group in the absence of said test compound and otherwise identical conditions; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or the number of biliary epithelial cells or bile duct epithelial cell populations S4;

(b) Comparing S1 and S3 detected in the previous step, and S2 and S4, thereby determining whether the test compound is one that promotes differentiation of pluripotent stem cells into hepscs; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or potential material of biliary epithelial cells or populations of biliary epithelial cells;

wherein if S1 is significantly higher than S3, and/or S2 is significantly higher than S4, it is indicative that the test compound promotes differentiation of pluripotent stem cells into hepscs; and/or a parenchymal hepatic cell or a population of parenchymal hepatic cells; and/or potential material of biliary epithelial cells or populations of biliary epithelial cells.

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