Method for preparing hepatoblast, hepatic progenitor cell, or mature hepatocyte

Abstract

The present invention belongs to the field of biopharmaceuticals, and relates to a method for preparing a hepatoblast, a hepatic progenitor cell, or a mature hepatocyte, and a combination of a culture media progenitor cell, or a mature hepatocyte, and a combination of culture media. Specifically, the present invention relates to a method for preparing a hepatoblast. The method comprises the following steps: inducing foregut cells to differentiate into hepatoblasts by means of a first-stage induction and a 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 hepatocyte is high in terms of efficiency and has good application prospects.

Description

Method for preparing hepatoblasts, hepatic precursor cells or mature hepatocytes

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the application with CN application number 202311133081.8 and application date September 4, 2023, and claims its priority. The entire content of the CN application is hereby introduced as a whole into this application. Technical Field

[0003] The present 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

[0004] The generation of induced human pluripotent stem cells provides a new approach for the in vitro differentiation and preparation of multiple functional cells, showing great application potential. 1 Recently, the inventors' team successfully induced human cells to become 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 .

[0005] Hepatocytes have important applications in drug screening, disease simulation, and cell therapy for end-stage liver diseases. In recent years, different strategies 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 to differentiate human induced 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 further mature into mature hepatocytes. 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 and enrichment processes, which prolongs the preparation cycle of hepatocytes and hinders their clinical application.

[0006] Currently, there is still a need to develop new methods for preparing mature hepatocytes.

[0007] Summary of the Invention

[0008] After extensive research and creative work, the present inventors have developed a method for preparing mature hepatocytes and a culture medium combination. The present inventors surprisingly discovered that the method of the present invention has a high efficiency in preparing mature hepatocytes. This provides the following invention:

[0009] One aspect of the present invention relates to a method for preparing hepatoblasts, comprising the following steps:

[0010] Through the first stage induction and the second stage induction, the foregut cells are induced to differentiate into hepatoblasts;

[0011] The culture medium used for the first stage of induction is different from the culture medium used for the second stage of induction.

[0012] 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;

[0013] Preferably, RPMI1640 medium is supplemented with B27, KGF, BMP2, BMP4, bFGF and Y27632 without vitamin A;

[0014] Preferably, B27 without vitamin A is 1.6%-2.4%, KGF is 18-22 ng / mL, BMP2 is 18-22 ng / mL, BMP4 is 80-120 ng / mL, bFGF is 8-12 ng / mL, and Y27632 is 8-12 μmol / L;

[0015] Preferably, the content of B27 without vitamin A is 2%, the content of KGF is 20 ng / mL, the content of BMP2 is 20 ng / mL, the content of BMP4 is 100 ng / mL, the content of bFGF is 10 ng / mL, and the content of Y27632 is 10 μmol / L.

[0016] 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;

[0017] Preferably, William's medium E is supplemented with B27, EGF, Foskolin and Y27632 without vitamin A;

[0018] Preferably, B27 without vitamin A is 1.6%-2.4%, EGF is 18-22 ng / mL, Foskolin is 23-28 μmol / L, and Y27632 is 8-12 μmol / L;

[0019] Preferably, the content of B27 without vitamin A is 2%, the content of EGF is 20 ng / mL, the content of Foskolin is 25 μmol / L, and the content of Y27632 is 10 μmol / L.

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

[0021] The induction time of the first stage induction is 3-6 days, preferably 3-4 days; and / or

[0022] The induction time of the second stage induction is 2-6 days, preferably 3 days.

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

[0024] The product of the first stage induction is digested into single cells, which are then inoculated using the same medium as that used for the first stage induction and cultured for 12-48 hours, preferably 24-36 hours; and then the second stage induction is performed.

[0025] In some embodiments of the present invention, in the method for preparing hepatoblasts, the foregut cells are obtained by inducing differentiation of endoderm cells.

[0026] In some embodiments of the present invention, the method for preparing hepatoblasts, wherein,

[0027] The culture medium used to induce endoderm cell differentiation to obtain foregut cells was RPMI1640;

[0028] Preferably, RPMI1640 medium is supplemented with B27, KGF, SB431542 and Y27632 without vitamin A;

[0029] 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;

[0030] Preferably, the content of B27 without vitamin A is 2%, the content of KGF is 20 ng / mL, the content of SB431542 is 2.5 μmol / L, and the content of Y27632 is 10 μmol / L.

[0031] In some embodiments of the present invention, the method for preparing hepatoblasts, wherein:

[0032] The time for inducing endoderm cells to differentiate into foregut cells is 1-3 days, preferably 2 days.

[0033] 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;

[0034] Preferably, the pluripotent stem cells are induced to differentiate into endoderm cells through stage I induction and stage II induction in sequence;

[0035] The culture medium used in the induction phase I contained CHIR99021, while the culture medium used in the induction phase II did not contain CHIR99021.

[0036] In some embodiments of the present invention, the method for preparing hepatoblasts, wherein,

[0037] The culture medium used to induce pluripotent stem cells to differentiate into endoderm cells was RPMI1640;

[0038] Preferably, RPMI1640 medium is supplemented with B27 without vitamin A, Activin A, BMP4, bFGF, Y27632, and optionally CHIR99021;

[0039] Preferably, the RPMI1640 medium in stage I contains CHIR99021, and the RPMI1640 medium in stage II does not contain CHIR99021;

[0040] 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;

[0041] 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;

[0042] Optionally, CHIR99021 is 2.5-3.5 μmol / L;

[0043] Optionally, CHIR99021 is 3 μmol / L.

[0044] In some embodiments of the present invention, the method for preparing hepatoblasts, wherein,

[0045] The time for inducing pluripotent stem cells to differentiate into endoderm cells is 3-6 days, preferably 4 days;

[0046] Preferably, stage I is 1 day and stage II is 2-5 days; preferably, stage II is 3 days.

[0047] In some embodiments of the present invention, in the method for preparing hepatoblasts, the pluripotent stem cells are human or mouse pluripotent stem cells;

[0048] 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.

[0049] In some embodiments of the present invention, the method for preparing hepatoblasts, wherein,

[0050] Pluripotent stem cells were pre-treated as follows:

[0051] Cultivate in mTeSR Plus medium for 12-48 hours, preferably 24-36 hours;

[0052] Preferably, mTeSR Plus medium is supplemented with Activin A, bFGF, and Y27632;

[0053] Preferably, Activin A is 8-12 ng / mL, bFGF is 8-12 ng / mL, and Y27632 is 8-12 μmol / L;

[0054] Preferably, Activin A is 10 ng / mL, bFGF is 10 ng / mL, and Y27632 is 10 μmol / L.

[0055] In some embodiments of the present invention, the method for preparing hepatoblasts, wherein,

[0056] The method further comprises the step of detecting molecular markers specifically expressed by the obtained endoderm cells;

[0057] Preferably, the detection is performed by flow cytometry;

[0058] 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%.

[0059] Another aspect of the present invention relates to a method for preparing hepatic progenitor cells, comprising the following steps:

[0060] preparing hepatoblasts by the method for preparing hepatoblasts according to any one of the present invention; and

[0061] Induce hepatoblasts to differentiate into hepatic progenitor cells.

[0062] In some embodiments of the present invention, the method for preparing hepatic progenitor cells, wherein:

[0063] The culture medium used to induce 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 basal 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 basal culture medium;

[0064] 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;

[0065] Preferably, the content of B27 containing vitamin A is 1.6%-2.4%, the content of Nicotinamide is 4-6 mmol / L, the content of 2-Phospho-L-ascorbic acid trisodium salt is 80-120 μmol / L, the content of 1-Oleoyl Lysophosphatidic Aci is 4-6 μmol / L, the content of D-erythro-Sphingosine-1-phosphate is 0.4-0.6 μmol / L, the content of SB431542 is 2.3-2.8 μmol / L, the content of CHIR99021 is 1.3-1.8 μmol / L, the content of Foskolin is 22-28 μmol / L, the content of EGF is 18-22 ng / mL, the content of Dexamethasone is 0.8-1.2 μmol / L, and the content of Heparin is 80-120 μg / mL.

[0066] 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.

[0067] In some embodiments of the present invention, the method for preparing hepatic progenitor cells, wherein:

[0068] The time for inducing hepatoblasts to differentiate into hepatic progenitor cells is 1-3 days, preferably 2 days.

[0069] In some embodiments of the present invention, the method for preparing hepatic progenitor cells, wherein:

[0070] The method further includes the step of detecting molecular markers specifically expressed by the obtained hepatic progenitor cells;

[0071] Preferably, the detection is performed by flow cytometry;

[0072] Preferably, the molecular marker specifically expressed by hepatic progenitor cells is alpha-fetoprotein (AFP); preferably, the proportion of alpha-fetoprotein-positive cells is ≥70%, ≥80%, ≥90% or ≥95%.

[0073] Another aspect of the present invention relates to a method for preparing mature hepatocytes, comprising the following steps:

[0074] preparing hepatic progenitor cells by the method for preparing hepatic progenitor cells according to any one of the present invention; and

[0075] Induce hepatic progenitor cells to differentiate into mature hepatocytes.

[0076] In some embodiments of the present invention, the method for maturing hepatocytes, wherein:

[0077] The culture medium used to induce differentiation of hepatic progenitor cells into mature hepatocytes was William's medium E;

[0078] Preferably, William's medium E is supplemented with B27 containing vitamin A, Foskolin and SB431542;

[0079] Preferably, the B27 containing vitamin A is 1.6%-2.4%, Foskolin is 23-28 μmol / L, and SB431542 is 2.3-2.8 μmol / L;

[0080] Preferably, the content of B27 containing vitamin A is 2%, the content of Foskolin is 25 μmol / L, and the content of SB431542 is 2.5 μmol / L.

[0081] In some embodiments of the present invention, the method for maturing hepatocytes, wherein:

[0082] The time for inducing hepatic progenitor cells to differentiate into mature hepatocytes is 5-12 days, preferably 9-10 days.

[0083] In some embodiments of the present invention, the method for maturing hepatocytes, wherein:

[0084] The method further includes the step of detecting molecular markers specifically expressed by the obtained mature hepatocytes;

[0085] Preferably, the detection is performed by flow cytometry;

[0086] 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%.

[0087] The present invention also relates to a method for preparing mature hepatocytes, comprising the following steps:

[0088] (1) Induce pluripotent stem cells to differentiate into endoderm cells;

[0089] (2) Inducing endoderm cells to differentiate into foregut cells;

[0090] (3) Inducing foregut cells to differentiate into hepatoblasts;

[0091] (4) inducing hepatoblasts to differentiate into hepatic progenitor cells; and

[0092] (5) Induce hepatic progenitor cells to differentiate into mature hepatocytes;

[0093] Wherein, step (3) comprises the following steps:

[0094] (3-1) Induction of the first stage of differentiation of foregut cells into hepatoblasts; and

[0095] (3-2) Induction of the second stage of differentiation of foregut cells into hepatoblasts;

[0096] Among them, the culture medium used in the first stage is different from the culture medium used in the second stage.

[0097] 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):

[0098] ① In step (1), the induction time is 3-6 days, preferably 4 days;

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

[0100] ③ In step (3-1), the induction time is 3-6 days; preferably 3-4 days;

[0101] ④ In step (3-2), the induction time is 2-6 days, preferably 3 days;

[0102] ⑤ In step (4), the induction time is 1-3 days; preferably 2 days; and

[0103] ⑥ In step (5), the induction time is 5-12 days; preferably 9-10 days.

[0104] In some embodiments of the present invention, the preparation method, wherein,

[0105] The induction of step (1) comprises two stages, the first stage being 1 day and the second stage being 2-5 days (eg 2, 3, 4 or 5 days); preferably, the second stage is 3 days.

[0106] In some embodiments of the present invention, the preparation method, wherein,

[0107] The culture medium in step (1) is RPMI1640;

[0108] Preferably, RPMI1640 medium is supplemented with B27 without vitamin A, Activin A, BMP4, bFGF, Y27632, and optionally CHIR99021;

[0109] 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;

[0110] 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;

[0111] 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;

[0112] Optionally, CHIR99021 is 2.5-3.5 μmol / L;

[0113] Optionally, CHIR99021 is 3 μmol / L.

[0114] In some embodiments of the present invention, the preparation method, wherein,

[0115] The culture medium in step (2) is RPMI1640;

[0116] Preferably, RPMI1640 medium is supplemented with B27, KGF, SB431542 and Y27632 without vitamin A;

[0117] 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;

[0118] Preferably, the content of B27 without vitamin A is 2%, the content of KGF is 20 ng / mL, the content of SB431542 is 2.5 μmol / L, and the content of Y27632 is 10 μmol / L.

[0119] In some embodiments of the present invention, the preparation method, wherein,

[0120] The culture medium in step (3-1) is RPMI1640;

[0121] Preferably, RPMI1640 medium is supplemented with B27, KGF, BMP2, BMP4, bFGF and Y27632 without vitamin A;

[0122] Preferably, B27 without vitamin A is 1.6%-2.4%, KGF is 18-22 ng / mL, BMP2 is 18-22 ng / mL, BMP4 is 80-120 ng / mL, bFGF is 8-12 ng / mL, and Y27632 is 8-12 μmol / L;

[0123] Preferably, the content of B27 without vitamin A is 2%, the content of KGF is 20 ng / mL, the content of BMP2 is 20 ng / mL, the content of BMP4 is 100 ng / mL, the content of bFGF is 10 ng / mL, and the content of Y27632 is 10 μmol / L.

[0124] In some embodiments of the present invention, the preparation method, wherein,

[0125] The culture medium in step (3-2) is William's medium E;

[0126] Preferably, William's medium E is supplemented with B27, EGF, Foskolin and Y27632 without vitamin A;

[0127] Preferably, B27 without vitamin A is 1.6%-2.4%, EGF is 18-22 ng / mL, Foskolin is 23-28 μmol / L, and Y27632 is 8-12 μmol / L;

[0128] Preferably, the content of B27 without vitamin A is 2%, the content of EGF is 20 ng / mL, the content of Foskolin is 25 μmol / L, and the content of Y27632 is 10 μmol / L.

[0129] In some embodiments of the present invention, the preparation method, wherein,

[0130] 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;

[0131] 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;

[0132] 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;

[0133] 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.

[0134] In some embodiments of the present invention, the preparation method, wherein,

[0135] The culture medium in step (5) is William's medium E;

[0136] Preferably, William's medium E is supplemented with B27 containing vitamin A, Foskolin and SB431542;

[0137] Preferably, the B27 containing vitamin A is 1.6%-2.4%, Foskolin is 23-28 μmol / L, and SB431542 is 2.3-2.8 μmol / L;

[0138] Preferably, the content of B27 containing vitamin A is 2%, the content of Foskolin is 25 μmol / L, and the content of SB431542 is 2.5 μmol / L.

[0139] In some embodiments of the present invention, the preparation method, wherein,

[0140] The pluripotent stem cells used in step (1) are pretreated as follows:

[0141] Cultivate in mTeSR Plus medium for 12-48 hours, preferably 24-36 hours;

[0142] Preferably, mTeSR Plus medium is supplemented with Activin A, bFGF, and Y27632;

[0143] Preferably, Activin A is 8-12 ng / mL, bFGF is 8-12 ng / mL, and Y27632 is 8-12 μmol / L;

[0144] Preferably, Activin A is 10 ng / mL, bFGF is 10 ng / mL, and Y27632 is 10 μmol / L.

[0145] In some embodiments of the present invention, the preparation method, wherein,

[0146] The pluripotent stem cells are human or mouse pluripotent stem cells;

[0147] 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.

[0148] In some embodiments of the present invention, the preparation method further comprises the step of detecting molecular markers specifically expressed by the cells obtained in step (1), step (4) and / or step (5);

[0149] Preferably, the detection is performed by flow cytometry;

[0150] 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%;

[0151] 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%;

[0152] 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%.

[0153] Another aspect of the present invention relates to a culture medium composition comprising:

[0154] 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

[0155] Vitamin A-free B27, EGF, Foskolin, KGF, BMP2, BMP4, bFGF, and Y27632.

[0156] In some embodiments of the present invention, the culture medium combination further includes DMEM / F12 culture medium; preferably, further includes mTeSR Plus culture medium.

[0157] In some embodiments of the present invention, the culture medium composition comprises:

[0158] Nicotinamide, 2-Phospho-L-ascorbic acid trisodium salt, 1-Oleoyl Lysophosphatidic Acid, D-erythro-Sphingosine-1-phosphate, SB431542, CHIR99021, Dexamethasone and Heparin.

[0159] In some embodiments of the present invention, the culture medium composition comprises:

[0160] 1) William's medium E, and B27 without vitamin A, EGF, Foskolin, and Y27632, mixed or not mixed with William's medium E; and

[0161] 2) RPMI1640 medium, and B27, KGF, BMP2, BMP4, bFGF, and Y27632 without vitamin A, mixed with or not mixed with RPMI1640 medium.

[0162] 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):

[0163] 3) RPMI1640 medium, and B27 without vitamin A, Activin A, BMP4, bFGF, Y27632, and CHIR99021 mixed or not mixed with RPMI1640 medium;

[0164] 4) RPMI1640 medium, and B27 without vitamin A, Activin A, BMP4, bFGF, and Y27632 mixed or not mixed with RPMI1640 medium;

[0165] 5) RPMI1640 medium, and B27, KGF, SB431542, and Y27632 without vitamin A, mixed or not mixed with RPMI1640 medium;

[0166] 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

[0167] B27 with 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 mixed with or without DMEM / F12 medium and William's medium E;

[0168] 7) William's medium E, and B27 containing vitamin A, Foskolin, and SB431542 mixed with or without William's medium E; and

[0169] 8) mTeSR Plus medium, and Activin A, bFGF, and Y27632 mixed or not mixed with mTeSR Plus medium.

[0170] Another aspect of the present invention relates to a kit comprising the culture medium combination according to any one of the present invention.

[0171] Y-27632 is a selective inhibitor of ROCK1 and ROCK2, with Ki values ​​of 140 nM and 300 nM, respectively, in cell-free assays. It is over 200-fold 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.

[0172] Unless otherwise specified, reagent B27 herein refers to a product of GIBCO, such as B-27 Serum-Free Supplement (50X) liquid, which includes B27 without vitamin A (Cat. No. 17504001) and B27 with vitamin A (Cat. No. 17504044). The concentration is 50X, preferably diluted 50-fold to the final concentration, in which case the concentration is expressed as 2% (vol / vol %).

[0173] In the present invention, unless otherwise specified, the "first" (e.g., the first stage) or "second" (e.g., the second stage) is merely for reference distinction and does not have a particular meaning of order.

[0174] 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.

[0175] Advantageous Effects of the Invention

[0176] The present invention achieves any one or more of the following technical effects:

[0177] (1) The method for preparing mature hepatocytes of the present invention has a high efficiency in preparing mature hepatocytes;

[0178] (2) The mature hepatocytes prepared by the present invention have a higher degree of compatibility with primary hepatocytes;

[0179] (3) The hepatoblasts prepared by the present invention have a higher degree of compatibility with the hepatoblasts in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0180] Figure 1A to Figure 1F: Induction and differentiation of chemically induced pluripotent stem cells (hCiPSCs) into hepatic progenitor cells (hiHPCs) and identification.

[0181] Figure 1A: Flowchart of the differentiation of human chemically induced pluripotent stem cells into hepatocytes.

[0182] Figure 1B: Flow cytometric analysis of the proportion of FOXA2 and SOX17 double-positive cells in the endoderm cell population.

[0183] Figure 1C: Flow cytometry analysis of the proportion of AFP-positive cells in the hepatic progenitor cell population. The curve on the left is the curve of the control sample (Ctrl), and the curve on the right is the curve of hiHPCs.

[0184] FIG1D : QPCR analysis of the expression of multiple progenitor cell-related genes in induced pluripotent stem cells (hCiPSCs), three differentiated hepatic progenitor cell lines (hiHPCs), a hepatoma cell line (HepG2), and primary hepatocytes (F-PHHs). n = 3.

[0185] Figure 1E: Immunofluorescence staining to detect the expression of AFP, CK18, HNF4A, and Ki67 in differentiated hepatic progenitor cells. Scale bar: 50 μm.

[0186] Figure 1F: Comparison of transcriptome analysis of induced pluripotent stem cells (hCiPSCs; marked with "●" in the figure), differentiated hepatic progenitor cells (hiHPCs; marked with "▲" in the figure), fetal liver cells (hFLCs; marked with "+" in the figure), and primary hepatocytes (F-PHHs; marked with "■" in the figure).

[0187] Figures 2A to 2I: Identification of hepatic progenitor cells (hiHPCs) after maturation and differentiation into mature hepatocytes (hiHEPs).

[0188] Figure 2A: Morphology of differentiated mature hepatocytes. Scale bar: 50 μm.

[0189] Figure 2B: Flow cytometry analysis of the proportion of ALB-positive cells in a mature hepatocyte population. The curve on the left is the curve of the control sample (Ctrl), and the curve on the right is the curve of hiHEPs.

[0190] Figure 2C: Immunofluorescence staining to detect the expression of ALB, CYP3A4, and HNF4A in differentiated mature hepatocytes. Scale bar: 50 μm.

[0191] Figure 2D: QPCR analysis of the expression of multiple liver-related genes in induced pluripotent stem cells (hCiPSCs), three differentiated mature hepatocyte lines (hiHEPs), and primary hepatocytes (F-PHHs). n = 3.

[0192] Figure 2E: Comparative analysis of transcriptomes (including genes related to different functions and cell states) of induced pluripotent stem cells, differentiated mature hepatocytes, and primary hepatocytes.

[0193] Figure 2F: Detection of the ability of differentiated mature hepatocytes to absorb low-density lipoprotein (left), synthesize glycogen (center), and store lipids (right). Scale bar: 50 μm.

[0194] FIG2G : ELISA assay for the albumin secretion capacity of induced pluripotent stem cells (hCiPSCs), differentiated mature hepatocytes (hiHEPs), hepatic progenitor cells (hiHPCs), and primary hepatocytes (F-PHHs). n=3.

[0195] FIG2H : 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.

[0196] FIG2I : Comparison of the metabolic capacity of the 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.

[0197] FIG3A : Flow cytometry comparison of the proportion of PROX1-positive cells in hepatoblasts generated by the original protocol (hepatoblast I) and the novel protocol of the present invention (hepatoblast II), n=3.

[0198] FIG3B : QPCR comparison of the expression of key hepatic attribute genes in hepatoblasts generated by the original protocol (hepatoblast I) and the novel protocol of the present invention (hepatoblast II), n=3.

[0199] Figure 4A: Yamanaka factors establish hiPSCs differentiation into endoderm cells.

[0200] Figure 4B: Differentiation of hCiPSCs into endoderm cells using small chemical molecules.

[0201] Figure 5A: Flow cytometric analysis of the proportion of AFP-positive cells in chemically reprogrammed hCiPSC-derived hiHPCs. The curve on the left is the curve of the control sample (CTR), and the curve on the right is the curve of hCiPSC-hiHPCs.

[0202] Figure 5B: Flow cytometric analysis of the proportion of AFP-positive cells in hiHPCs derived from hiPSCs induced by Yamanaka factors. The curve on the left is the curve of the control sample (CTR), and the curve on the right is the curve of hiPSCs-hiHPCs.

[0203] Figure 6: QPCR detection of the expression of multiple progenitor cell-related genes.

[0204] Figure 7A: Flow cytometric analysis of the proportion of ALB-positive cells in hiHEPs derived from hCiPSCs. The curve on the left is the curve of the control sample (CTR), and the curve on the right is the curve of hCiPSCs-hiHEPs.

[0205] Figure 7B: Flow cytometric analysis of the proportion of ALB-positive cells in hiHEPs derived from hiPSCs induced by Yamanaka factors. The curve on the left is the curve of the control sample (CTR), and the curve on the right is the curve of hiPSCs-hiHEPs.

[0206] Figure 8: QPCR detection of the expression of multiple liver property-related genes.

[0207] Figure 9A: Albumin secretion levels.

[0208] Figure 9B: Urea synthesis levels. DETAILED DESCRIPTION

[0209] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0210] Some of the reagents involved in the present invention are shown in Table A below.

[0211] Table A

[0212] Example 1: Preparation of Hepatocytes from Human Chemically Induced Pluripotent Stem Cells The flow chart for the differentiation of human chemically induced pluripotent stem cells into hepatocytes is shown in FIG1A . The specific steps are as follows:

[0213] (1) Pretreatment of pluripotent stem cells before differentiation to promote their subsequent induced differentiation

[0214] Chemically induced human pluripotent stem cells 8 When 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 into a 12-well plate coated with a matrix such as Matrigel. The inoculation medium is mTeSR Plus supplemented with the following components: Activin A (10 ng / mL), bFGF (10 ng / mL), and Y27632 (10 μmol / L).

[0215] Differentiation was initiated 24 hours after seeding.

[0216] (2) The first stage of differentiation: inducing pre-treated pluripotent stem cells to differentiate into endoderm cells

[0217] 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).

[0218] The induction medium composition from the second to the fourth day was: 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).

[0219] After induction of endoderm differentiation, flow cytometry was used to detect the expression of endoderm-specific molecular markers FOXA2 and SOX17, as shown in Figure 1B. The results showed that the proportion of FOXA2 and SOX17 double-positive cells was approximately 80%.

[0220] (3) The second stage of differentiation: inducing the differentiation of endoderm cells into foregut cells

[0221] The induction medium composition at this stage was: 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).

[0222] After 2 days of induction in this stage, the next induction stage begins.

[0223] (4) The third stage of differentiation: induction of the first stage of differentiation of foregut cells into hepatoblasts

[0224] 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).

[0225] 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.

[0226] The next induction phase began 24 hours after inoculation.

[0227] (5) Differentiation stage 4: Induction of stage 2 differentiation of foregut cells into hepatoblasts

[0228] The induction medium composition at this stage was as follows: William's medium E as the basal medium, supplemented with 2% B27 (without vitamin A), EGF (20 ng / mL), Foskolin (25 μmol / L), and Y27632 (10 μmol / L).

[0229] After 3 days of induction, the cells entered the next induction phase.

[0230] (6) The fifth stage of differentiation: inducing the differentiation of hepatoblasts into hepatic progenitor cells

[0231] The induction medium at this stage consists of a 1:1 mixture of DMEM / F12 medium and William's medium E medium as the basal medium, supplemented with 2% B27 (containing vitamin A), Nicotinamide (5 mmol / L), 2-Phospho-L-ascorbic acid trisodium salt (PVC, 100 μmol / L), 1-Oleoyl Lysophosphatidic Acid (LPA, 5 μmol / L), D-erythro-Sphingosine-1-phosphate (S1P, 0.5 μmol / L), SB431542 (2.5 μmol / L), CHIR99021 (1.5 μmol / L), Foskolin (25 μmol / L), EGF (20 ng / mL), Dexamethasone (1 μmol / L), and Heparin (100 μg / mL).

[0232] After two days of induction, the cells were digested with the culture medium at a 1:1 ratio and seeded into matrigel-coated 12-well plates to obtain first-generation hepatic progenitor cells (hiHPCs). hiHPCs can be passaged and expanded multiple times under these culture conditions.

[0233] To identify the properties of hepatic progenitor cells, flow cytometry was first used to measure the proportion of hepatic progenitor cells expressing AFP (alpha-fetoprotein), a liver-specific molecular marker. The results are shown in Figure 1C. The results showed that the proportion of AFP-positive cells reached 97.67%, demonstrating the high purity of the hepatic progenitor cells.

[0234] Then, QPCR was used to detect the expression of liver-attribute-related genes in hepatic progenitor cells, and the results are shown in Figure 1D. The results showed that hepatic progenitor cells highly expressed liver-attribute-specific genes such as AFP, ALB, FOXA2, GATA4, CEBPA, PROX1, DLK1, HHEX, and HNF4A.

[0235] To further demonstrate the expression of hepatic progenitor cell-specific genes at the protein level, immunofluorescence staining was performed for AFP, HNF4A, CK8, and Ki67, as shown in Figure 1E. The results showed that all differentiated hepatic progenitor cells expressed the aforementioned proteins associated with hepatic progenitor cell properties.

[0236] In addition, to compare the transcriptome similarity of these hepatic progenitor cells with that of fetal liver cells, RNA-seq was used to analyze the transcriptomes of pluripotent stem cells, hepatic progenitor cells, and fetal liver cells. The results are shown in Figure 1F. The results showed that the transcriptome of the differentiated hepatic progenitor cells was similar to that of fetal liver cells, but significantly different from the pluripotent stem cells from which they originated.

[0237] (7) Differentiation stage 6: Inducing differentiation of hepatic progenitor cells into mature hepatocytes

[0238] When the hepatic progenitor cells reached near 100% confluence, they were cultured for 9 days in a maturation induction medium to further induce differentiation into mature hepatocytes. The maturation induction medium consisted of Williams' medium E as the basal medium supplemented with 2% B27 (containing vitamin A), Foskolin (25 μmol / L), and SB431542 (2.5 μmol / L).

[0239] After maturation induction, mature hepatocytes had the typical polygonal morphology of primary hepatocytes (Figure 2A). Flow cytometry analysis showed that more than 96% of the differentiated cells expressed the key hepatocyte marker ALB (Figure 2B). Immunofluorescence analysis 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).

[0240] QPCR confirmed that differentiated and mature hepatocytes highly expressed 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 (Figure 2D).

[0241] Furthermore, transcriptome sequencing results showed that hiHEPs generated by maturation of hCiPSCs and primary hepatocytes had similar gene expression profiles in multiple key hepatocyte functions, such as drug metabolism, fatty acids, glucose, and lipid cholesterol (Figure 2E).

[0242] Furthermore, multiple functions of the differentiated hepatocytes were tested, and the results are shown in Figure 2F. The results showed that hiHEPs differentiated from hCiPSCs had functions such as absorbing low-density lipoprotein (LDL), accumulating lipid droplets, and synthesizing glycogen. In addition, the levels of albumin secretion (Figure 2G) and urea synthesis in hiHEPs differentiated from hCiPSCs were comparable to those of primary hepatocytes (Figure 2H); the metabolic activity of CYP3A4, a key drug-metabolizing enzyme, in differentiated hiHEPs was also comparable to that of primary hepatocytes (Figure 2I).

[0243] Example 2: Experimental validation of the high efficiency of the protocol for differentiating pluripotent stem cells into hepatocytes

[0244] Originally established differentiation program 9 Containing only one stage of hepatoblast differentiation, the efficiency of hepatoblast production is not high and the gene expression network is not perfect. The proportion of PROX1-positive cells in the hepatoblasts produced 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. 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 show that the hepatoblast differentiation strategy of the new scheme of the present invention obtained a higher proportion of PROX1-positive hepatoblasts.

[0245] Further QPCR analysis was performed to compare the expression of key liver-attribute genes in hepatoblasts generated by the previously established differentiation protocol and those generated by the novel differentiation protocol of the present invention. The results are shown in Figure 3B. The results showed that the expression levels of several key liver-attribute genes, such as CK7, CK8, CK18, SOX9, SPINK1, and PROX1, were significantly higher in hepatoblasts generated by the novel differentiation protocol of the present invention than in hepatoblasts generated by the previously established differentiation protocol. This indicates that the hepatoblasts induced by the novel differentiation protocol of the present invention have a more complete liver-attribute gene expression network and are more compatible with hepatoblasts in vivo.

[0246] Example 3: Universal validation experiment of different sources of human pluripotent stem cells (hiPSC) to differentiate into hepatocytes

[0247] (1) Directed differentiation of endoderm (obtained endoderm cells)

[0248] The two hiPSC samples are as follows:

[0249] Yamanaka factors establish hiPSCs: xeno-free hiPS Cell-1 (cauliscell, 200101)

[0250] The preparation method of hCiPSCs with chemical small molecules can refer to the existing technology, such as the reference Guan J et al. 10 .

[0251] Referring to step (2) in Example 1, two hiPSC samples from different established lineages were differentiated into endoderm cells. Flow cytometry was used to detect the expression of high proportions of endoderm marker genes FOXA2 (ordinate) and SOX17 (abscissa) in the differentiated cells. The results are shown in Figures 4A and 4B. The results show that high-purity endoderm cells can be obtained from the pluripotent stem cells of the two induced established lineages through the induction process of step 2 in Example 1. The obtained endoderm cells were used for subsequent operations.

[0252] (2) Obtaining a high proportion of hepatic progenitor cells

[0253] 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. The expression of the liver-specific molecular marker AFP (alpha-fetoprotein) was detected, and the results are shown in Figures 5A and 5B. The results showed that the proportion of AFP-positive cells was >95%, demonstrating the high purity of the hepatic progenitor cells.

[0254] (3) Major gene expression in hepatic progenitor cells

[0255] The endoderm cells obtained in step (1) of Example 2 were further differentiated according to steps (3) to (6) in Example 1 to obtain hepatic progenitor cells. In order to further prove at the RNA level that the hepatic attribute gene expression levels of the hepatic progenitor cells differentiated from hiPSCs of different established lines were comparable, hCiPSCs were used as negative controls to compare the gene expression of hiHPCs obtained by differentiation of hiPSCs established with Yamanaka factors and hiHPCs obtained by differentiation of three cell lines 10.7, C16, and C19 of hCiPSCs established with chemical small molecules. The results are shown in Figure 6. The results show that hepatic progenitor cells with similar expression of hepatic attribute-related genes can be obtained from the two induced established lineage pluripotent stem cells through the continuous induction process of steps (2) to (6) in Example 1.

[0256] (4) Obtaining a high proportion of hepatocytes

[0257] The hepatic progenitor cells obtained in step (2) of Example 2 were further differentiated into mature hepatocytes according to step (7) of Example 1. The expression ratio of ALB, a key hepatocyte marker, was detected by flow cytometry. The results are shown in Figures 7A and 7B. The results showed that the proportion of hepatocytes expressing ALB was >85% after multi-step differentiation of hiPSCs from different established lines.

[0258] (5) Major gene expression in hepatocytes

[0259] 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 demonstrate at the RNA level that the hepatocytes differentiated from hiPSCs of different established lineages had comparable expression levels of hepatic attribute genes, hCiPSCs and hiHPCs differentiated from hCiPSCs were used as negative controls, and PHH was used as a positive control to compare the gene expression of hiHEPs differentiated from three hCiPSC cell lines 10.7, C16, and C19 with that of hiHEPs differentiated from hiPSCs of Yamanaka factors established lineages. The results are shown in FIG8 . The results show that the two induced established lineages derived pluripotent stem cells can be used to obtain mature hepatocytes with similar expression levels of multiple key hepatic attribute genes, including key hepatocyte transcription factor genes, urea cycle-related genes, drug metabolism and coagulation-related genes, through the continuous induction process of steps (2) to (7) in Example 1, and their expression levels are comparable to those of primary hepatocytes.

[0260] (6) Function of mature hepatocytes

[0261] At the same time, the albumin secretion level and urea synthesis level of hiHEPs differentiated from hiPSCs of different established lineages obtained previously were compared. The negative controls were hCiPSCs and hiHPCs differentiated from hCiPSCs, the positive control was PHH, and the experimental groups were hiHEPs differentiated from three hCiPSC cell lines 0.7, C16, and C19, and hiHEPs differentiated from Yamanaka factors established lineage hiPSCs. The results are shown in Figures 9A and 9B. The results show that the albumin secretion level of mature hepatocytes obtained from the two induced established lineages of pluripotent stem cells after the continuous induction process of steps (2)-(7) in Example 1 is comparable to that of primary hepatocytes, and the urea secretion level is not weaker than that of primary hepatocytes.

[0262] References:

[0263] 1.Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131(5):861-872.

[0264] 2.Raab S,Klingenstein M,Liebau S,Linta L.A Comparative View on Human Somatic Cell Sources for iPSC Generation.Stem Cells Int.2014;2014:768391.

[0265] 3.Thomson JA.Embryonic stem cell lines derived from human blastocysts(vol 282,pg 1147,1998).Science.1998;282(5395):1827-1827.

[0266] 4.Liuyang S,Wang G,Wang Y,et al.Highly efficient and rapid generation of human pluripotent stem cells by chemical reprogramming.Cell Stem Cell.2023;30(4):450-459e459.

[0267] 5.Du YY,Liang Z,Wang S,et al.Human pluripotent stem-cell-derived islets ameliorate diabetes in non-human primates.Nature Medicine.2022.

[0268] 6.Si-Tayeb K,Noto FK,Nagaoka M,et al.Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells.Hepatology.2010;51(1):297-305.

[0269] 7. Yamashita T, Takayama K, Sakurai F, Mizuguchi H. Billion-scale production of hepatocyte-like cells from human induced pluripotent stem cells. Biochem Biophys Res Commun. 2018; 496(4): 1269-1275.

[0270] 8. Ng S, Schwartz RE, March S, et al. Human iPSC-derived hepatocyte-like cells support Plasmodium liver-stage infection in vitro. Stem Cell Reports. 2015; 4(3): 348-359.

[0271] 9. Chen S, Wang J, Ren H, et al. Hepatic spheroids derived from human induced pluripotent stem cells in bio-artificial liver rescue porcine acute liver failure. Cell research. 2020; 30(1):95-97.

[0272] 10.Guan J, Wang G, Wang J, et al. Chemical reprogramming of human somatic cells to pluripotent stem cells. Nature. 2022May; 605(7909): 325-331.

[0273] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full scope of the present invention is given by the appended 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 products of the first stage induction were digested into single cells and then inoculated with the same medium as the first stage induction. The cells are seeded and cultured for 12-48 hours, preferably 24-36 hours; and then the second stage of 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-OleoylLysophosphatidic Acid 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, alpha-fetoprotein positive The proportion of 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-Oleoyl Lysophosphatidic 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-Oleoyl Lysophosphatidic 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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