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

Abstract

The present invention provides a method for establishing a hepatic stem cell line HepSC with bidirectional differentiation potential using pluripotent stem cells and its application. Specifically, the present invention provides a method for inducing human pluripotent stem cells to differentiate into HepSC, comprising the steps of: culturing pluripotent stem cells in a culture system to obtain functional HepSC, and continuing to culture HepSC to obtain functional hepatocytes or hepatocytes; and / or bile duct epithelial cells or bile duct epithelial cells. The functional HepSC, hepatocytes or hepatocytes; and / or bile duct epithelial cells or bile duct epithelial cells of the present invention have a very high differentiation rate and purity, and the HepSC, hepatocytes or hepatocytes; and / or bile duct epithelial cells or bile duct epithelial cells of the present invention can be used as an in vitro model for drug screening.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and cell therapy, and in particular to a method for establishing a liver stem cell line with bidirectional differentiation potential using pluripotent stem cells and its application. Background Art

[0002] Liver diseases have a high incidence and mortality rate worldwide. In China, the impact of liver diseases is far greater than in other countries. The number of patients with viral hepatitis, drug-induced hepatitis, liver cancer, hereditary liver diseases, etc. has reached 300 million so far, and the number of people who die from liver diseases each year is about 300,000 to 400,000, which has a huge impact on the health of the Chinese people and the social economy.

[0003] Take end-stage liver disease and bile duct disease as examples.

[0004] For end-stage liver disease, the best treatment is liver transplantation. However, donor livers are very scarce, so hepatocyte transplantation therapy was developed. Hepatocyte transplantation refers to a treatment method that replaces liver function by transplanting liver cells. Compared with liver transplantation, cell transplantation has the following advantages: 1) primary hepatocytes from one donor can be used to treat multiple patients; 2) cell transplantation is simpler than complex surgical operations; 3) primary hepatocytes can be frozen, which also makes cell transplantation often applicable to emergency situations; 4) multiple repeated transplantation treatments can be performed; 5) the cost of cell transplantation is much lower than that of liver transplantation. However, the source of mature hepatocytes for transplantation has always restricted this treatment method.

[0005] Targeting bile duct diseases. Biliary duct diseases are mainly divided into two types: PSC and PBC (Banales et al., 2019). PSC and PBC are two rare but important causes of chronic liver disease. Both cause inflammation of the bile duct, which can lead to the process of biliary cirrhosis, portal hypertension and liver failure. Biliary duct diseases are often an important cause of liver diseases such as cystic fibrosis (CF) and Alagille syndrome. For more complex biliary diseases, such as primary sclerosing cholangitis and biliary atresia, there are very few therapeutic drugs available due to the lack of appropriate models to understand their pathophysiology and screen drug preparations.

[0006] The common problem of these two types of diseases is the shortage of donor cells.

[0007] ES (Endocytic stem cell) cells and iPS (induced pluripotent stem cell) cells have the ability to proliferate indefinitely in vitro, so in theory, sufficient amounts of hepatocytes and bile duct epithelial cells can be obtained. Using iPS cells as a starting point for hepatocyte differentiation can avoid various ethical issues on the one hand, and on the other hand, it is easy to obtain patient-specific iPS cells for differentiation, thereby solving the problem of immune rejection. However, the cells obtained by differentiation of ES or iPS cells are often mixed with ES or iPS cells, which can easily cause teratomas when transplanted into the body.

[0008] Therefore, there is an urgent need in the art to develop a new method for establishing a hepatic stem cell line with bidirectional differentiation potential. Summary of the invention

[0009] The invention discloses a new method capable of establishing a liver stem cell line with bidirectional differentiation potential.

[0010] In a first aspect of the present invention, a method for inducing pluripotent stem cells to differentiate into a hepatic stem cell line is provided, comprising the steps of:

[0011] (a) Cultivating pluripotent stem cells in a culture system under a first culture condition to obtain a hepatic stem cell line (HepSC); wherein the culture system comprises: L-glutamine, Ascorbic Acid, MTG, bFGF, EGF, VEGF, BMP4, A8301, and CHIR99021.

[0012] In another preferred embodiment, step (a) includes step (b1), culturing pluripotent stem cells in a culture system under conditions suitable for culture, thereby obtaining definitive endoderm cells.

[0013] In another preferred embodiment, step (a) includes step (b2), in which the definitive endoderm cells obtained in step (b1) are cultured in a culture system under a first culture condition to obtain a hepatic stem cell line (HepSC), wherein the culture system includes: L-glutamine, Ascorbic Acid, MTG, bFGF, EGF, VEGF, BMP4, A8301, and CHIR99021.

[0014] In another preferred example, the first culture condition contains a first culture medium.

[0015] In another preferred example, the first culture medium is selected from the group consisting of RPMI medium 1640, SFD, Ham's F12, or a combination thereof.

[0016] In another preferred embodiment, in step (b1), the culture system is a culture solution containing a second culture medium and additives, and the additives include L-glutamine, MTG, Ascorbic Acid, bFGF, VEGF, Activin A, CHIR99021, and BMP4.

[0017] In another preferred embodiment, the second culture medium is selected from the group consisting of RPMI medium 1640, SFD, Ham's F12, or a combination thereof.

[0018] In another preferred example, the second culture medium includes a second A culture medium, a second B culture medium and a second C culture medium.

[0019] In another preferred embodiment, the second A culture medium is a DE (Definitive endoderm) differentiation first stage culture medium.

[0020] In another preferred embodiment, the second B culture medium is a DE differentiation second stage culture medium.

[0021] In another preferred embodiment, the second C culture medium is a DE differentiation third stage culture medium.

[0022] In another preferred example, the second culture medium A includes RPMI medium 1640.

[0023] In another preferred embodiment, the second culture medium A contains one or more additives selected from the following group: L-glutamine, MTG, Activin A and CHIR99021.

[0024] In another preferred example, the second B culture medium includes RPMI medium 1640.

[0025] In another preferred embodiment, the second B culture medium contains one or more additives selected from the following group: L-glutamine, Ascorbic Acid, MTG, bFGF, VEGF, Activin A and BMP4.

[0026] In another preferred example, the second C culture medium includes Serum free medium (SFD).

[0027] In another preferred embodiment, the second C culture medium contains one or more additives selected from the following group: L-glutamine, Ascorbic Acid, MTG, bFGF, VEGF, Activin A and BMP4.

[0028] In another preferred embodiment, the pluripotent stem cells are derived from human embryonic stem cells or human induced pluripotent stem cells.

[0029] In another preferred embodiment, the pluripotent stem cells are selected from the following groups: human embryonic stem cell lines (such as H9-ES, HES2-ES, 9-3-ES), human induced pluripotent stem cell lines (such as iPS17, WT6-iPS), or a combination thereof.

[0030] In another preferred embodiment, the pluripotent stem cells are selected from the following group: H9-ES, 9-3-ES, iPS17, or a combination thereof.

[0031] In another preferred embodiment, in step (a), the pluripotent stem cells are cultured under the first culture condition for 2-15 days, preferably, 3-10 days, more preferably, 6-7 days.

[0032] In another preferred embodiment, in step (b1), the pluripotent stem cells are cultured in the second culture medium for 5-6 days.

[0033] In another preferred embodiment, in step (b2), the definitive endoderm cells are cultured under the first culture condition for 4-8 days, preferably 8-12 days, and more preferably 12-20 days.

[0034] In another preferred embodiment, the method further comprises step (c), culturing the HepSC obtained in step (a) in a culture system under a second culture condition, thereby obtaining hepatocytes; wherein the culture system comprises: bFGF, BMP4, IWP2, and A8301.

[0035] In another preferred embodiment, the hepatocytes are mature hepatocytes.

[0036] In another preferred example, the second culture condition contains a third culture medium.

[0037] In another preferred example, the third culture medium is selected from the following group: SFD, HCM-cAMP, Ham's F12, or a combination thereof.

[0038] In another preferred embodiment, step (c) includes step (c1), wherein the HepSC obtained in step (a) is cultured in a culture system under a second culture condition to obtain hepatoblasts, wherein the culture system includes: bFGF, BMP4, IWP2, and A8301.

[0039] In another preferred embodiment, in step (c1), the culture system further contains one or more additives selected from the following group: L-glutamine, Ascorbic Acid, MTG, EGF, VEGF, ROCKi and CHIR99021.

[0040] In another preferred embodiment, step (c) includes step (c2), wherein the hepatoblasts obtained in step (c1) are cultured in a culture system under suitable culture conditions, thereby obtaining immature hepatocytes.

[0041] In another preferred embodiment, step (c) includes step (c3), wherein the immature hepatocytes obtained in step (c2) are cultured in a culture system under suitable culture conditions, thereby obtaining mature hepatocytes.

[0042] In another preferred embodiment, in step (c2), the culture system is a culture solution containing a fourth culture medium and additives, and the additives include one or more selected from the following group: L-glutamine, Ascorbic Acid, MTG, HGF, OSM, DEXAMETHASONE, Vitamin K1, CE, A8301 and EGFi.

[0043] In another preferred example, the fourth culture medium is selected from the following group: SFD, HCM-cAMP, Ham's F12, or a combination thereof.

[0044] In another preferred embodiment, in step (c3), the culture system is a culture solution containing the fifth culture medium and additives, and the additives include one or more selected from the following group: L-glutamine, Ascorbic Acid, MTG, HGF, OSM, DEXAMETHASONE, Vitamin K1, CE, A8301 and EGFi.

[0045] In another preferred example, the fifth culture medium is selected from the following group: SFD, HCM-cAMP, Ham's F12, or a combination thereof.

[0046] In another preferred embodiment, in step (c), the HepSCs are cultured under the second culture condition for 10-40 days, preferably 12-30 days, and more preferably 15-25 days.

[0047] In another preferred embodiment, in step (c1), HepSCs are cultured under the second culture condition for 3-15 days, preferably 4-12 days, more preferably 5-10 days.

[0048] In another preferred embodiment, in step (c2), the hepatoblasts are cultured in the fourth culture medium for 3-15 days, preferably 4-12 days, and more preferably 5-10 days.

[0049] In another preferred embodiment, in step (c3), the immature hepatocytes are cultured in the fifth culture medium for 3-15 days, preferably 4-12 days, and more preferably 5-10 days.

[0050] In another preferred embodiment, the method further comprises step (d), culturing the HepSC obtained in step (a) in a culture system under a third culture condition, thereby obtaining bile duct epithelial cells; wherein the culture system comprises: bFGF, CHIR99021, Noggin, and TGFβ2.

[0051] In another preferred example, the third culture condition contains a sixth culture medium.

[0052] In another preferred embodiment, the sixth culture medium is selected from the following group: SFD, Ham's F12, or a combination thereof.

[0053] In another preferred embodiment, step (d) includes step (d1), culturing the HepSC obtained in step (a) in a culture system under a third culture condition to obtain bile duct precursor cells, wherein the culture system includes: bFGF, CHIR99021, Noggin, and TGFβ2.

[0054] In another preferred embodiment, step (d) includes step (d2), wherein the bile duct precursor cells obtained in step (d1) are cultured under suitable culture conditions to obtain bile duct epithelial cells.

[0055] In another preferred embodiment, the bile duct cells are mature bile duct epithelial cells.

[0056] In another preferred embodiment, in step (d1), the culture system further contains one or more additives selected from the following group: L-glutamine, Ascorbic Acid, MTG, HGF, EGF and TGFβ2.

[0057] In another preferred embodiment, in step (d2), the culture system is a culture solution containing the seventh culture medium and additives, and the additives include one or more selected from the following group: L-glutamine, Ascorbic Acid, MTG, HGF, EGF and TGFβ2.

[0058] In another preferred example, the seventh culture medium is selected from the following group: SFD, Ham's F12, or a combination thereof.

[0059] In another preferred embodiment, in step (d), the HepSCs are cultured under the third culture condition for 10-30 days, preferably 11-20 days, and more preferably 12-19 days.

[0060] In another preferred embodiment, in step (d1), HepSCs are cultured under the third culture condition for 3-15 days, preferably 4-12 days, more preferably 5-10 days.

[0061] In another preferred embodiment, in step (d2), the bile duct precursor cells are cultured in the seventh culture medium for 3-15 days, preferably 4-12 days, and more preferably 5-10 days.

[0062] In another preferred embodiment, the method has one or more characteristics selected from the following group:

[0063] (i) high HepSC differentiation rate, the differentiation rate is 50%-80%, preferably, 70%-80%;

[0064] (ii) high hepatocyte differentiation rate, the differentiation rate is 50%-80%, preferably, 70%-80%;

[0065] (iii) high cholangiocyte differentiation rate, the differentiation rate being 70% to 90%, preferably 80% to 90%;

[0066] (iv) During the culture process, 2 × 10 5 pluripotent stem cells, which can generate 1.8×10 6 HepSCs;

[0067] (v) During the culture process, 2 × 10 5 pluripotent stem cells, which can generate 2×10 6 Hepatocytes;

[0068] (vi) During the culture process, 2×10 5 pluripotent stem cells, which can generate 2×10 6 bile duct cells;

[0069] (vii) HepSCs have some genes specifically expressed by hepatocytes and bile duct epithelial cells (such as HNF4A, AFP, TBX3, CEBPA, SOX9, HNF1β, CK19), and maintain unlimited proliferation ability. They can be induced to differentiate into hepatocytes and bile duct epithelial cells in vitro;

[0070] (viii) the hepatocytes are in the form of a closed single-layer cell balloon with a hollow structure, wherein the diameter of the hepatocyte balloon is 300-500 μm, the size of the hepatocytes on the balloon surface is 80-100 μm, and the thickness of the cell layer is 8-15 μm;

[0071] (ix) The bile duct epithelial cells are vacuolar in shape and connect to each other to gradually form a network structure. The diameter of the bile duct network is 20-40 μm, and the thickness varies. The thickness of the cell layer is 8-12 μm.

[0072] In another preferred embodiment, in steps (a) to (d), the culture system further contains other substances that promote differentiation, selected from the following group: Matrigel, laminin, basement membrane extracts, or a combination thereof.

[0073] In another preferred embodiment, the method includes both therapeutic and non-therapeutic methods.

[0074] In another preferred embodiment, in the culture system, the density of pluripotent stem cells is 1×10 5 -5×10 5 cells / ml, preferably, 2×10 5 -4×10 5 cells / ml.

[0075] In another preferred embodiment, the culture system has a volume of 50-125 ml, preferably 80-125 ml, and most preferably 80-100 ml.

[0076] In another preferred embodiment, the ratio M2 / M1 of the number M2 of the obtained HepSCs to the number M1 of the pluripotent stem cells is 1-3, preferably 1-2, and more preferably 1-1.5.

[0077] In another preferred embodiment, the ratio M3 / M2 of the number M3 of the obtained hepatocytes to the number M2 of the HepSCs is 1-3, preferably 1-2, and more preferably 1-1.5.

[0078] In another preferred embodiment, the ratio M4 / M2 of the number M4 of the obtained bile duct epithelial cells to the number M2 of the HepSCs is 1-3, preferably 1-2, and more preferably 1-1.5.

[0079] In another preferred embodiment, the HepSC is a functional liver stem cell line.

[0080] In another preferred embodiment, the hepatocytes are functional hepatocytes.

[0081] In another preferred embodiment, the bile duct epithelial cells are functional bile duct epithelial cells.

[0082] In another preferred embodiment, in steps (a)-(d), the culture is suspension culture.

[0083] In another preferred embodiment, the method further comprises:

[0084] (e) Detection of cell phenotype of formed HepSCs.

[0085] The second aspect of the present invention provides a hepatic stem cell line (HepSC), wherein the HepSC is differentiated from pluripotent stem cells and has the phenotype of HNF4A+, SOX9+, AFP+, and ALB-.

[0086] In another preferred embodiment, the hepatic stem cell line has the phenotype of HNF4A+, TBX3+, SOX9+, AFP+, ALB-, and CYP3A7-.

[0087] In another preferred embodiment, the HepSC has one or more of the following characteristics:

[0088] It has the differentiation potential into hepatocytes and bile duct epithelial cells in vivo and in vitro;

[0089] Expresses liver-specific transcription factor HNF4A and hepatocyte-specific protein AFP;

[0090] Expression of bile duct-specific transcription factors SOX9 and HNF1β;

[0091] Maintain unlimited proliferation ability.

[0092] In another preferred embodiment, the hepatic stem cell line is a human hepatic stem cell line.

[0093] In another preferred embodiment, the HepSC has one or more characteristics selected from the following group:

[0094] (i) More than 99% of cells have the surface antigen CD99;

[0095] (ii) High purity, ≥95-99%;

[0096] (iii) low animal origin, low dependence on Matrigel, and independent of mouse fibroblasts;

[0097] (iv) unlimited expansion in vitro;

[0098] (v) no tumor formation in the body;

[0099] (vi) low cultivation cost;

[0100] (vii) Efficient in vitro differentiation into functional hepatocytes and bile duct epithelial cells;

[0101] (viii) During the process of forming bile duct epithelial cells in vitro, they can self-assemble into a tubular network structure through three-dimensional culture.

[0102] In another preferred embodiment, the HepSCs have the ability to differentiate into hepatocytes.

[0103] In another preferred embodiment, the HepSCs have the ability to differentiate into bile duct epithelial cells.

[0104] The third aspect of the present invention provides a hepatocyte and / or a hepatocyte group, wherein the hepatocyte and / or the hepatocyte group is a closed single-layer cell balloon with a hollow structure, wherein the diameter of the hepatocyte balloon is 300-500 μm, the size of the hepatocytes on the balloon surface is 80-100 μm, and the thickness of the cell layer is 8-15 μm.

[0105] In another preferred embodiment, the hepatocytes and / or hepatocyte populations have one or more characteristics selected from the following:

[0106] (a) 60% of cells are ALBUMIN+;

[0107] (b) 97% of cells were AFP positive (AFP+);

[0108] (c) 99% of cells were positive for HNF4A expression (HNF4A+).

[0109] In another preferred embodiment, the hepatocytes and / or hepatocyte population are prepared by the method described in the first aspect of the present invention.

[0110] In another preferred embodiment, the hepatocytes and / or hepatocyte population are obtained by inducing HepSCs.

[0111] The fourth aspect of the present invention provides a bile duct epithelial cell or a bile duct epithelial cell group, wherein the bile duct epithelial cell or the bile duct epithelial cell group is vacuolar in shape, connected to each other, and gradually forms a tube network structure, wherein the bile duct network has a diameter of 20-40 μm and varies in thickness, and the cell layer thickness is 8-12 μm.

[0112] In another preferred embodiment, the bile duct epithelial cells or bile duct epithelial cell population have one or more characteristics selected from the following:

[0113] (a) 99% of cells are CK19 positive (CK19+);

[0114] (b) 90% of cells are positive for CK7 expression (CK7+);

[0115] (c) 80% of cells were HNF1Β positive (HNF1Β+);

[0116] (d) 99% of the cells showed down-regulation of liver-specific gene expression.

[0117] In another preferred embodiment, the liver-specific gene is selected from the following group: HNF4A, AFP, ALB, CYP3A7, CYP3A4, or a combination thereof.

[0118] In another preferred embodiment, the bile duct epithelial cells or bile duct epithelial cell population are prepared by the method described in the first aspect of the present invention.

[0119] In another preferred embodiment, the bile duct epithelial cells or bile duct epithelial cell population are obtained by inducing HepSC.

[0120] The fifth aspect of the present invention provides a use of the HepSCs described in the second aspect of the present invention, the hepatocytes and / or hepatocyte groups described in the third aspect of the present invention, or the bile duct epithelial cells or bile duct epithelial cell groups described in the fourth aspect of the present invention, for (i) screening drugs that promote the differentiation of HepSCs, hepatocytes and / or hepatocyte groups, or bile duct epithelial cells or bile duct epithelial cell groups; and / or (ii) preventing and / or treating liver-related diseases.

[0121] In another preferred embodiment, the liver-related disease is selected from the following group: bile duct-related diseases, chronic liver failure, or a combination thereof.

[0122] In another preferred embodiment, the bile duct-related disease includes bile duct injury disease.

[0123] In another preferred embodiment, the bile duct-related disease includes bile duct injury disease induced by DDC (3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine).

[0124] The sixth aspect of the present invention provides a pharmaceutical composition for preventing and / or treating liver-related diseases, characterized in that the pharmaceutical composition comprises: an effective amount of HepSCs described in the second aspect of the present invention, the hepatocytes and / or hepatocyte populations described in the third aspect of the present invention, or the bile duct epithelial cells or bile duct epithelial cell populations described in the fourth aspect of the present invention, and a pharmaceutically acceptable carrier.

[0125] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0126] In another preferred embodiment, the pharmaceutical composition is a cell preparation.

[0127] In another preferred embodiment, the pharmaceutical composition is an intravenous injection agent.

[0128] In another preferred embodiment, the pharmaceutically acceptable carrier includes (but is not limited to): saline, buffer, glucose and combinations thereof.

[0129] In another preferred embodiment, the concentration of the HepSC, hepatocytes and / or hepatocyte populations or bile duct epithelial cells or bile duct epithelial cell populations is 1×10 3 / ml-1×10 7 / ml, preferably 1×10 4 -1×10 6 / ml, more preferably 1×10 5 / ml-9.9×10 5 Pieces / ml.

[0130] In another preferred embodiment, the liver disease is selected from the following group: bile duct related diseases, chronic liver failure or a combination thereof.

[0131] The seventh aspect of the present invention provides an induction medium, which contains a basal medium and additives; wherein the basal medium is selected from the following group: RPMI medium 1640, SFD, HCM-cAMP, Ham'sF12, or a combination thereof; and the additives include BMP4, A8301 and CHIR99021.

[0132] In another preferred embodiment, the additives further include bFGF, EGF and VEGF.

[0133] In another preferred embodiment, the additives further include L-glutamine, Ascorbic Acid and MTG.

[0134] The eighth aspect of the present invention provides an induction composition, comprising:

[0135] (a) a first induction factor, wherein the first induction factor comprises L-glutamine, Ascorbic Acid, MTG, bFGF, EGF, VEGF, BMP4, A8301, and CHIR99021;

[0136] (b) an optional second induction factor, wherein the second induction factor comprises bFGF, BMP4, IWP2, A8301;

[0137] (c) an optional third induction factor, said third induction factor comprising bFGF, CHIR99021, Noggin, TGFβ2; and

[0138] (d) other substances that promote differentiation, wherein the other substances that promote differentiation are selected from the following group: Matrigel, laminin, basement membrane extracts, or a combination thereof.

[0139] The ninth aspect of the present invention provides a use of the induction composition described in the eighth aspect of the present invention, for inducing pluripotent stem cells to differentiate into the HepSC described in the second aspect of the present invention; and / or the hepatocytes or hepatocyte populations described in the third aspect of the present invention; and / or the bile duct epithelial cells or bile duct epithelial cell populations described in the fourth aspect of the present invention.

[0140] The tenth aspect of the present invention provides a method for screening or determining a potential substance that promotes the differentiation of pluripotent stem cells into HepSCs; and / or hepatocytes or hepatocyte populations; and / or bile duct epithelial cells or bile duct epithelial cell populations, comprising the steps of:

[0141] (a) in a test group, in a culture system, in the presence of a test compound, culturing pluripotent stem cells for a period of time T1, and detecting the number S1 of HepSCs; and / or the number S2 of hepatocytes or hepatocyte populations; and / or the number S2 of bile duct epithelial cells or bile duct epithelial cell populations in the culture system of the test group;

[0142] And in a control group without the test compound and with the same other conditions, the number of HepSCs in the culture system of the control group S3; and / or the number of hepatocytes or hepatocytes; and / or the number of bile duct epithelial cells or bile duct epithelial cells S4 is detected;

[0143] (b) comparing S1 and S3 detected in the previous step, and comparing S2 and S4, so as to determine whether the test compound is a potential substance that promotes the differentiation of pluripotent stem cells into HepSCs; and / or hepatocytes or hepatocyte populations; and / or bile duct epithelial cells or bile duct epithelial cell populations;

[0144] Among them, if S1 is significantly higher than S3, and / or S2 is significantly higher than S4, it means that the test compound can promote the differentiation of pluripotent stem cells into HepSC; and / or hepatocytes or hepatocyte populations; and / or bile duct epithelial cells or bile duct epithelial cell populations.

[0145] In another preferred embodiment, the “significantly higher than” refers to S1 / S3 ≥ 2, preferably ≥ 3, and more preferably ≥ 4.

[0146] In another preferred embodiment, the “significantly higher than” refers to S2 / S4 ≥ 2, preferably ≥ 3, and more preferably ≥ 4.

[0147] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0148] In another preferred embodiment, the pluripotent stem cells are cells cultured in vitro.

[0149] In another preferred embodiment, the method is an in vitro method.

[0150] The eleventh aspect of the present invention provides a method for treating liver disease, comprising the steps of:

[0151] The HepSCs described in the second aspect of the present invention, the hepatocytes or hepatocytes described in the third aspect of the present invention, or the bile duct epithelial cells or bile duct epithelial cells described in the fourth aspect of the present invention are administered to a subject in need, or a pharmaceutical composition containing the HepSCs described in the second aspect of the present invention, the hepatocytes or hepatocytes described in the third aspect of the present invention, or the bile duct epithelial cells or bile duct epithelial cells described in the fourth aspect of the present invention is administered.

[0152] In another preferred embodiment, the subject is a human or a non-human mammal, preferably a human.

[0153] In another preferred embodiment, the administration site is into the portal vein of the subject.

[0154] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0155] Figure 1 The establishment of a new human hepatic stem cell line was demonstrated; among them,

[0156] (A) Schematic diagram of the establishment and maintenance of HepSCs.

[0157] (B) Morphology of HepSCs at passage 15. Scale bar: 200 μm.

[0158] (C) qRT-PCR was used to detect the expression of HepSC characteristic genes. The data are relative values ​​to the expression of the housekeeping gene TBP. HepSC downregulated the definitive endoderm characteristic genes EOMEs and SOX17, upregulated the liver characteristic genes HNF4A, TBX3 and AFP, and did not express the mature liver characteristic gene ALB; upregulated the bile duct epithelial cell characteristic genes HNF1β, SOX9 and CK19, but did not express the mature bile duct epithelial cell characteristic gene CK7.

[0159] (D) Flow cytometry was used to detect the expression of HepSC characteristic genes at the protein level. HepSC is a group of CK19, SOX9, and HNF4A positive, ALB negative, and AFP expression has a certain heterogeneity in HepSC. This indicates that the cells have characteristic genes of both hepatocytes and bile duct epithelial cells and have a certain heterogeneity.

[0160] (E) Cell immunofluorescence detection of HepSC characteristic gene expression at the protein level. HepSC is a group of FOXA1, FOXA2, CK19, SOX9, HNF4A positive, SOX17 and ALB negative. This shows that the cells have characteristic genes of both hepatocytes and bile duct epithelial cells, and do not express characteristic genes of mature hepatocytes or definitive endoderm. This is consistent with the qPCR data in Figure (C).

[0161] (F) Chromosome karyotype identification map of HepSC cells at passage 30, indicating that the cells still maintain a diploid karyotype stability.

[0162] (G) Proliferation curves of HepSCs from the first to the 30th passages.

[0163] (H) Experimental verification of HepSC tumorigenicity. HepSC were transplanted under the skin of the neck or into the inner thigh muscle of immunodeficient Scid Beige mice. The data showed that HepSC did not have tumorigenicity compared with the control group.

[0164] (I) Verification of HepSC self-differentiation potential. The transplanted cells in (H) were taken out after about 4 weeks, and frozen sections were performed for immunofluorescence staining. The data showed that the cell mass retained the bile duct characteristic gene SOX9 at the transplantation site and expressed the characteristic gene CFTR of mature bile duct; at the same time, it lost the characteristic gene HNF4A of hepatocytes. This shows that HepSC will form bile duct epithelial cells after self-differentiation in vivo.

[0165] Figure 2 The study showed that a novel human hepatic stem cell line can be efficiently induced to differentiate into functional hepatocytes in vitro, among which

[0166] (A) Schematic diagram of the hepatic differentiation of HepSCs.

[0167] (B) Morphology of hepatocytes induced by HepSCs. Scale bar: 200um.

[0168] (C) qRT-PCR was used to detect the expression of characteristic genes of hepatocytes induced by HepSC. The data are relative values ​​to the expression of the housekeeping gene TBP. When HepSC were induced to form hepatoblasts, the characteristic genes of hepatocytes AFP, ALB and HNF4A were upregulated; the characteristic genes of bile ducts HNF1β, SOX9, etc. were downregulated. When HepSC were induced to form mature hepatocytes, the immature liver characteristic gene AFP was downregulated, and the characteristic genes of mature hepatocytes, such as ALB, HNF4A and CYP3A7, were maintained or improved.

[0169] (D) Flow cytometry was used to detect the expression of characteristic genes of hepatocytes induced by HepSC at the protein level, indicating that the hepatocytes derived from HepSC are AFP+ALB+ (efficiency is 60-70%).

[0170] (E) Cell immunofluorescence detection of the expression of characteristic genes of hepatocytes induced by HepSC at the protein level. The data showed that the induced hepatocytes expressed the characteristic genes of hepatocytes HNF4A, AFP and ALB, and the expression of polarity protein E-cadherin also indicated that the three-dimensional hepatocyte spheres had a certain polarity. Scale bar: 100um.

[0171] Figure 3 The study showed that a novel human hepatic stem cell line can be efficiently induced to differentiate into functional bile duct epithelial cells in vitro, among which,

[0172] (A) Schematic diagram of the directed differentiation of HepSC into biliary epithelial cells.

[0173] (B) Morphology of HepSC-induced bile duct epithelial cells. The image shows that the cells gradually connect into a tubular network structure in the matrix gel, forming a morphology similar to the intrahepatic bile duct network. Scale bar: 200um.

[0174] (C) qRT-PCR was used to detect the expression of characteristic genes of HepSC-induced biliary epithelial cells. The data are relative values ​​to the expression of the housekeeping gene TBP. When HepSC were induced to form bile duct precursor cells, the liver characteristic genes HNF4A and AFP were downregulated, and the bile duct characteristic genes SOX9 and CK19 were upregulated, but the bile duct maturation genes were still not expressed; when further induced to form bile duct epithelial cells, the cells further downregulated the liver characteristic genes HNF4A, AFP and ALB, and maintained the bile duct characteristic genes SOX9, HNF1β, CK19 and other genes. In addition, the related genes HES1 and NOTCH2 of the NOTCH signaling pathway, which are relatively important in bile duct development, were upregulated. This shows that HepSC does have the potential for bile duct differentiation in vitro.

[0175] (D) Flow cytometry was used to detect the expression of characteristic genes of bile duct epithelial cells induced by HepSC at the protein level, indicating that the hepatocytes derived from HepSC are SOX9+CK7+ (efficiency is 80-90%).

[0176] (E) Cell immunofluorescence detection of the expression of characteristic genes of bile duct epithelial cells induced by HepSC at the protein level. The data showed that the characteristic genes of primary bile duct epithelial cells SOX9 and CK7 were expressed in the induced bile duct network structure. Scale bar: 100um

[0177] Figure 4The integration of a novel human hepatic stem cell line into the liver of mice with bile duct injury was shown.

[0178] (A) Schematic diagram of mouse bile duct injury model construction and cell transplantation.

[0179] (B) Fluorescence imaging of liver tissue. The image shows that HepSCs are well integrated in mouse liver tissue.

[0180] (C) Immunofluorescence staining of frozen sections of mouse liver tissue. The image shows that the integrated HepSCs formed a good luminal structure in the mouse liver, and the integrated cells did not express the liver-specific gene HNF4A. Scale bar: 200um. DETAILED DESCRIPTION

[0181] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that, under the first culture condition of the present invention, by culturing pluripotent stem cells in a culture system, a liver stem cell line HepSC with a specific cell phenotype and bidirectional differentiation potential to the liver and bile duct can be obtained, and HepSC can be directed to differentiate into mature functional hepatocytes and bile duct epithelial cells with specific structures in vitro through a combination of small molecules and cytokines. The hepatocytes of the present invention can be used for specific treatment of liver-related diseases or screening of drugs for the treatment of liver-related diseases. The bile duct epithelial cells of the present invention can be used for the construction of tissue engineering bile duct networks and cell therapy of bile duct diseases.

[0182] Furthermore, the present invention unexpectedly found that the differentiation rate of the HepSC of the present invention is greater than 50%, the differentiation rate of mature hepatocytes is greater than 60%, and the differentiation rate of bile duct epithelial cells is greater than 86%. On this basis, the present inventors completed the present invention.

[0183] Specifically, starting from human ES or iPS cells, they are differentiated into definitive endoderm, and then under the culture conditions of bFGF, EGF, VEGF, BMP4, A8301 and CHIR99021, the definitive endoderm cells can be induced to differentiate and maintain a certain state to establish a cell line.

[0184] Taking HepSC as the starting point, a three-dimensional suspension differentiation system can be used in vitro to induce differentiation into mature hepatocytes. A three-dimensional "gel dripping" differentiation system can also be used to induce differentiation into bile duct epithelial cells. HepSC is of great significance for liver tissue engineering and cell therapy of liver diseases.

[0185] the term

[0186] Hepatoblast

[0187] In the present invention, HepSCs are formed into bipotential hepatic progenitor cells with the ability to generate bile duct epithelial cells and hepatic parenchymal cells under the influence of hepatic specific factors.

[0188] The hepatoblasts of the present invention are closed single-layer cell balloons with a hollow structure, wherein the diameter of the hepatoblast balloon is 300-500 μm, the size of the hepatoblasts on the balloon surface is 60-80 μm, and the thickness of the cell layer is 6-12 μm.

[0189] Hepatocytes

[0190] As used herein, the terms "hepatocytes" and "hepatocyte populations" are used interchangeably unless specifically referring to a single cell.

[0191] In the present invention, hepatoblasts are transformed into parenchymal cells having liver functions under the action of hepatocyte maturation-inducing factors.

[0192] The hepatocytes of the present invention are closed single-layer cell balloons with a hollow structure, wherein the balloon diameter of the hepatocytes is 300-500 μm, the hepatocytes on the balloon surface are 80-100 μm in size, and the cell layer thickness is 8-15 μm.

[0193] Bile duct precursor cells

[0194] In the present invention, HepSCs are formed into progenitor cells with the potential to generate bile duct epithelial cells under the induction of specific bile duct-directed factors.

[0195] The bile duct precursor cells of the present invention are vacuolar in shape, connected to each other, and gradually form a tube network structure, wherein the diameter of the bile duct network is 20-40 μm, and the thickness varies, and the thickness of the cell layer is 8-12 μm.

[0196] Bile duct epithelial cells

[0197] As used herein, the terms "biliary epithelial cells" and "biliary epithelial cell population" are used interchangeably unless specifically referring to a single cell.

[0198] In the present invention, bile duct precursor cells form a bile duct network having the functions and molecular characteristics of mature primary bile ducts under the action of bile duct cell maturation inducing factors.

[0199] The bile duct precursor cells of the present invention are vacuolar in shape, connected to each other, and gradually form a tube network structure, wherein the diameter of the bile duct network is 20-40 μm, and the thickness varies, and the thickness of the cell layer is 8-12 μm.

[0200] As used herein, the term "A8301" has a chemical formula of C25H19N5S and a CAS number of 909910-43-6. The chemical formula of "CE" is C27H24F2N4O3 and a CAS number of 209986-17-4. The chemical formula of "EGFi" is C22H23N3O4.HCl and a CAS number of 183319-69-9. The chemical formula of "CHIR99021" is C22H18Cl2N8 and a CAS number of 252917-06-9. The chemical formula of "IWP2" is C22H18N4O2S3 and a CAS number of 686770-61-6. The chemical formula of "ROCKi" is C14H21N3O and a CAS number of 146986-50-7.

[0201] Pluripotent stem cells

[0202] The starting seed cells for establishing HepSC described in the present invention. Pluripotent stem cells (Pluripotent StemCells) are a type of multipotent cells with the ability to self-renew and self-replicate. Under certain conditions, pluripotent stem cells (HSC) have the potential to differentiate into a variety of cell tissues. There are two sources, one is the cells derived from the inner cell mass of the human embryonic blastocyst stage; the other is the induced pluripotent stem cells obtained by reprogramming adult cells.

[0203] Induction and culture method of hepatic stem cell line, hepatocytes and bile duct epithelial cells

[0204] The starting cells of the liver stem cell line of the present invention are human pluripotent stem cells, which are cultured under the first culture conditions to obtain the liver stem cell line, wherein the culture system under the first culture conditions (such as containing the first culture medium) includes L-glutamine, Ascorbic Acid, MTG, bFGF, EGF, VEGF, BMP4, A8301, and CHIR99021.

[0205] Next, the HepSCs are further cultured under suitable culture conditions (such as under conditions containing a third culture medium and additives (including bFGF, BMP4, IWP2, and A8301)) to obtain hepatocytes; or

[0206] HepSCs are cultured under suitable culture conditions (eg, under conditions containing the sixth culture medium and additives (including bFGF, CHIR99021, Noggin, TGFβ2)) to obtain bile duct epithelial cells.

[0207] In a preferred embodiment, the culture system of the present invention further contains other substances that promote differentiation, selected from the following group: Matrigel, laminin, basement membrane extracts, or a combination thereof.

[0208] In a preferred embodiment, the method of inducing human pluripotent stem cells to differentiate into HepSC lines of the present invention is described in General Materials and Methods 8.

[0209] In a preferred embodiment, RPMI medium 1640 is commercially available (from GIBCO).

[0210] Ham's F12 medium is commercially available (from Cellgro Corporation).

[0211] Method for screening or identifying potential substances that promote the differentiation of pluripotent stem cells into hepatic stem cell lines; and / or hepatocytes or hepatocyte populations; and / or bile duct epithelial cells or bile duct epithelial cell populations

[0212] In the present invention, a method for screening or determining a potential substance that promotes the differentiation of pluripotent stem cells into HepSCs; and / or hepatocytes or hepatocyte populations; and / or bile duct epithelial cells or bile duct epithelial cell populations is provided, comprising the steps of:

[0213] (a) in a test group, in a culture system, in the presence of a test compound, culturing pluripotent stem cells for a period of time T1, and detecting the number S1 of HepSCs; and / or the number S2 of hepatocytes or hepatocyte populations; and / or the number S2 of bile duct epithelial cells or bile duct epithelial cell populations in the culture system of the test group;

[0214] And in a control group without the test compound and with the same other conditions, the number of HepSCs in the culture system of the control group S3; and / or the number of hepatocytes or hepatocytes; and / or the number of bile duct epithelial cells or bile duct epithelial cells S4 is detected;

[0215] (b) comparing S1 and S3 detected in the previous step, and comparing S2 and S4, so as to determine whether the test compound combination is a potential reagent combination for promoting the differentiation of pluripotent stem cells into HepSCs; and / or hepatocytes or hepatocyte populations; and / or bile duct epithelial cells or bile duct epithelial cell populations;

[0216] Among them, if S1 is significantly higher than S3, and S2 is significantly higher than S4, it means that the test compound can promote the differentiation of pluripotent stem cells into HepSC; and / or hepatocytes or hepatocyte populations; and / or bile duct epithelial cells or bile duct epithelial cell populations, which is a potential reagent combination.

[0217] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0218] The main advantages of the present invention include:

[0219] (1) The present invention first discovered that by culturing pluripotent stem cells in a culture system, functional HepSCs with extremely high differentiation rates (up to 80-90%) can be obtained; and / or hepatocytes or hepatocyte groups; and / or bile duct epithelial cells or bile duct epithelial cell groups, and the HepSCs of the present invention have a specific cell phenotype. The hepatocytes and / or hepatocyte groups are closed single-layer cell balloons with a hollow structure, wherein the balloon diameter of the hepatocytes is 300-500 μm, the size of the hepatocytes on the balloon surface is 80-100 μm, and the thickness of the cell layer is 8-15 μm. The bile duct epithelial cells or bile duct epithelial cell groups are vacuolar in shape, connected to each other, and gradually form a tube network structure. Among them, the diameter of the bile duct network is 20-40 μm, and the thickness varies, and the cell layer thickness is 8-12 μm.

[0220] Furthermore, the purity of the functional HepSCs; and / or hepatocytes or hepatocyte populations; and / or bile duct epithelial cells or bile duct epithelial cell populations of the present invention is also very high.

[0221] (2) The present invention established the HepSC line in vitro for the first time. The stem cell line retains the differentiation potential of hepatocytes and bile duct epithelial cells in vitro, and has unlimited proliferation ability. After 30 generations of culture, the chromosome diploid karyotype remains stable. It is independent of mouse fibroblasts, has low culture cost and high safety, is closer to hepatocytes and bile duct epithelial cells in the development path, and only has the differentiation potential of hepatocytes and bile duct epithelial cells, which is a good starting point for the differentiation of hepatocytes and bile duct epithelial cells.

[0222] (3) The present invention is the first to induce differentiation of new HepSCs to obtain mature hepatocytes, providing a good cell source for liver tissue engineering and cell therapy and drug screening for liver diseases.

[0223] (4) The present invention induced differentiation of new HepSCs to obtain mature bile duct epithelial cells for the first time, and constructed a continuous bile duct network in vitro for the first time, providing a good cell source for liver tissue engineering and cell therapy and drug screening for bile duct diseases.

[0224] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0225] Unless otherwise stated, the reagents and materials used in the examples of the present invention are commercially available products.

[0226] General Materials and Methods

[0227] 1. Experimental Animals

[0228] All animals involved in this chapter were kept in SPF animal rooms. SCID-Beige mice were purchased from Shanghai Lingchang. All animal experiments were carried out in strict accordance with the regulations of the Animal Management Committee of the Institute of Biochemistry and Cell Biology.

[0229] 2. Construction of mouse bile duct injury model and cell transplantation

[0230] RG mice aged 6-8 weeks were fed with DDC-containing feed four days before cell transplantation, and were kept fed with DDC feed in the later period. Cell transplantation was performed by spleen transplantation. The cells (transplanted cells were HepSC with green fluorescent protein EGFP) were resuspended in HepSC cell culture medium and 1uM ROCKi was added. The cell concentration was controlled at 1-2x10 6 Cells / 100ul, 1-2x 10 cells / 100ul per mouse 6 Cells. And at around the 4th week, the mice were sacrificed and liver tissues were taken for analysis.

[0231] During cell transplantation, mice were anesthetized with isoflurane gas, and their livers were exposed after opening the abdomen. The spleen was exposed by pulling the fat attached to the spleen, and the front end of the spleen was ligated with silk thread. The cell suspension was injected from the spleen with an insulin needle. After the injection was completed, the spleen and other organs were put back in place and the abdomen was sutured and closed.

[0232] 3. Tissue Cryosectioning

[0233] Fresh liver was fixed with 4% PFA at 4°C overnight, then washed with DPBS on a shaker for 30 minutes, then transferred to 30% sucrose solution (diluted with DPBS) for dehydration at 4°C overnight, then washed with DPBS on a shaker for 30 minutes, embedded with OCT in a tissue embedding frame, and frozen at -80°C. Then sliced ​​with an ice cutter, with a thickness of 8-10um, attached to a slide, and stored at -80°C. When staining, take out the slide, bake at 55°C for 15 minutes, then wash with PBST (0.1% Triton-X-100inPBS) at room temperature for 20 minutes. Block with blocking solution 2% BSA (diluted with PBST) at room temperature for 1 hour. Subsequent staining is the same as immunofluorescence staining.

[0234] 4.qPCR detection

[0235] All cell samples or tissue samples were extracted with RNA using a micro kit (purchased from Tiangen Biotechnology) and converted into cDNA (Promaga), and qPCR experiments were performed using the SYBR method (Rhoche).

[0236] 5. Flow Cytometry Analysis

[0237] For cell surface protein staining, after the cell samples are digested with trypsin into single cells, 5×104 to 1×105 cells are directly resuspended with the corresponding antibody working solution, incubated on ice for 30 minutes, washed 3 times with FACS buffer, resuspended with 300μl FACS buffer, kept on ice away from light, and loaded onto the machine as soon as possible. Most of the surface protein antibodies available in the laboratory are directly labeled with fluorescein. If secondary antibody labeling is required, continue to incubate with secondary antibody working solution after incubation with primary antibody and washing, and the subsequent operations are consistent.

[0238] The ratio of antibodies used in cell flow cytometry: CK7 antibody is used at a concentration of 1:100, CK19 antibody is used at a concentration of 1:100, SOX9 antibody is used at a concentration of 1:300, HNF4A antibody is used at a concentration of 1:200, AFP antibody is used at a concentration of 1:2000, and Albumin antibody is used at a concentration of 1:200. The antibody diluent is a 0.1% BSA DPBS solution.

[0239] 6. Intracellular protein staining

[0240] After the cell samples were digested into single cells with trypsin, 1×106 cells were fixed with 1.6% PFA at 37°C for 30 minutes, then washed 3 times with FACS buffer, resuspended in 500μl FACS buffer and stored at 4°C (fixed cell samples can be stored for up to 1 month). 1×Saponin buffer was used during the staining process, and all antibodies were diluted with 1×Saponin buffer. The primary antibody was incubated at room temperature for 30 minutes, washed 3 times with 20 1×Saponin buffer, and the secondary antibody was incubated in the dark for 30 minutes, washed 3 times with 1×Saponin buffer, resuspended in 300μl FACS buffer, kept on ice and protected from light, and loaded on the machine as soon as possible. All intracellular protein stainings were set with isotype controls. The flow cytometer was purchased from BD, model LSRⅡ. Flow data were all analyzed using Flowjo software.

[0241] 7. Immunofluorescence Staining

[0242] Cells or tissues were first fixed with 4% PFA at 37°C for 1 hour, and then incubated with PBS buffer containing 0.5% TritonX-100 (purchased from Sigma) and 0.05% BSA at room temperature for 10 minutes. Then washed 3 times with PBS, and blocked with 3% BSA solution at room temperature for 1 hour. After incubation with primary antibody at 4°C overnight or at room temperature for 2 to 3 hours, washed three times with PBS, and incubated with secondary antibody for 1 hour. After washing 3 times with PBS, stain with instant DAPI for 5 minutes. Photographs were taken using a Leica TCS SP5 laser confocal microscope.

[0243] Immunofluorescence antibody usage ratio:

[0244] The concentration of FOXA1 antibody was 1:200, the concentration of FOXA2 antibody was 1:200, the concentration of SOX17 antibody was 1:100, the concentration of E-cadherin antibody was 1:200, the concentration of CK19 antibody was 1:100, the concentration of CFTR antibody was 1:200, the concentration of CK7 antibody was 1:100, the concentration of SOX9 antibody was 1:500, the concentration of HNF4A antibody was 1:200, the concentration of AFP antibody was 1:2000, and the concentration of ALB antibody was 1:400. The antibody diluent was 0.1% BSA in DPBS.

[0245] 8. Establishment of HepSCs

[0246] The starting cells are human pluripotent stem cell lines. First, use 1:3 Matrigel to spread cells in a six-well plate without MEF. The human pluripotent stem cells that fill the six-well plate are passaged at a ratio of 1:3 and transferred to the above six-well plate. After passage, culture for 2-3 days, and when the ESC / iPSC clones grow to 80-90% density, they can be used for DE differentiation. The specific culture conditions are as follows:

[0247] From day 1 to day 2, the second A culture medium (such as RPMI 1640 culture medium) is supplemented with L-glutamine (1X, Cellgro), MTG (4.5×10-4 M), Activin A (10 ng / ml), and CHIR99021 (3 uM).

[0248] From day 2 to day 4, the second B culture medium (such as RPMI 1640 medium) is supplemented with L-glutamine (1X, Cellgro), Ascorbic Acid (50 μg / ml, Wako, cat#013-19641), MTG (4.5×10 -4 M), bFGF (5ng / ml), hVEGF (10ng / ml), Activin A (10ng / ml), hBMP4 (0.25ng / ml).

[0249] From day 4 to day 6, the second C medium (such as SFD medium) was supplemented with L-glutamine (1X, Cellgro), Ascorbic Acid (50 μg / ml, Wako, cat#013-19641), MTG (4.5×10 -4 M), bFGF (5ng / ml), hVEGF (10ng / ml), Activin A (10ng / ml), hBMP4 (0.25ng / ml).

[0250] DE differentiation to obtain definitive endoderm cells. Starting from definitive endoderm cells, HepSCs can be established under specific culture conditions. The specific method is as follows:

[0251] First, use 1:4-1:6 Matrigel to spread cells in a six-well plate, and then plate the definitive endoderm cells at 2×10 5 The cells were inoculated into the culture plate at a density of 100 cells / well and then transferred to the above six-well plate. The culture medium was SFD medium supplemented with L-glutamine (1X, Cellgro), Ascorbic Acid (50 μg / ml, Wako, cat#013-19641), MTG (4.5×10 -4M), bFGF (10ng / ml), hVEGF (10ng / ml), hEGF (10ng / ml), hBMP4 (50ng / ml), A8301 (5uM), CHIR99021 (3uM). After 37°C culture for 3-4 days, the clones were passaged when they grew to 80-90% density. When the cells were passaged, they were digested with 0.25% Trypsin at 37°C for 3-5 minutes, and the digestion was terminated with 1 / 2 volume of FBS of the digestion enzyme. After being blown into single cells, they were centrifuged at 1300rpm for 3 minutes, the supernatant was removed, and the cells were resuspended with fresh culture medium and inoculated into the culture plate described above to complete the passage. After 3-5 passages, HepSC was stably established.

[0252] 9. Directed differentiation of hepatocytes

[0253] New HepSC (1.2-2×10 6 Cells / plate) were cultured in Matrigel (Corning, cat#354230) coated cell culture plates with HepSC medium. After HepSCs filled the cell culture plates, they were digested with 0.25% trypsin at 37°C for 5-7 minutes, and then mechanically blown into single cells, which were then seeded in low-adhesion six-well plates (Corning, cat#3471) with 1X10 cells per well. 6 cells / ml, 3ml culture medium per well, add 10ul / ml Matrigel to the culture medium. Place the cell plate on a shaker at 150rpm and culture in a low-oxygen incubator. Change the medium every 2 days.

[0254] From day 1 to day 6, SFD medium was supplemented with L-glutamine (1X, Cellgro), Ascorbic Acid (50 μg / ml, Wako, cat#013-19641), MTG (4.5×10 -4 M), hBMP4 (50ng / ml), bFGF (10ng / ml), IWP2 ((0.5mM), A8301 (5uM), ROCKi (1uM). Directed differentiation into hepatoblasts. The hepatoblasts are closed single-layer cell balloons with a hollow structure, wherein the balloon diameter of the hepatoblasts is 400μm, the hepatoblast size on the balloon surface is 70μm, and the cell layer thickness is 8μm.

[0255] From day 6 to day 12, SFD medium was supplemented with L-glutamine (1X, Cellgro), Ascorbic Acid (50 μg / ml, Wako, cat#013-19641), MTG (4.5×10 -4M), HGF (25ng / ml), OSM (20ng / ml), Dexamethasone (40ng / ml), Vitamin K1 (6g / ml, Sangon), CE (0.1uM), A83-01 (0.5uM), EGFi (2uM) to differentiate into immature hepatocytes.

[0256] From day 12 to day 18, HCM medium (Lonza) was supplemented with L-glutamine (1X, Cellgro), Ascorbic Acid (50 μg / ml, Wako, cat#013-19641), MTG (4.5×10 -4 M), HGF (25ng / ml), OSM (20ng / ml), Dexamethasone (40ng / ml), Vitamin K1 (6g / ml, Sangon), CE (0.1uM), A83-01 (0.5uM), EGFi (2uM), differentiated into hepatocytes, and finally obtained 6×10 6 The hepatocytes are in the form of a closed single-layer cell balloon with a hollow structure, wherein the balloon diameter of the hepatocytes is 400 μm, the hepatocytes on the balloon surface are 90 μm in size, and the cell layer thickness is 10 μm.

[0257] All the small molecule compounds involved were purchased from Selleck, and the small molecule compounds were prepared according to the instructions. Cytokines were purchased from RD, and the dissolution method was according to the instructions.

[0258] In the present invention, the basic culture medium is SFD and HCM (hepatocyte culture medium). SFD (Serumfree differentiation medium) includes the following components: 75% Iscove's modified Dulbecco's medium (IMDM) (Cellgro), supplemented with 1% N2 and B27 supplements (Gibco-BRL), 1% penicillin / streptomycin, 25% Ham's F12 medium (Cellgro) with 0.05% bovine serum albumin.

[0259] HCM culture medium is commercially available (Commercial medium: Lonza: 15CC-4182) (Ogawa, S., Surapisitchat, J., Virtanen, C., Ogawa, M., Niapour, M., Sugamori, KS, ... & Zhao, B. (2013). Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes. Development, 140 (15), 3285-3296.).

[0260] 10. Directed differentiation of bile duct epithelial cells

[0261] New HepSC (1.2-2×10 6 Cells / plate) were cultured in Matrigel (Corning, cat#354230) coated cell culture plates with HepSC medium. After HepSCs filled the cell culture plates, they were digested with 0.25% trypsin at 37°C for 5-7 minutes, then mechanically blown into single cells, resuspended in Matrigel at a density of 15x106 cells per ml, and each drop was controlled at 20 microliters. After solidification at 37 degrees, they were cultured in a low oxygen incubator. The medium was changed every 2 days.

[0262] From day 1 to day 2, SFD medium was supplemented with L-glutamine (1X, Cellgro), Ascorbic Acid (50 μg / ml, Wako, cat#013-19641), MTG (4.5×10 -4 M), hBMP4 (50ng / ml), bFGF (10ng / ml), hVEGF (10ng / ml), hEGF (10ng / ml), A8301 (5uM), CHIR99021 (3uM), ROCKi (1uM).

[0263] From day 2 to day 8, SFD medium was supplemented with L-glutamine (1X, Cellgro), Ascorbic Acid (50 μg / ml, Wako, cat#013-19641), MTG (4.5×10 -4 M), bFGF (10 ng / ml), Noggin (100 ng / ml), CHIR99021 (3 uM), TGFβ2 (0.1 ng / ml) to obtain bile duct precursor cells.

[0264] From day 8 to day 16, SFD medium was supplemented with L-glutamine (1X, Cellgro), Ascorbic Acid (50 μg / ml, Wako, cat#013-19641), MTG (4.5×10 -4 M), HGF (25ng / ml), EGF (50ng / ml), TGFβ2 (0.1ng / ml). The bile duct epithelial cells were obtained, and the cells formed vacuolar morphology, connected to each other, and gradually formed a tube network structure. Among them, the diameter of the bile duct network was 20-40μm, and the thickness was different, and the cell layer thickness was 8-12μm.

[0265] All the small molecule compounds involved were purchased from Selleck, and the small molecule compounds were prepared according to the instructions. Cytokines were purchased from RD, and the dissolution method was according to the instructions.

[0266] Example 1 Establishment and characterization of HepSC cell line

[0267] HepSC cell lines are derived from human pluripotent stem cells ( Figure 1 .AB); Under culture conditions that do not rely on any trophoblast cells and Matrigel, the combination of special small molecules such as CHIR99021 and cytokines such as bFGF can maintain HepSCs at a developmental stage similar to hepatoblasts ( Figure 1 .CE); HepSCs express key liver transcription factors HNF4A and TBX3, hepatoblast marker AFP, bile duct transcription factors CK19, SOX9 and HNF1B, but do not express mature liver parenchyma or bile duct cell markers ALB or P450 family proteins, nor do they express mature bile duct cell marker genes ASBT or GGT1, etc. ( Figure 1 .CE). HepSCs can proliferate indefinitely in vitro (>P70) and maintain phenotypic and genomic stability for a long time ( Figure 1 .FG), and subclonal lines can be established from single HepSC cells, which are true stem cells. HepSC has the characteristics of not forming tumors in vivo ( Figure 1 .H), and in immunodeficient mice, they can self-differentiate to form luminal structures. These luminal structures do not express the liver-specific transcription factor HNF4A, but on the basis of retaining the bile duct epithelial cell transcription factor SOX9, they upregulate the expression of bile duct epithelial cell-specific expression genes CFTR, ASBT and AQP1, etc. Figure 1 .I)

[0268] Example 2 Establishment of an in vitro hepatic differentiation system of HepSCs

[0269] At present, the applicant has established an in vitro liver differentiation system for HepSC, which can efficiently prepare liver parenchyma ( Figure 2-3 ).

[0270] First, we established a three-dimensional suspension differentiation system of HepSCs into hepatocytes ( Figure 2 .AB), the differentiation process includes two main stages: hepatoblasts and hepatocytes. As differentiation progresses, HepSCs gradually upregulate hepatocyte characteristic genes AFP, ALB and CYP3A7 while maintaining the key liver transcription factor HNF4A, and downregulate bile duct transcription factors HNF1B and SOX9; hepatoblast characteristic factor AFP is significantly downregulated in mature hepatocytes ( Figure 2 .C). In the final stage, AFP+ALB+ hepatocytes accounted for 60% of the total, and there were no AFP-ALB+ mature hepatocytes, indicating that the efficiency of obtaining mature hepatocytes in this system needs to be improved ( Figure 2 .DE).

[0271] Secondly, we established a bile duct-directed differentiation system of HepSCs in vitro ( Figure 3 .A), using Matrigel-wrapped cell culture method ( Figure 3 .A), HepSCs spontaneously form dense bile duct network structures during differentiation ( Figure 3 .B). HepSC-derived biliary epithelial cells significantly downregulated liver marker genes HNF4A, AFP and ALB, and upregulated or maintained biliary epithelial cell marker genes SOX9, HNF1B, CK19, CK7, GGT1 and AE2 ( Figure 3 .CE).

[0272] Example 3 Integration of HepSCs in a DDC-induced bile duct injury mouse model

[0273] Previous data showed that HepSCs have the differentiation potential to hepatic parenchymal and bile duct epithelial cells in vitro, and can spontaneously differentiate into bile duct epithelial-like cells in vivo in the subcutaneous tissue or muscle of immunodeficient mice ( Figure 1 .I). We then used the DDC-induced chronic bile duct injury mouse model to evaluate the integration and differentiation ability of HepSCs after transplantation ( Figure 4 .A). Observing the mouse liver one month after transplantation under a fluorescence microscope, it was found that HepSCs could be efficiently integrated into the host and form bile duct-like structures ( Figure 4 .B). Integrated GFP+ cells do not express the key liver transcription factor HNF4A ( Figure 4 .C). These data preliminarily indicate that HepSCs have the ability to integrate and differentiate after being transplanted into mice with chronic bile duct injury, and thus have the potential to be directly used for cell replacement therapy of liver disease.

[0274] discuss

[0275] We have established and maintained a new HepSC in vitro for the first time. Compared with the existing endoderm cell lines maintained in vitro, it has many advantages, such as it does not rely on many animal-derived matrix gels and auxiliary cells, does not have tumorigenicity, and is closer to adult hepatic parenchymal cells and bile duct epithelial cells in the developmental path, so the time period for obtaining terminal adult cells is shorter. Therefore, this cell line has great value and significance both in the field of tissue engineering and in the field of cell therapy.

[0276] The data of the present invention show that the HepSC, in addition to expressing the characteristic gene or transcription factor HNF4A of hepatocytes, also retains the characteristic genes or transcription factors SOX9, HNF1β, CK19, etc. of bile duct epithelial cells. However, it does not express the characteristic functional genes of mature liver parenchyma, such as ALB and CYP enzyme genes. At the same time, Ki67 is almost all positive, indicating that the cells are in the cell cycle and can proliferate. This also corresponds to the molecular characteristics of HepSC. Furthermore, we hope to understand what state the cell line is in at the single-cell level, whether there is a cell population in such a cell state in the fetal liver or adult liver. And what are the similarities and differences in its maintenance mechanism compared to the existing endoderm cell lines maintained in vitro.

[0277] The potential of this new HepSC to differentiate into hepatic parenchyma also has great clinical value. At present, liver transplantation is the most effective means of treating severe liver disease, and hepatic parenchymal cell transplantation as an alternative means has also shown positive therapeutic effects in the treatment of various liver diseases. Human primary hepatic parenchymal cells are currently the only cell type used for cell replacement therapy for liver disease. However, the quality and quantity of donor livers have seriously hindered the development of primary hepatic parenchymal cell transplantation. Therefore, how to obtain large-scale hepatic parenchymal cells in vitro is still a problem that needs to be solved urgently. Even though the terminal hepatic parenchymal cells obtained by the differentiation of this HepSC are still a certain distance away from the primary liver, the unlimited capacity of this new HepSC and its low maintenance cost also provide a good idea for solving this problem in the future.

[0278] The research field of bile duct is currently in a relatively new stage, and the main research work is concentrated in two aspects: one is the problem of the cell source of bile duct epithelial cells, which involves the establishment of bile duct disease models in vitro for drug screening or cell therapy; the other is how to achieve hepatobiliary when constructing engineered tissue liver, which also involves bile duct tissue engineering. The HepSC established by the present invention can well cater to these two problems.

[0279] First, the HepSC is derived from human pluripotent stem cells, which can avoid ethical issues; secondly, whether it is the pluripotent stem cells from which it originates or the HepSC itself, both have unlimited proliferation capabilities, and the cost of culturing the cell line in vitro is very low, so it can well solve the problem of large-scale cell culture; the cell has good bile duct differentiation potential in vitro, combined with the convenience of large-scale expansion, it can well solve the problem of the source of donor bile duct cells, which is conducive to the construction of bile duct-related disease models in vitro and used for drug screening.

[0280] Our data show that HepSC has good bile duct epithelial cell differentiation potential and integration ability in the mouse in vivo environment, which also provides us with an idea for using this stem cell directly as a cell therapy for bile duct diseases. At present, we hope that HepSC can treat two types of bile duct diseases. One is drug-induced bile duct cell damage; the other is genetic diseases, such as congenital biliary atresia syndrome. In the former, bile duct epithelial cells are damaged, the bile duct system is damaged, and bile cannot be excreted, causing liver damage; in the latter, the bile duct system is missing and bile cannot be excreted. Both require bile duct epithelial cells that can be integrated to repair or even reconstruct the bile duct system. Here, HepSC is a good source of cells.

[0281] The data of the present invention show that the cell line has a good ability to self-assemble into a tube network structure in vitro, which is also for us to further use tissue chips or microfluidics or 3D printing technology to achieve the purpose of in vitro construction of complex bile duct networks. At present, liver tissue engineering is more focused on the construction of hepatocytes themselves, and rarely involves the bile duct system. However, the intrahepatic bile duct plays an important role in assisting proliferation and differentiation and excreting bile during development and physiological processes. Therefore, it is of great significance to integrate the bile duct system into tissue-engineered liver.

[0282] At present, there are still some defects in the in vitro differentiation of HepSC in the bile duct, that is, the maturity of the terminal bile duct epithelial cells obtained is still a certain distance away from that of the primary bile duct epithelial cells, which requires us to further optimize the differentiation conditions. However, its differentiation potential in vivo and its integration ability in vivo make this cell line have very high potential for clinical use.

[0283] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for inducing pluripotent stem cells to differentiate into a hepatic stem cell line HepSC, wherein the hepatic stem cell line is differentiated from pluripotent stem cells and has the phenotypes of HNF4A+, SOX9+, AFP+, and ALB-, and has the differentiation potential of hepatic parenchymal cells and bile duct epithelial cells, and has unlimited proliferation ability, and AFP expression is heterogeneous in the hepatic stem cell line, and the hepatic stem cell line has characteristic genes of hepatic parenchymal cells and bile duct epithelial cells, characterized in that: Includes steps: (a) culturing pluripotent stem cells in a culture system under a first culture condition to obtain a liver stem cell line; wherein the culture system comprises: L-glutamine, Ascorbic Acid, MTG, bFGF, EGF, VEGF, BMP4, A8301, and CHIR99021; In step (a), including step (b1), culturing pluripotent stem cells in a culture system to obtain definitive endoderm cells; In step (b1), the culture system is a culture solution containing a second culture medium and additives, and the additives include L-glutamine, MTG, Ascorbic Acid, bFGF, VEGF, Activin A, CHIR99021, and BMP4; and the second culture medium includes a second A culture medium, a second B culture medium, and a second C culture medium; The second culture medium A comprises RPMI medium 1640, and the second culture medium A contains the following additives: 1X L-glutamine, 4.5×10 -4 M of MTG, 10 ng / ml of Activin A and 3 uM of CHIR99021, and cultured with the second A medium from day 1 to day 2; The second B culture medium includes RPMI medium 1640, and the second B culture medium contains the following additives: 1X L-glutamine, 50 μg / ml Ascorbic Acid, 4.5×10 -4 5 ng / ml of MTG, 5 ng / ml of bFGF, 10 ng / ml of VEGF, 10 ng / ml of Activin A and 0.25 ng / ml of BMP4; and from day 2 to day 4, cultured with the second B medium; The second C culture medium includes Serum free medium, and the second C culture medium contains the following additives: 1X L-glutamine, 50 μg / ml Ascorbic Acid, 4.5×10 -4 M of MTG, 5 ng / ml of bFGF, 10 ng / ml of VEGF, 10 ng / ml of Activin A, and 0.25 ng / ml of BMP4; In step (a), the method further comprises step (b2), wherein the definitive endoderm cells obtained in step (b1) are cultured in a culture system under a first culture condition to obtain a hepatic stem cell line, wherein the culture system comprises: 1X L-glutamine, 50 μg / ml Ascorbic Acid, 4.5×10 -4 The first culture condition comprises: 10 ng / ml MTG, 10 ng / ml bFGF, 10 ng / ml EGF, 10 ng / ml VEGF, 50 ng / ml BMP4, 5 μM A8301, and 3 uM CHIR99021, and the first culture condition contains a first culture medium, and the first culture medium is selected from the following group: RPMI medium 1640, Serum free medium, Ham's F12, or a combination thereof.

2. An in vitro differentiation method for hepatocytes and / or hepatocyte groups, wherein the hepatocytes and / or hepatocyte groups are closed single-layer cell balloons with a hollow structure, wherein: The hepatocyte balloon has a diameter of 300-500 μm, the hepatocytes on the balloon surface have a size of 80-100 μm, and the thickness of the cell layer is 8-15 μm, and includes: (i) under a second culture condition, culturing the liver stem cell line of claim 1 in a culture system to obtain hepatoblasts, wherein the culture system comprises: 10 ng / ml bFGF, 50 ng / ml BMP4, 0.5 mM IWP2, 5 μM A8301, and the culture system further comprises the following additives: 1X L-glutamine, 50 μg / ml Ascorbic Acid, MTG, ROCKi, and the second culture condition comprises a third culture medium, and the third culture medium is selected from the group consisting of Serum free medium, Ham's F12, or a combination thereof; (ii) culturing the hepatoblasts obtained in step (i) in a culture system to obtain immature hepatocytes; the culture system is a culture solution containing a fourth culture medium and additives, the additives comprising: 1X L-glutamine, 50 μg / ml Ascorbic Acid, MTG, HGF, OSM, DEXAMETHASONE, Vitamin K1, CE, 0.5 μM A8301 and EGFi, and the fourth culture medium is selected from the group consisting of Serum free medium, Ham's F12, or a combination thereof; (iii) culturing the immature hepatocytes obtained in step (ii) in a culture system to obtain mature hepatocytes, wherein the culture system is a culture solution containing a fifth culture medium and additives, wherein the additives include: 1X L-glutamine, 50 μg / ml Ascorbic Acid, MTG, HGF, OSM, DEXAMETHASONE, Vitamin K1, CE, 0.5 μM A8301 and EGFi, and the fifth culture medium is HCM; The chemical formula of the CE is C27H24F2N4O3.

3. A method for in vitro differentiation of bile duct epithelial cells or bile duct epithelial cell groups, wherein the bile duct epithelial cells or bile duct epithelial cell groups are vacuolar in shape, connected to each other, and gradually form a tube network structure; wherein: The bile duct network has a diameter of 20-40 μm and varies in thickness. The cell layer is 8-12 μm thick and includes: (i) Under the third culture condition, the HepSCs described in claim 1 are cultured in a culture system to obtain bile duct precursor cells, wherein, from day 1 to day 2, the cells are cultured in a culture system containing 1X L-glutamine, 50 μg / ml Ascorbic Acid, MTG, 50 ng / ml hBMP4, 10 ng / ml bFGF, 10 ng / ml hVEGF, 10 ng / ml hEGF, 5 μM A8301, 3 μM CHIR99021, and 1 μM ROCKi; from day 2 to day 8, the cells are cultured in a culture system containing 1X L-glutamine, 50 μg / ml Ascorbic Acid, MTG, 10 ng / ml bFGF, 100 ng / ml Noggin, 3 μM CHIR99021, and 0.1 ng / ml TGFβ2, and the third culture condition contains a sixth culture medium, and the sixth culture medium is selected from the group consisting of Serum free medium, Ham's F12, or a combination thereof; (ii) culturing the bile duct precursor cells obtained in step (i) to obtain bile duct epithelial cells, wherein the culture system is a culture solution containing a seventh culture medium and additives, wherein the additives include: 1X L-glutamine, 50 μg / ml Ascorbic Acid, MTG, HGF, 50 ng / ml EGF and TGFβ2, and the seventh culture medium is selected from the following group: Serum free medium, Ham's F12, or a combination thereof.

4. A method for screening or determining a potential substance that promotes the differentiation of pluripotent stem cells into HepSCs as claimed in claim 1, characterized in that: Includes steps: (a) in a test group, in a culture system, in the presence of a test compound, culturing pluripotent stem cells for a period of time T1, and detecting the number S1 of HepSCs in the culture system of the test group; And in a control group without the test compound and with other conditions being the same, detecting the number of HepSCs in the culture system of the control group S3; (b) comparing S1 and S3 detected in the previous step to determine whether the test compound is a potential substance that promotes the differentiation of pluripotent stem cells into HepSCs; If S1 is significantly higher than S3, it indicates that the test compound is a potential substance that can promote the differentiation of pluripotent stem cells into HepSCs.

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