Method for preparing hepatocytes, hepatoprogenitor cells or mature hepatocytes

Abstract

The invention belongs to the field of biological medicine, and relates to a method for preparing hepatoblasts, hepatic precursor cells or mature hepatocytes and a culture medium combination. Specifically, the invention relates to a method for preparing hepatoblasts, which comprises the following steps: inducing pregut cells to differentiate into hepatoblasts through first-stage induction and second-stage induction in sequence, wherein the culture medium used in the first-stage induction is different from the culture medium used in the second-stage induction. The method for preparing the mature hepatocytes is relatively high in efficiency and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a method for preparing hepatoblasts, hepatic precursor cells or mature hepatocytes and a culture medium combination. Background Art

[0002] The generation of induced human pluripotent stem cells provides a new idea for the preparation of multiple functional cells through in vitro differentiation, showing great application potential 1 Recently, the inventor's team successfully induced human cells to transform into chemically induced pluripotent stem cells (hCiPSCs) through small molecule-based chemical reprogramming. 2 Chemical reprogramming is easy to quantify and standardize, does not integrate with the genome, and is low-cost, making hCiPSCs prepared by chemical reprogramming more suitable for clinical applications. The inventors have demonstrated the feasibility of hCiPSC differentiation into functional pancreatic islets, which can improve diabetes in non-human primates. 3 .

[0003] Hepatocytes have important applications in drug screening, disease simulation, and cell therapy for end-stage liver disease. In recent years, different protocols for differentiating pluripotent stem cells into hepatocytes have been established by simulating liver development. 4 This type of program can be roughly divided into three stages: differentiation to produce endoderm, hepatoblast specialization and further differentiation to mature hepatocytes. 5 The inventors' team established a protocol for differentiating induced human pluripotent stem cells into mature hepatocytes (hiHEPs) 6 , so that a large number of mature hepatocytes can be prepared in vitro. Hepatoblasts are a key cell stage in the process of liver organogenesis in vivo. 7 , hepatoblasts can differentiate into hepatic progenitor cells through further development, and then hepatic progenitor cells can differentiate into mature hepatocytes through further maturation. However, the efficiency of hepatoblast differentiation into hepatic progenitor cells (hiHPCs) is still limited, and the purity of hepatic progenitor cells needs to be improved through multiple passages of enrichment, which prolongs the preparation cycle of hepatocytes and hinders their clinical application.

[0004] Currently, there is still a need to develop new methods for preparing mature hepatocytes. Summary of the invention

[0005] After in-depth research and creative work, the inventors have obtained a method for preparing mature hepatocytes and a culture medium combination. The inventors surprisingly found that the method for preparing mature hepatocytes of the present invention has a high efficiency in preparing mature hepatocytes. The following invention is provided:

[0006] One aspect of the present invention relates to a method for preparing hepatoblasts, comprising the following steps:

[0007] Through the first stage induction and the second stage induction, the foregut cells are induced to differentiate into hepatoblasts;

[0008] The culture medium used for the first stage of induction is different from the culture medium used for the second stage of induction.

[0009] In some embodiments of the present invention, in the method for preparing hepatoblasts, the culture medium used in the first stage of induction is RPMI1640;

[0010] Preferably, the RPMI1640 medium is supplemented with B27, KGF, BMP2, BMP4, bFGF and Y27632 without vitamin A;

[0011] Preferably, B27 without vitamin A is 1.6%-2.4%, KGF is 18-22ng / mL, BMP2 is 18-22ng / mL, BMP4 is 80-120ng / mL, bFGF is 8-12ng / mL, and Y27632 is 8-12μmol / L;

[0012] Preferably, B27 without vitamin A is 2%, KGF is 20 ng / mL, BMP2 is 20 ng / mL, BMP4 is 100 ng / mL, bFGF is 10 ng / mL, and Y27632 is 10 μmol / L.

[0013] In some embodiments of the present invention, in the method for preparing hepatoblasts, the culture medium used in the second stage of induction is William's medium E;

[0014] Preferably, William's medium E culture medium is supplemented with B27, EGF, Foskolin and Y27632 without vitamin A;

[0015] Preferably, B27 without vitamin A is 1.6%-2.4%, EGF is 18-22ng / mL, Foskolin is 23-28μmol / L, and Y27632 is 8-12μmol / L;

[0016] Preferably, B27 without vitamin A is 2%, EGF is 20 ng / mL, Foskolin is 25 μmol / L, and Y27632 is 10 μmol / L.

[0017] In some embodiments of the present invention, the method for preparing hepatoblasts, wherein:

[0018] The induction time of the first stage induction is 3-6 days, preferably 3-4 days; and / or

[0019] The induction time of the second stage induction is 2-6 days, preferably 3 days.

[0020] In some embodiments of the present invention, the method for preparing hepatoblasts, wherein:

[0021] The product of the first stage induction is digested into single cells, and then inoculated with the same medium as that for the first stage induction and cultured for 12-48 hours, preferably 24-36 hours; then the second stage induction is performed.

[0022] In some embodiments of the present invention, in the method for preparing hepatoblasts, the foregut cells are obtained by inducing differentiation of endoderm cells.

[0023] In some embodiments of the present invention, the method for preparing hepatoblasts, wherein:

[0024] The culture medium used to induce endoderm cell differentiation to obtain foregut cells was RPMI1640;

[0025] Preferably, the RPMI1640 medium is supplemented with B27, KGF, SB431542 and Y27632 without vitamin A;

[0026] Preferably, B27 without vitamin A is 1.6%-2.4%, KGF is 18-22 ng / mL, SB431542 is 2.3-2.8 μmol / L, and Y27632 is 8-12 μmol / L;

[0027] Preferably, B27 without vitamin A is 2%, KGF is 20 ng / mL, SB431542 is 2.5 μmol / L, and Y27632 is 10 μmol / L.

[0028] In some embodiments of the present invention, the method for preparing hepatoblasts, wherein:

[0029] The time for inducing endoderm cells to differentiate into foregut cells is 1-3 days, preferably 2 days.

[0030] In some embodiments of the present invention, the method for preparing hepatoblasts, wherein the endoderm cells are obtained by inducing differentiation of pluripotent stem cells;

[0031] Preferably, the pluripotent stem cells are induced to differentiate into endoderm cells through stage I induction and stage II induction in sequence;

[0032] The culture medium used in the induction of stage I contains CHIR99021, while the culture medium used in the induction of stage II does not contain CHIR99021.

[0033] In some embodiments of the present invention, the method for preparing hepatoblasts, wherein:

[0034] The culture medium used to induce pluripotent stem cells to differentiate into endoderm cells was RPMI1640;

[0035] Preferably, RPMI1640 medium is supplemented with B27 without vitamin A, Activin A, BMP4, bFGF, Y27632, and optionally CHIR99021;

[0036] Preferably, the RPMI1640 medium in stage I contains CHIR99021, and the RPMI1640 medium in stage II does not contain CHIR99021;

[0037] Preferably, B27 without vitamin A is 1.6%-2.4%, Activin A is 80-120 ng / mL, BMP4 is 0.4-0.6 ng / mL, bFGF is 4-6 ng / mL, and Y27632 is 8-12 μmol / L;

[0038] Preferably, B27 without vitamin A is 2%, Activin A is 100 ng / mL, BMP4 is 0.5 ng / mL, bFGF is 5 ng / mL, and Y27632 is 10 μmol / L;

[0039] Optionally, CHIR99021 is 2.5-3.5 μmol / L;

[0040] Optionally, CHIR99021 is 3 μmol / L.

[0041] In some embodiments of the present invention, the method for preparing hepatoblasts, wherein:

[0042] The time for inducing pluripotent stem cells to differentiate into endoderm cells is 3-6 days, preferably 4 days;

[0043] Preferably, Phase I lasts for 1 day, and Phase II lasts for 2-5 days; preferably, Phase II lasts for 3 days.

[0044] In some embodiments of the present invention, in the method for preparing hepatoblasts, the pluripotent stem cells are human or mouse pluripotent stem cells;

[0045] Preferably, the pluripotent stem cells are chemically induced pluripotent stem cells, such as human chemically induced pluripotent stem cells or mouse chemically induced pluripotent stem cells.

[0046] In some embodiments of the present invention, the method for preparing hepatoblasts, wherein:

[0047] Pluripotent stem cells were pre-treated as follows:

[0048] Cultivate in mTeSR Plus medium for 12-48 hours, preferably 24-36 hours;

[0049] Preferably, mTeSR Plus medium is supplemented with Activin A, bFGF and Y27632;

[0050] Preferably, Activin A is 8-12 ng / mL, bFGF is 8-12 ng / mL, and Y27632 is 8-12 μmol / L;

[0051] Preferably, Activin A is 10 ng / mL, bFGF is 10 ng / mL, and Y27632 is 10 μmol / L.

[0052] In some embodiments of the present invention, the method for preparing hepatoblasts, wherein:

[0053] The method also includes the step of detecting molecular markers specifically expressed by the obtained endoderm cells;

[0054] Preferably, the detection is performed by flow cytometry;

[0055] Preferably, the molecular markers specifically expressed by endoderm cells are FOXA2 and SOX17; preferably, the proportion of FOXA2 and SOX17 double-positive cells is ≥60%, ≥70% or ≥80%.

[0056] Another aspect of the present invention relates to a method for preparing hepatic progenitor cells, comprising the following steps:

[0057] Prepare hepatoblasts by the method for preparing hepatoblasts according to any one of the present invention; and

[0058] Induce hepatoblasts to differentiate into hepatic progenitor cells.

[0059] In some embodiments of the present invention, the method for preparing hepatic progenitor cells comprises:

[0060] The culture medium used for inducing hepatoblasts to differentiate into hepatic progenitor cells is: DMEM / F12 culture medium and William's medium E culture medium are mixed in a mass ratio of (1:2) to (2:1) as the basic culture medium; preferably, DMEM / F12 culture medium and William's medium E culture medium are mixed in a mass ratio of 1:1 as the basic culture medium;

[0061] Preferably, the basal medium is supplemented with B27 containing vitamin A, Nicotinamide, 2-Phospho-L-ascorbic acid trisodium salt, 1-Oleoyl Lysophosphatidic Acid, D-erythro-Sphingosine-1-phosphate, SB431542, CHIR99021, Foskolin, EGF, Dexamethasone and Heparin;

[0062] Preferably, B27 containing vitamin A is 1.6%-2.4%, Nicotinamide is 4-6mmol / L, 2-Phospho-L-ascorbic acid trisodium salt is 80-120μmol / L, 1-Oleoyl Lysophosphatidic Aci is 4-6μmol / L, D-erythro-Sphingosine-1-phosphate is 0.4-0.6μmol / L, SB431542 is 2.3-2.8μmol / L, CHIR99021 is 1.3-1.8μmol / L, Foskolin is 22-28μmol / L, EGF is 18-22ng / mL, Dexamethasone is 0.8-1.2μmol / L, and Heparin is 80-120μg / mL;

[0063] Preferably, B27 containing vitamin A is 2%, Nicotinamide is 5mmol / L, 2-Phospho-L-ascorbic acid trisodium salt is 100μmol / L, 1-Oleoyl Lysophosphatidic Aci is 5μmol / L, D-erythro-Sphingosine-1-phosphate is 0.5μmol / L, SB431542 is 2.5μmol / L, CHIR99021 is 1.5μmol / L, Foskolin is 25μmol / L, EGF is 20ng / mL, Dexamethasone is 1μmol / L, and Heparin is 100μg / mL.

[0064] In some embodiments of the present invention, the method for preparing hepatic progenitor cells comprises:

[0065] The time for inducing hepatoblasts to differentiate into hepatic progenitor cells is 1-3 days, preferably 2 days.

[0066] In some embodiments of the present invention, the method for preparing hepatic progenitor cells comprises:

[0067] The method also includes the step of detecting the molecular markers specifically expressed by the obtained liver precursor cells;

[0068] Preferably, the detection is performed by flow cytometry;

[0069] Preferably, the molecular marker specifically expressed by hepatic precursor cells is alpha-fetoprotein (AFP); preferably, the proportion of alpha-fetoprotein-positive cells is ≥70%, ≥80%, ≥90% or ≥95%.

[0070] Another aspect of the present invention relates to a method for preparing mature hepatocytes, comprising the following steps:

[0071] Prepare hepatic progenitor cells by the method for preparing hepatic progenitor cells according to any one of the present invention; and

[0072] Induce hepatic progenitor cells to differentiate into mature hepatocytes.

[0073] In some embodiments of the present invention, the method for maturing hepatocytes, wherein:

[0074] The medium used to induce differentiation of hepatic progenitor cells into mature hepatocytes was William's medium E;

[0075] Preferably, William's medium E is supplemented with B27 containing vitamin A, Foskolin and SB431542;

[0076] Preferably, B27 containing vitamin A is 1.6%-2.4%, Foskolin is 23-28 μmol / L, and SB431542 is 2.3-2.8 μmol / L;

[0077] Preferably, the B27 containing vitamin A is 2%, the Foskolin is 25 μmol / L, and the SB431542 is 2.5 μmol / L.

[0078] In some embodiments of the present invention, the method for maturing hepatocytes, wherein:

[0079] The time for inducing hepatic progenitor cells to differentiate into mature hepatocytes is 5-12 days, preferably 9-10 days.

[0080] In some embodiments of the present invention, the method for maturing hepatocytes, wherein:

[0081] The method also includes the step of detecting molecular markers specifically expressed by the obtained mature hepatocytes;

[0082] Preferably, the detection is performed by flow cytometry;

[0083] Preferably, the molecular marker specifically expressed by mature hepatocytes is albumin (ALB). Preferably, the proportion of albumin-positive cells is ≥70%, ≥80%, ≥90% or ≥95%.

[0084] The present invention also relates to a method for preparing mature hepatocytes, comprising the following steps:

[0085] (1) Induce pluripotent stem cells to differentiate into endoderm cells;

[0086] (2) Induce endoderm cells to differentiate into foregut cells;

[0087] (3) Inducing foregut cells to differentiate into hepatoblasts;

[0088] (4) inducing hepatoblasts to differentiate into hepatic progenitor cells; and

[0089] (5) Induce hepatic progenitor cells to differentiate into mature hepatocytes;

[0090] Wherein, step (3) comprises the following steps:

[0091] (3-1) Induction of the first stage of differentiation of foregut cells into hepatoblasts; and

[0092] (3-2) Induction of the second stage of differentiation of foregut cells into hepatoblasts;

[0093] Among them, the culture medium used in the first stage is different from the culture medium used in the second stage.

[0094] In some embodiments of the present invention, the preparation method is characterized by any one or more of the following items ① to ⑥ (for example, any 2, 3, 4, 5 or all 6):

[0095] ① In step (1), the induction time is 3-6 days; preferably 4 days;

[0096] ② In step (2), the induction time is 1-3 days; preferably 2 days;

[0097] ③ In step (3-1), the induction time is 3-6 days; preferably 3-4 days;

[0098] ④ In step (3-2), the induction time is 2-6 days; preferably 3 days;

[0099] ⑤ In step (4), the induction time is 1-3 days; preferably 2 days; and

[0100] ⑥ In step (5), the induction time is 5-12 days; preferably 9-10 days.

[0101] In some embodiments of the present invention, the preparation method, wherein:

[0102] The induction of step (1) includes two stages, the first stage is 1 day, and the second stage is 2-5 days (eg, 2, 3, 4 or 5 days); preferably, the second stage is 3 days.

[0103] In some embodiments of the present invention, the preparation method, wherein:

[0104] The culture medium in step (1) is RPMI1640;

[0105] Preferably, RPMI1640 medium is supplemented with B27 without vitamin A, Activin A, BMP4, bFGF, Y27632, and optionally CHIR99021;

[0106] Preferably, the RPMI1640 medium in the first stage of induction in step (1) contains CHIR99021, and the RPMI1640 medium in the second stage does not contain CHIR99021;

[0107] Preferably, B27 without vitamin A is 1.6%-2.4%, Activin A is 80-120 ng / mL, BMP4 is 0.4-0.6 ng / mL, bFGF is 4-6 ng / mL, and Y27632 is 8-12 μmol / L;

[0108] Preferably, B27 without vitamin A is 2%, Activin A is 100 ng / mL, BMP4 is 0.5 ng / mL, bFGF is 5 ng / mL, and Y27632 is 10 μmol / L;

[0109] Optionally, CHIR99021 is 2.5-3.5 μmol / L;

[0110] Optionally, CHIR99021 is 3 μmol / L.

[0111] In some embodiments of the present invention, the preparation method, wherein:

[0112] The culture medium in step (2) is RPMI1640;

[0113] Preferably, the RPMI1640 medium is supplemented with B27, KGF, SB431542 and Y27632 without vitamin A;

[0114] Preferably, B27 without vitamin A is 1.6%-2.4%, KGF is 18-22 ng / mL, SB431542 is 2.3-2.8 μmol / L, and Y27632 is 8-12 μmol / L;

[0115] Preferably, B27 without vitamin A is 2%, KGF is 20 ng / mL, SB431542 is 2.5 μmol / L, and Y27632 is 10 μmol / L.

[0116] In some embodiments of the present invention, the preparation method, wherein:

[0117] The culture medium in step (3-1) is RPMI1640;

[0118] Preferably, the RPMI1640 medium is supplemented with B27, KGF, BMP2, BMP4, bFGF and Y27632 without vitamin A;

[0119] Preferably, B27 without vitamin A is 1.6%-2.4%, KGF is 18-22ng / mL, BMP2 is 18-22ng / mL, BMP4 is 80-120ng / mL, bFGF is 8-12ng / mL, and Y27632 is 8-12μmol / L;

[0120] Preferably, B27 without vitamin A is 2%, KGF is 20 ng / mL, BMP2 is 20 ng / mL, BMP4 is 100 ng / mL, bFGF is 10 ng / mL, and Y27632 is 10 μmol / L.

[0121] In some embodiments of the present invention, the preparation method, wherein:

[0122] The culture medium in step (3-2) is William's medium E;

[0123] Preferably, William's medium E culture medium is supplemented with B27, EGF, Foskolin and Y27632 without vitamin A;

[0124] Preferably, B27 without vitamin A is 1.6%-2.4%, EGF is 18-22ng / mL, Foskolin is 23-28μmol / L, and Y27632 is 8-12μmol / L;

[0125] Preferably, B27 without vitamin A is 2%, EGF is 20 ng / mL, Foskolin is 25 μmol / L, and Y27632 is 10 μmol / L.

[0126] In some embodiments of the present invention, the preparation method, wherein:

[0127] The culture medium in step (4) is: DMEM / F12 culture medium and William's medium E culture medium mixed in a ratio of 1:1 as the basal culture medium;

[0128] Preferably, the basal medium is supplemented with B27 containing vitamin A, Nicotinamide, 2-Phospho-L-ascorbic acid trisodium salt, 1-Oleoyl Lysophosphatidic Acid, D-erythro-Sphingosine-1-phosphate, SB431542, CHIR99021, Foskolin, EGF, Dexamethasone and Heparin;

[0129] Preferably, B27 containing vitamin A is 1.6%-2.4%, Nicotinamide is 4-6mmol / L, 2-Phospho-L-ascorbic acid trisodium salt is 80-120μmol / L, 1-Oleoyl Lysophosphatidic Aci is 4-6μmol / L, D-erythro-Sphingosine-1-phosphate is 0.4-0.6μmol / L, SB431542 is 2.3-2.8μmol / L, CHIR99021 is 1.3-1.8μmol / L, Foskolin is 22-28μmol / L, EGF is 18-22ng / mL, Dexamethasone is 0.8-1.2μmol / L, and Heparin is 80-120μg / mL;

[0130] Preferably, B27 containing vitamin A is 2%, Nicotinamide is 5mmol / L, 2-Phospho-L-ascorbic acid trisodium salt is 100μmol / L, 1-Oleoyl Lysophosphatidic Aci is 5μmol / L, D-erythro-Sphingosine-1-phosphate is 0.5μmol / L, SB431542 is 2.5μmol / L, CHIR99021 is 1.5μmol / L, Foskolin is 25μmol / L, EGF is 20ng / mL, Dexamethasone is 1μmol / L, and Heparin is 100μg / mL.

[0131] In some embodiments of the present invention, the preparation method, wherein:

[0132] The culture medium in step (5) is William's medium E;

[0133] Preferably, William's medium E is supplemented with B27 containing vitamin A, Foskolin and SB431542;

[0134] Preferably, B27 containing vitamin A is 1.6%-2.4%, Foskolin is 23-28 μmol / L, and SB431542 is 2.3-2.8 μmol / L;

[0135] Preferably, the B27 containing vitamin A is 2%, the Foskolin is 25 μmol / L, and the SB431542 is 2.5 μmol / L.

[0136] In some embodiments of the present invention, the preparation method, wherein:

[0137] The pluripotent stem cells used in step (1) are pretreated as follows:

[0138] Cultivate in mTeSR Plus medium for 12-48 hours, preferably 24-36 hours;

[0139] Preferably, mTeSR Plus medium is supplemented with Activin A, bFGF and Y27632;

[0140] Preferably, Activin A is 8-12 ng / mL, bFGF is 8-12 ng / mL, and Y27632 is 8-12 μmol / L;

[0141] Preferably, Activin A is 10 ng / mL, bFGF is 10 ng / mL, and Y27632 is 10 μmol / L.

[0142] In some embodiments of the present invention, the preparation method, wherein:

[0143] The pluripotent stem cells are human or mouse pluripotent stem cells;

[0144] Preferably, the pluripotent stem cells are chemically induced pluripotent stem cells, such as human chemically induced pluripotent stem cells or mouse chemically induced pluripotent stem cells.

[0145] In some embodiments of the present invention, the preparation method further comprises the step of detecting the molecular markers specifically expressed by the cells obtained in step (1), step (4) and / or step (5);

[0146] Preferably, the detection is performed by flow cytometry;

[0147] Preferably, the molecular markers specifically expressed by the cells obtained in step (1) are FOXA2 and SOX17; preferably, the proportion of FOXA2 and SOX17 double-positive cells is ≥60%, ≥70% or ≥80%;

[0148] Preferably, the molecular marker specifically expressed by the cells obtained in step (4) is alpha-fetoprotein (AFP); preferably, the proportion of alpha-fetoprotein-positive cells is ≥70%, ≥80%, ≥90% or ≥95%;

[0149] Preferably, the molecular marker specifically expressed by the cells obtained in step (5) is albumin (ALB). Preferably, the proportion of albumin-positive cells is ≥70%, ≥80%, ≥90% or ≥95%.

[0150] Another aspect of the present invention relates to a culture medium composition comprising:

[0151] William's medium E culture medium and RPMI1640 culture medium, wherein the William's medium E culture medium and the RPMI1640 culture medium are not mixed; and

[0152] Vitamin A-free B27, EGF, Foskolin, KGF, BMP2, BMP4, bFGF and Y27632.

[0153] In some embodiments of the present invention, the culture medium combination further includes DMEM / F12 culture medium; preferably, it also includes mTeSR Plus culture medium.

[0154] In some embodiments of the present invention, the culture medium composition comprises:

[0155] Nicotinamide, 2-Phospho-L-ascorbic acid trisodium salt, 1-OleoylLysophosphatidic Acid, D-erythro-Sphingosine-1-phosphate, SB431542, CHIR99021, Dexamethasone and Heparin.

[0156] In some embodiments of the present invention, the culture medium composition comprises:

[0157] 1) William's medium E, and B27 without vitamin A, EGF, Foskolin, and Y27632 mixed or not mixed with William's medium E; and

[0158] 2) RPMI1640 medium, and B27, KGF, BMP2, BMP4, bFGF and Y27632 without vitamin A, mixed or not mixed with RPMI1640 medium.

[0159] In some embodiments of the present invention, the culture medium combination further comprises any one or more selected from the following items 3) to 8):

[0160] 3) RPMI1640 medium, and B27 without vitamin A, Activin A, BMP4, bFGF, Y27632 and CHIR99021 mixed or not mixed with RPMI1640 medium;

[0161] 4) RPMI1640 medium, and B27 without vitamin A, Activin A, BMP4, bFGF and Y27632 mixed or not mixed with RPMI1640 medium;

[0162] 5) RPMI1640 medium, and B27, KGF, SB431542 and Y27632 without vitamin A mixed or not mixed with RPMI1640 medium;

[0163] 6) DMEM / F12 medium and William's medium E medium, wherein the DMEM / F12 medium and William's medium E medium are mixed or not mixed; and

[0164] B27 with vitamin A, Nicotinamide, 2-Phospho-L-ascorbic acid trisodium salt, 1-OleoylLysophosphatidic Acid, D-erythro-Sphingosine-1-phosphate, SB431542, CHIR99021, Foskolin, EGF, Dexamethasone and Heparin mixed or not with DMEM / F12 medium and William's medium E medium;

[0165] 7) William's medium E, and B27 containing vitamin A, Foskolin, and SB431542 mixed or not mixed with William's medium E; and

[0166] 8) mTeSR Plus medium, and Activin A, bFGF, and Y27632 mixed or not mixed with mTeSR Plus medium.

[0167] Another aspect of the present invention relates to a kit comprising the culture medium combination according to any one of the present invention.

[0168] Y-27632 is a selective ROCK1 and ROCK2 inhibitor with Ki values ​​of 140 nM and 300 nM, respectively, in a cell-free assay, which is more than 200 times more potent than other kinases including PKC, cAMP-dependent protein kinase, MLCK and PAK. The structural formula of Y-27632 is shown in Formula I below.

[0169]

[0170] In the present invention, if not otherwise specified, the reagent B27 is a product of GIBCO, such as B-27 Serum-Free Supplement (50X) liquid; it includes B27 without vitamin A (Cat. No. 17504001) and B27 with vitamin A (Cat. No. 17504044). Its concentration is 50X, preferably 50 times diluted according to the final concentration, at which time the concentration is expressed as 2% (volume / volume %).

[0171] In the present invention, unless otherwise specified, the “first” (eg, first stage) or “second” (eg, second stage) is merely for reference distinction and does not have a particular meaning of order.

[0172] In the present invention, unless otherwise specified, the "I" (e.g., stage I) or "II" (e.g., stage II) is merely for reference and does not have a particular meaning of order.

[0173] Advantageous Effects of the Invention

[0174] The present invention achieves any one or more of the following technical effects:

[0175] (1) The method for preparing mature hepatocytes of the present invention has a high efficiency in preparing mature hepatocytes;

[0176] (2) The mature hepatocytes prepared by the present invention have a higher degree of compatibility with primary hepatocytes;

[0177] (3) The hepatoblasts prepared by the present invention have a higher degree of compatibility with the hepatoblasts in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0178] Figures 1A to 1F :Induction and differentiation of chemically induced pluripotent stem cells (hCiPSCs) into hepatic progenitor cells (hiHPCs) and identification. Among them:

[0179] Figure 1A :Flowchart of differentiation of human chemically induced pluripotent stem cells into hepatocytes.

[0180] Figure 1B : Flow cytometry analysis of the proportion of FOXA2 and SOX17 double-positive cells in the endoderm cell population.

[0181] Figure 1C : Flow cytometry analysis of the proportion of AFP-positive cells in the hepatic progenitor cell population.

[0182] Figure 1D : QPCR was used to detect the expression of multiple precursor cell-related genes in induced pluripotent stem cells (hCiPSCs), three differentiated hepatic progenitor cells (hiHPCs), hepatoma cell line (HepG2) and primary hepatocytes (F-PHHs). n=3.

[0183] Figure 1E :Immunofluorescence staining was used to detect the expression of AFP, CK18, HNF4A, and Ki67 in differentiated hepatic progenitor cells. Scale bar: 50 μm.

[0184] Figure 1F : Comparative transcriptome analysis of induced pluripotent stem cells (hCiPSCs), differentiation-derived hepatic progenitor cells (hiHPCs), fetal liver cells (hFLCs), and primary hepatocytes (F-PHHs).

[0185] FIG. 2A to FIG. 2I :Identification of mature hepatocytes (hiHEPs) after maturation and differentiation of hepatic progenitor cells (hiHPCs).

[0186] Figure 2A :Morphology of differentiated mature hepatocytes. Scale bar: 50 μm.

[0187] Figure 2B : Flow cytometry analysis of the proportion of ALB-positive cells in the mature hepatocyte population.

[0188] Figure 2C :Immunofluorescence staining was used to detect the expression of ALB, CYP3A4, and HNF4A in differentiated mature hepatocytes. Scale bar: 50 μm.

[0189] Figure 2D : QPCR was used to detect the expression of multiple liver-related genes in induced pluripotent stem cells (hCiPSCs), three differentiated mature hepatocytes (hiHEPs) and primary hepatocytes (F-PHHs). n=3.

[0190] Figure 2E : Comparative transcriptome analysis of induced pluripotent stem cells, differentiated mature hepatocytes, and primary hepatocytes.

[0191] Figure 2F :Detection of the ability of differentiated mature hepatocytes to absorb low-density lipoprotein (left), synthesize glycogen (middle), and store lipids (right). Scale bar: 50 μm.

[0192] Figure 2G : ELISA was used to detect the ability of induced pluripotent stem cells (hCiPSCs), differentiated mature hepatocytes (hiHEPs), hepatic progenitor cells (hiHPCs) and primary hepatocytes (F-PHHs) to secrete albumin. n=3.

[0193] Figure 2H : Comparison of urea synthesis capacity of induced pluripotent stem cells (hCiPSCs), differentiated mature hepatocytes (hiHEPs), hepatic progenitor cells (hiHPCs) and primary hepatocytes (F-PHHs). n=3.

[0194] Fig.2I : Comparison of the metabolic capacity of drug metabolizing enzyme CYP3A4 in induced pluripotent stem cells (hCiPSCs), differentiated mature hepatocytes (hiHEPs), hepatic progenitor cells (hiHPCs), and primary hepatocytes (F-PHHs). n=3.

[0195] Figure 3A : Flow cytometry comparison of the proportion of PROX1 positive cells in hepatoblasts produced by the original protocol (hepatoblast I) and the new protocol of the present invention (hepatoblast II), n=3.

[0196] Figure 3B : QPCR comparison of the expression of key hepatic attribute genes in hepatoblasts generated by the original protocol (hepatoblast I) and the new protocol of the present invention (hepatoblast II), n=3.

[0197] Figure 4A :Yamanaka factors establish hiPSCs differentiation into endoderm cells.

[0198] Figure 4B :Construction of hCiPSCs differentiation into endoderm cells using small chemical molecules.

[0199] Figure 5A :hCiPSCs-hiHPCs.

[0200] Figure 5B : Yamanaka factors-hiPSCs-hiHPC.

[0201] Figure 6 :QPCR detected the expression of multiple progenitor cell-related genes.

[0202] Fig. 7A :hCiPSCs were differentiated into hiHEPs.

[0203] Figure 7B :Yamanaka factors were used to establish hiPSCs and differentiate them into hiHEPs.

[0204] Figure 8 :QPCR detected the expression of multiple liver property-related genes.

[0205] Fig. 9A : Albumin secretion level.

[0206] Fig. 9B : Urea synthesis level. DETAILED DESCRIPTION

[0207] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0208] Some of the reagents involved in the present invention are shown in Table A below.

[0209] Table A

[0210]

[0211]

[0212] Example 1: Preparation of hepatocytes from human chemically induced pluripotent stem cells

[0213] Flow chart of human chemically induced pluripotent stem cells differentiation into hepatocytes Figure 1A The specific steps are as follows:

[0214] (1) Pretreatment of pluripotent stem cells before differentiation to promote their subsequent induced differentiation

[0215] Chemically induced human pluripotent stem cells 8When the confluence reaches 70% to 80%, the cells are digested with Accutase to become single cells and seeded at a density of 300,000 cells / well in a 12-well plate coated with a matrix such as Matrigel. The seeding medium is mTeSR Plus supplemented with the following components: Activin A (10 ng / mL), bFGF (10 ng / mL), and Y27632 (10 μmol / L).

[0216] Differentiation was initiated 24 hours after seeding.

[0217] (2) The first stage of differentiation: inducing the pretreated pluripotent stem cells to differentiate into endoderm cells

[0218] The composition of the induction medium on the first day: RPMI1640 as the basic medium, supplemented with 2% B27 (without vitamin A), Activin A (100 ng / mL), BMP4 (0.5 ng / mL), bFGF (5 ng / mL), CHIR99021 (3 μmol / L), and Y27632 (10 μmol / L).

[0219] The composition of the induction medium from the second day to the fourth day: RPMI1640 as the basic medium, supplemented with 2% B27 (without vitamin A), Activin A (100 ng / mL), BMP4 (0.5 ng / mL), bFGF (5 ng / mL), and Y27632 (10 μmol / L).

[0220] After the induction of endoderm differentiation was completed, flow cytometry was used to detect the molecular markers FOXA2 and SOX17 specifically expressed by endoderm cells. The results were as follows: Figure 1B The results showed that the proportion of FOXA2 and SOX17 double-positive cells was about 80%.

[0221] (3) The second stage of differentiation: inducing the differentiation of endoderm cells into foregut cells

[0222] The composition of the induction medium at this stage: RPMI1640 as the basic medium, supplemented with 2% B27 (without vitamin A), KGF (20 ng / mL), SB431542 (2.5 μmol / L), and Y27632 (10 μmol / L).

[0223] After 2 days of induction in this stage, the next induction stage begins.

[0224] (4) The third stage of differentiation: induction of the first stage of differentiation of foregut cells into hepatoblasts

[0225] The components of the induction medium from the first to the third day of this stage: RPMI1640 as the basic medium, supplemented with 2% B27 (without vitamin A), KGF (20 ng / mL), BMP2 (20 ng / mL), BMP4 (100 ng / mL), bFGF (10 ng / mL), and Y27632 (10 μmol / L).

[0226] After 3 days of induction, the cells were digested into single cells using Accutase and then seeded into Matrigel-coated 12-well plates at a 1:1 ratio using the same culture medium.

[0227] The next induction phase began 24 hours after inoculation.

[0228] (5) Differentiation stage 4: Induction of stage 2 differentiation of foregut cells into hepatoblasts

[0229] The composition of the induction medium at this stage: William's medium E as the basic medium, supplemented with 2% B27 (without vitamin A), EGF (20 ng / mL), Foskolin (25 μmol / L), and Y27632 (10 μmol / L).

[0230] After 3 days of induction, the cells entered the next induction phase.

[0231] (6) Differentiation stage 5: Inducing differentiation of hepatoblasts into hepatic progenitor cells

[0232] The components of the induction medium at this stage: DMEM / F12 medium and William's medium E medium were mixed in a ratio of 1:1 as the basic medium, supplemented with 2% B27 (containing vitamin A), Nicotinamide (5mmol / L), 2-Phospho-L-ascorbic acid trisodium salt (PVC, 100μmol / L), 1-Oleoyl Lysophosphatidic Acid (LPA, 5μmol / L), D-erythro-Sphingosine-1-phosphate (S1P, 0.5μmol / L), SB431542 (2.5μmol / L), CHIR99021 (1.5μmol / L), Foskolin (25μmol / L), EGF (20ng / mL), Dexamethasone (1μmol / L), and Heparin (100μg / mL).

[0233] After 2 days of induction, the culture medium was used for digestion at a 1:1 ratio and seeded into a 12-well plate coated with matrigel to obtain the first generation of hepatic progenitor cells (hiHPCs). hiHPCs can be passaged and expanded multiple times under this culture condition.

[0234] To identify the properties of hepatic progenitor cells, flow cytometry was first used to detect the proportion of hepatic progenitor cells expressing the liver-specific molecular marker AFP (alpha-fetoprotein). Figure 1C The results showed that the proportion of AFP-positive cells reached 97.67%, proving that the purity of liver progenitor cells was high.

[0235] Then, QPCR was used to detect the expression of liver attribute-related genes in liver progenitor cells. The results were as follows: Figure 1D The results showed that hepatic progenitor cells highly expressed liver-specific genes such as AFP, ALB, FOXA2, GATA4, CEBPA, PROX1, DLK1, HHEX and HNF4A.

[0236] To further demonstrate the expression of liver progenitor cell-specific genes at the protein level, AFP, HNF4A, CK8, and Ki67 were detected by immunofluorescence staining. Figure 1E The results showed that the differentiated hepatic progenitor cells all expressed the above-mentioned hepatic progenitor cell attribute-related proteins.

[0237] In addition, in order to compare the similarity of the hepatic progenitor cells with fetal liver cells at the transcriptome level, the transcriptomes of pluripotent stem cells, hepatic progenitor cells and fetal liver cells were analyzed by RNA-seq. Figure 1F The results showed that the differentiated hepatic progenitor cells were similar to fetal liver cells at the transcriptome level, but were significantly different from the pluripotent stem cells from which they originated.

[0238] (7) Differentiation stage 6: Inducing differentiation of hepatic progenitor cells into mature hepatocytes

[0239] When the hepatic progenitor cells reached a confluence of 100%, they were cultured for 9 days using a maturation induction medium to further induce differentiation into mature hepatocytes. The composition of the maturation induction medium: William's medium E as the basal medium, supplemented with 2% B27 (containing vitamin A), Foskolin (25 μmol / L), and SB431542 (2.5 μmol / L).

[0240] After maturation induction, mature hepatocytes have the typical polygonal morphology of primary hepatocytes ( Figure 2A Flow cytometry analysis showed that more than 96% of the differentiated cells expressed ALB, a key marker of hepatocytes ( Figure 2BImmunofluorescence detection results showed that these differentiated and mature hepatocytes co-expressed ALB, the key hepatocyte transcription factor HNF4A and the key drug metabolizing enzyme CYP3A4 ( Figure 2C ).

[0241] QPCR confirmed that differentiated and mature hepatocytes highly expressed multiple key liver-attribute genes, including key hepatocyte transcription factor genes, urea cycle-related genes, drug metabolism and coagulation-related genes, and their expression levels were comparable to those of primary hepatocytes ( Figure 2D ).

[0242] In addition, transcriptome sequencing results showed that hiHEPs generated by hCiPSCs differentiation and maturation had similar gene expression profiles to primary hepatocytes in multiple key hepatocyte functions (such as drug metabolism, fatty acids, glucose, and lipid cholesterol) ( Figure 2E ).

[0243] Furthermore, multiple functions of differentiated hepatocytes were tested, and the results were as follows: Figure 2F The results showed that hiHEPs differentiated from hCiPSCs have the functions of absorbing low-density lipoprotein (LDL), accumulating lipid droplets and synthesizing glycogen. In addition, the albumin secretion level in hiHEPs differentiated from hCiPSCs ( Figure 2G ) and urea synthesis levels were also comparable to those of primary hepatocytes ( Figure 2H ); The metabolic activity of the key drug-metabolizing enzyme CYP3A4 in differentiated hiHEPs is also comparable to that in primary hepatocytes ( Fig.2I ).

[0244] Example 2: Experimental verification of the high efficiency of the differentiation scheme of pluripotent stem cells into hepatocytes

[0245] Originally established differentiation scheme 9 The method contains only one stage of hepatoblast differentiation, resulting in low efficiency of hepatoblast generation and an imperfect gene expression network. The ratio of PROX1-positive cells in the hepatoblasts generated by the original differentiation scheme and the hepatoblasts obtained in step (5) of Example 1 was analyzed by flow cytometry. The results are shown in Figure 3A As shown. The results showed that the proportion of PROX1-positive hepatoblasts in the original differentiation scheme was about 60%, and the proportion of PROX1-positive hepatoblasts in the new scheme of the present invention was about 80%. The results showed that the hepatoblast differentiation strategy of the new scheme of the present invention obtained a higher proportion of PROX1-positive hepatoblasts.

[0246] The expression of key liver attribute-related genes in the hepatoblasts produced by the previously established differentiation scheme and the hepatoblasts produced by the new differentiation scheme of the present invention were further compared by QPCR analysis. The results are as follows: Figure 3BThe results showed that the expression levels of multiple key liver-attribute genes such as CK7, CK8, CK18, SOX9, SPINK1, and PROX1 in the hepatoblasts produced by the new differentiation scheme of the present invention were significantly higher than those in the hepatoblasts produced by the previously established differentiation scheme, indicating that the hepatoblasts induced by the new scheme of the present invention have a more complete liver-attribute gene expression network and are more compatible with the hepatoblasts in vivo.

[0247] Example 3: Universal validation experiment of different sources of human pluripotent stem cells (hiPSC) to differentiate into hepatocytes

[0248] (1) Directed differentiation of endoderm (obtained endoderm cells)

[0249] The two hiPSC samples are as follows:

[0250] Yamanaka factors establish hiPSCs: xeno-free hiPS Cell-1 (cauliscell, 200101)

[0251] The method for preparing hCiPSCs by chemical small molecule construction can refer to the prior art, such as the reference GuanJ et.al. 10 .

[0252] Referring to step (2) in Example 1, two hiPSC samples from different established lineages were differentiated into endoderm cells, and flow cytometry was used to detect whether the differentiated cells expressed high proportions of endoderm marker genes FOXA2 (ordinate) and SOX17 (abscissa). FIG. 4A to FIG. 4B The results show that high-purity endoderm cells can be obtained from the pluripotent stem cells from the two induced lineages through the induction process in step 2 of Example 1. The obtained endoderm cells are used for the following operations.

[0253] (2) Obtaining a high proportion of hepatic progenitor cells

[0254] The endoderm cells obtained in step (1) of Example 2 were further differentiated according to steps (3) to (6) of Example 1 to obtain liver progenitor cells, and the expression of liver-specific molecular marker AFP (alpha-fetoprotein) was detected. The results were as follows: Figure 5A and Figure 5B The results showed that the proportion of AFP-positive cells was >95%, proving that the purity of hepatic progenitor cells was high.

[0255] (3) Major gene expression in hepatic progenitor cells

[0256] The endoderm cells obtained in step (1) of Example 2 were further differentiated according to steps (3) to (6) of Example 1 to obtain hepatic progenitor cells. To further prove at the RNA level that the hepatic progenitor cells differentiated from hiPSCs of different established sources have comparable hepatic gene expression levels, hCiPSCs were used as negative controls to compare the gene expression of hiHPCs differentiated from Yamanaka factors-derived hiPSCs and hiHPCs differentiated from chemical small molecule-derived hCiPSCs in three cell lines: 10.7, C16, and C19. The results are shown in Figure 2. Figure 6 The results show that the two induced pluripotent stem cells can be used to obtain hepatic progenitor cells with similar expression of liver-related genes through the continuous induction process of steps (2) to (6) in Example 1.

[0257] (4) Obtaining a high proportion of hepatocytes

[0258] The hepatic progenitor cells obtained in step (2) of Example 2 were further differentiated into mature hepatocytes according to step (7) of Example 1, and the expression ratio of ALB, a key marker of hepatocytes, was detected by flow cytometry. The results were as follows: Fig. 7A and Figure 7B The results showed that the proportion of hepatocytes expressing ALB was greater than 85% after multi-step differentiation of hiPSCs from different established lines.

[0259] (5) Major gene expression in hepatocytes

[0260] The hepatic progenitor cells obtained in step (2) of Example 2 were further differentiated into mature hepatocytes according to step (7) of Example 1. To further prove at the RNA level that the hepatocytes differentiated from hiPSCs of different established lines had similar expression levels of hepatic attribute genes, the gene expression of hiHEPs differentiated from three hCiPSC lines 10.7, C16, and C19 was compared with that of hiHEPs differentiated from hiPSCs of Yamanaka factors established lines using hCiPSCs and hiHPCs differentiated from hCiPSCs as negative controls and PHH as positive controls. Figure 8 The results showed that the two induced lineage-derived pluripotent stem cells could be used to obtain mature hepatocytes with similar expression levels of multiple key liver-attribute genes, including key hepatocyte transcription factor genes, urea cycle-related genes, and drug metabolism and coagulation-related genes, and their expression levels were comparable to those of primary hepatocytes after the continuous induction process of steps (2) to (7) in Example 1.

[0261] (6) Function of mature hepatocytes

[0262] At the same time, the albumin secretion level and urea synthesis level of hiHEPs differentiated from hiPSCs of different established lines were compared. The negative control was hCiPSCs and hiHPCs differentiated from hCiPSCs, the positive control was PHH, and the experimental group was hiHEPs differentiated from three hCiPSC cell lines 0.7, C16, and C19, and hiHEPs differentiated from Yamanaka factors established hiPSCs. The results are shown in Fig. 9A and Fig. 9B The results showed that the albumin secretion level of mature hepatocytes obtained from the two induced pluripotent stem cells through the continuous induction process of steps (2) to (7) in Example 1 was comparable to that of primary hepatocytes, and the urea secretion level was not weaker than that of primary hepatocytes.

[0263] References:

[0264] 1.Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stemcells from adult human fibroblasts by defined factors. Cell. 2007; 131(5):861-872.

[0265] 2. Raab S, Klingenstein M, Liebau S, Linta LA Comparative View on HumanSomatic Cell Sources for iPSC Generation. Stem Cells Int. 2014; 2014:768391.

[0266] 3. Thomson JA. Embryonic stem cell lines derived from human blastocysts (vol 282, pg 1147, 1998). Science. 1998; 282 (5395): 1827-1827.

[0267] 4. Liuyang S, Wang G, Wang Y, et al. Highly efficient and rapid generation of human pluripotent stem cells by chemical reprogramming. Cell StemCell. 2023; 30(4):450-459 e459.

[0268] 5.Du YY,Liang Z,Wang S,et al.Human pluripotent stem-cell-derivedislets ameliorate diabetes in non-human primates.Nature Medicine.2022.

[0269] 6.Si-Tayeb K,Noto FK,Nagaoka M,et al.Highly efficient generation ofhuman hepatocyte-like cells from induced pluripotent stemcells.Hepatology.2010;51(1):297-305.

[0270] 7.Yamashita T,Takayama K,Sakurai F,Mizuguchi H.Billion-scaleproduction of hepatocyte-like cells from human induced pluripotent stemcells.Biochem Biophys Res Commun.2018;496(4):1269-1275.

[0271] 8.Ng S,Schwartz RE,March S,et al.Human iPSC-derived hepatocyte-likecells support Plasmodium liver-stage infection in vitro.Stem CellReports.2015;4(3):348-359.

[0272] 9.Chen S,Wang J,Ren H,et al.Hepatic spheroids derived from humaninduced pluripotent stem cells in bio-artificial liver rescue porcine acuteliver failure.Cell research.2020;30(1):95-97.

[0273] 10.Guan J, Wang G, Wang J, et al. Chemical reprogramming of human somaticcells to pluripotent stem cells. Nature. 2022May; 605(7909):325-331.

[0274] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all the teachings disclosed, various modifications and replacements can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A method for preparing hepatoblasts, comprising the following steps: Through the first stage induction and the second stage induction, the foregut cells are induced to differentiate into hepatoblasts; in, The culture medium used for the first stage of induction was different from that used for the second stage of induction.

2. The method for preparing hepatoblasts according to claim 1, wherein: The medium used for the first stage of induction was RPMI1640; Preferably, the RPMI1640 medium is supplemented with B27, KGF, BMP2, BMP4, bFGF and Y27632 without vitamin A; Preferably, B27 without vitamin A is 1.6%-2.4%, KGF is 18-22ng / mL, BMP2 is 18-22ng / mL, BMP4 is 80-120ng / mL, bFGF is 8-12ng / mL, and Y27632 is 8-12μmol / L; Preferably, B27 without vitamin A is 2%, KGF is 20 ng / mL, BMP2 is 20 ng / mL, BMP4 is 100 ng / mL, bFGF is 10 ng / mL, and Y27632 is 10 μmol / L.

3. The method for preparing hepatoblasts according to any one of claims 1 to 2, wherein: The medium used for the second stage of induction was William's medium E; Preferably, William's medium E culture medium is supplemented with B27, EGF, Foskolin and Y27632 without vitamin A; Preferably, B27 without vitamin A is 1.6%-2.4%, EGF is 18-22ng / mL, Foskolin is 23-28μmol / L, and Y27632 is 8-12μmol / L; Preferably, B27 without vitamin A is 2%, EGF is 20 ng / mL, Foskolin is 25 μmol / L, and Y27632 is 10 μmol / L.

4. The method for preparing hepatoblasts according to any one of claims 1 to 3, wherein: The induction time of the first stage induction is 3-6 days, preferably 3-4 days; and / or The induction time of the second stage induction is 2-6 days, preferably 3 days.

5. The method for preparing hepatoblasts according to any one of claims 1 to 4, wherein: The product of the first stage induction is digested into single cells, and then inoculated with the same medium as that for the first stage induction and cultured for 12-48 hours, preferably 24-36 hours; then the second stage induction is performed.

6. The method for preparing hepatoblasts according to any one of claims 1 to 5, wherein: The foregut cells are obtained by inducing differentiation of endoderm cells.

7. The method for preparing hepatoblasts according to claim 6, wherein: The culture medium used to induce endoderm cell differentiation to obtain foregut cells was RPMI1640; Preferably, the RPMI1640 medium is supplemented with B27, KGF, SB431542 and Y27632 without vitamin A; Preferably, B27 without vitamin A is 1.6%-2.4%, KGF is 18-22 ng / mL, SB431542 is 2.3-2.8 μmol / L, and Y27632 is 8-12 μmol / L; Preferably, B27 without vitamin A is 2%, KGF is 20 ng / mL, SB431542 is 2.5 μmol / L, and Y27632 is 10 μmol / L.

8. The method for preparing hepatoblasts according to any one of claims 6 to 7, wherein: The time for inducing endoderm cells to differentiate into foregut cells is 1-3 days, preferably 2 days.

9. The method for preparing hepatoblasts according to any one of claims 6 to 8, wherein: Obtaining the endoderm cells by inducing differentiation of pluripotent stem cells; Preferably, inducing pluripotent stem cells to differentiate into endoderm cells through stage I induction and stage II induction in sequence; The culture medium used in the induction of stage I contains CHIR99021, while the culture medium used in the induction of stage II does not contain CHIR99021.

10. The method for preparing hepatoblasts according to claim 9, wherein: The culture medium used to induce pluripotent stem cells to differentiate into endoderm cells was RPMI1640; Preferably, RPMI1640 medium is supplemented with B27 without vitamin A, Activin A, BMP4, bFGF, Y27632, and optionally CHIR99021; Preferably, the RPMI1640 medium in stage I contains CHIR99021, and the RPMI1640 medium in stage II does not contain CHIR99021; Preferably, B27 without vitamin A is 1.6%-2.4%, Activin A is 80-120 ng / mL, BMP4 is 0.4-0.6 ng / mL, bFGF is 4-6 ng / mL, and Y27632 is 8-12 μmol / L; Preferably, B27 without vitamin A is 2%, Activin A is 100 ng / mL, BMP4 is 0.5 ng / mL, bFGF is 5 ng / mL, and Y27632 is 10 μmol / L; Optionally, CHIR99021 is 2.5-3.5 μmol / L; Optionally, CHIR99021 is 3 μmol / L.

11. The method for preparing hepatoblasts according to any one of claims 9 to 10, wherein: The time for inducing pluripotent stem cells to differentiate into endoderm cells is 3-6 days, preferably 4 days; Preferably, Phase I lasts for 1 day and Phase II lasts for 2-5 days; preferably, Phase II lasts for 3 days.

12. The method for preparing hepatoblasts according to any one of claims 9 to 11, wherein: The pluripotent stem cells are human or mouse pluripotent stem cells; Preferably, the pluripotent stem cells are chemically induced pluripotent stem cells, such as human chemically induced pluripotent stem cells or mouse chemically induced pluripotent stem cells.

13. The method for preparing hepatoblasts according to any one of claims 9 to 12, wherein: Pluripotent stem cells were pre-treated as follows: Cultivate in mTeSR Plus medium for 12-48 hours, preferably 24-36 hours; Preferably, mTeSR Plus medium is supplemented with Activin A, bFGF and Y27632; Preferably, Activin A is 8-12 ng / mL, bFGF is 8-12 ng / mL, and Y27632 is 8-12 μmol / L; Preferably, Activin A is 10 ng / mL, bFGF is 10 ng / mL, and Y27632 is 10 μmol / L.

14. The method for preparing hepatoblasts according to any one of claims 9 to 13, wherein: The method also includes the step of detecting molecular markers specifically expressed by the obtained endoderm cells; Preferably, the detection is performed by flow cytometry; Preferably, the molecular markers specifically expressed by endoderm cells are FOXA2 and SOX17; preferably, the proportion of FOXA2 and SOX17 double-positive cells is ≥60%, ≥70% or ≥80%.

15. A method for preparing hepatic progenitor cells, comprising the following steps: Preparing hepatoblasts by the method for preparing hepatoblasts according to any one of claims 1 to 14; and inducing the hepatoblasts to differentiate into hepatic progenitor cells.

16. The method for preparing hepatic progenitor cells according to claim 15, wherein: The culture medium used for inducing hepatoblasts to differentiate into hepatic progenitor cells is: DMEM / F12 culture medium and William's medium E culture medium are mixed in a mass ratio of (1:2) to (2:1) as the basic culture medium; preferably, DMEM / F12 culture medium and William's medium E culture medium are mixed in a mass ratio of 1:1 as the basic culture medium; Preferably, the basal medium is supplemented with B27 containing vitamin A, Nicotinamide, 2-Phospho-L-ascorbic acid trisodium salt, 1-Oleoyl Lysophosphatidic Acid, D-erythro-Sphingosine-1-phosphate, SB431542, CHIR99021, Foskolin, EGF, Dexamethasone and Heparin; Preferably, B27 containing vitamin A is 1.6%-2.4%, Nicotinamide is 4-6mmol / L, 2-Phospho-L-ascorbic acid trisodium salt is 80-120μmol / L, 1-Oleoyl Lysophosphatidic Aci is 4-6μmol / L, D-erythro-Sphingosine-1-phosphate is 0.4-0.6μmol / L, SB431542 is 2.3-2.8μmol / L, CHIR99021 is 1.3-1.8μmol / L, Foskolin is 22-28μmol / L, EGF is 18-22ng / mL, Dexamethasone is 0.8-1.2μmol / L, and Heparin is 80-120μg / mL; Preferably, B27 containing vitamin A is 2%, Nicotinamide is 5mmol / L, 2-Phospho-L-ascorbic acid trisodium salt is 100μmol / L, 1-Oleoyl Lysophosphatidic Aci is 5μmol / L, D-erythro-Sphingosine-1-phosphate is 0.5μmol / L, SB431542 is 2.5μmol / L, CHIR99021 is 1.5μmol / L, Foskolin is 25μmol / L, EGF is 20ng / mL, Dexamethasone is 1μmol / L, and Heparin is 100μg / mL.

17. The method for preparing hepatic progenitor cells according to any one of claims 15 to 16, wherein: The time for inducing hepatoblasts to differentiate into hepatic progenitor cells is 1-3 days, preferably 2 days.

18. The method for preparing hepatic progenitor cells according to any one of claims 15 to 17, wherein: The method also includes the step of detecting the molecular markers specifically expressed by the obtained liver precursor cells; Preferably, the detection is performed by flow cytometry; Preferably, the molecular marker specifically expressed by hepatic progenitor cells is alpha-fetoprotein; preferably, the proportion of alpha-fetoprotein-positive cells is ≥70%, ≥80%, ≥90% or ≥95%.

19. A method for preparing mature hepatocytes, comprising the following steps: preparing hepatic progenitor cells by the method for preparing hepatic progenitor cells according to any one of claims 15 to 18; and Induce hepatic progenitor cells to differentiate into mature hepatocytes.

20. The method for mature hepatocytes according to claim 19, wherein: The medium used to induce differentiation of hepatic progenitor cells into mature hepatocytes was William's medium E; Preferably, William's medium E is supplemented with B27 containing vitamin A, Foskolin and SB431542; Preferably, B27 containing vitamin A is 1.6%-2.4%, Foskolin is 23-28 μmol / L, and SB431542 is 2.3-2.8 μmol / L; Preferably, the B27 containing vitamin A is 2%, the Foskolin is 25 μmol / L, and the SB431542 is 2.5 μmol / L.

21. The method for maturing hepatocytes according to any one of claims 19 to 20, wherein: The time for inducing hepatic progenitor cells to differentiate into mature hepatocytes is 5-12 days, preferably 9-10 days.

22. The method for maturing hepatocytes according to any one of claims 19 to 21, wherein: The method also includes the step of detecting molecular markers specifically expressed by the obtained mature hepatocytes; Preferably, the detection is performed by flow cytometry; Preferably, the molecular marker specifically expressed by mature hepatocytes is albumin. Preferably, the proportion of albumin-positive cells is ≥70%, ≥80%, ≥90% or ≥95%.

23. A culture medium composition comprising: William's medium E culture medium and RPMI1640 culture medium, wherein the William's medium E culture medium and the RPMI1640 culture medium are not mixed; as well as B27 without vitamin A, B27 with vitamin A, EGF, Foskolin, KGF, BMP2, BMP4, bFGF, and Y27632.

24. The culture medium combination according to claim 23, further comprising DMEM / F12 culture medium; preferably, further comprising mTeSR Plus culture medium.

25. The culture medium combination according to any one of claims 23 to 24, comprising: Nicotinamide, 2-Phospho-L-ascorbic acid trisodium salt, 1-OleoylLysophosphatidic Acid, D-erythro-Sphingosine-1-phosphate, SB431542, CHIR99021, Dexamethasone and Heparin.

26. The culture medium combination according to any one of claims 23 to 25, comprising: 1) William's medium E, and B27 without vitamin A, EGF, Foskolin, and Y27632 mixed or not mixed with William's medium E; and 2) RPMI1640 medium, and B27, KGF, BMP2, BMP4, bFGF and Y27632 without vitamin A, mixed or not mixed with RPMI1640 medium.

27. The culture medium combination according to claim 26, further comprising any one or more selected from the following items 3) to 8): 3) RPMI1640 medium, and B27 without vitamin A, Activin A, BMP4, bFGF, Y27632 and CHIR99021 mixed or not mixed with RPMI1640 medium; 4) RPMI1640 medium, and B27 without vitamin A, Activin A, BMP4, bFGF and Y27632 mixed or not mixed with RPMI1640 medium; 5) RPMI1640 medium, and B27, KGF, SB431542 and Y27632 without vitamin A mixed or not mixed with RPMI1640 medium; 6) DMEM / F12 medium and William's medium E medium, wherein the DMEM / F12 medium and William's medium E medium are mixed or not mixed; and B27 with vitamin A, Nicotinamide, 2-Phospho-L-ascorbic acid trisodium salt, 1-OleoylLysophosphatidic Acid, D-erythro-Sphingosine-1-phosphate, SB431542, CHIR99021, Foskolin, EGF, Dexamethasone and Heparin mixed or not with DMEM / F12 medium and William's medium E medium; 7) William's medium E, and B27 containing vitamin A, Foskolin and SB431542 mixed or not mixed with William's medium E; and 8) mTeSR Plus medium, and Activin A, bFGF, and Y27632 mixed or not mixed with mTeSR Plus medium.

28. A kit comprising the culture medium combination according to any one of claims 23 to 27.

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