CN113265373A

CN113265373A - Culture medium and method for differentiating human pluripotent stem cells into hepatic progenitor cells and application of culture medium

Info

Publication number: CN113265373A
Application number: CN202110811610.XA
Authority: CN
Inventors: 不公告发明人
Original assignee: Beijing Mingcheng Innovation Medical Research Institute Co ltd
Current assignee: Beijing Mingcheng Innovation Medical Research Institute Co ltd
Priority date: 2021-07-19
Filing date: 2021-07-19
Publication date: 2021-08-17

Abstract

The invention relates to the technical field of cell culture, in particular to a culture medium and a method for differentiating human pluripotent stem cells into hepatic progenitor cells. The culture medium provided by the invention comprises a first culture medium and a second culture medium for differentiating human pluripotent stem cells into endoderm cells and a third culture medium for inducing the differentiation of endoderm cells into hepatic progenitor cells. The culture medium provided by the invention can induce and differentiate the pluripotent stem cells into the hepatic progenitor cells, and the differentiation efficiency and the survival rate of the hepatic progenitor cells are both high. Experiments show that the differentiation efficiency of the hepatic progenitor cells reaches more than 84 percent, and the survival rate reaches more than 90 percent. Meanwhile, the culture medium is free of serum and animal-derived components, and has definite components and high safety. The invention also provides a method for inducing differentiation of the hepatic progenitor cells, which utilizes the culture medium to induce differentiation and carries out sectional digestion on the obtained hepatic progenitor cells, thereby further improving the differentiation efficiency and the survival rate of the hepatic progenitor cells and having lower production cost.

Description

Culture medium and method for differentiating human pluripotent stem cells into hepatic progenitor cells and application of culture medium

Technical Field

The invention relates to the technical field of cell culture, in particular to a culture medium and a method for differentiating human pluripotent stem cells into hepatic progenitor cells and application thereof.

Background

It is estimated that over one fifth of the population in China is afflicted with liver disease, and the number of patients with liver disease is constantly increasing. Liver disease can be caused by lifestyle problems such as obesity, virus, alcohol abuse, and also by non-lifestyle problems such as autoimmune and gene-mediated diseases.

Currently, the most effective method for treating severe liver diseases such as liver cirrhosis and hepatitis is liver transplantation, however, life-long complications may occur after liver transplantation, and the demand for donor organs far exceeds the increasing demand.

The liver cell transplantation is to obtain complete normal liver or partial liver tissue cut off by operation, to separate and purify in vitro, to implant the separated and purified liver cell into body, to restore or rebuild liver function. The method has proven to be an effective treatment for acute and chronic liver failure, cirrhosis, metabolic liver disease.

Researchers from the western Kaempferia medical college, USA, found that human embryonic stem cells can differentiate into a previously unknown liver progenitor cell (liver progenitor cell), wherein the liver progenitor cell is an early progeny of the stem cell and can produce mature functional hepatocytes. At present, the commonly used culture medium and culture method for differentiating pluripotent stem cells into hepatic progenitor cells mainly adopt animal-derived components such as FBS, KOSR, BSA and the like, and have certain limitations, such as: a) key indexes of industrial production, including cost control and stability, cannot be met. b) The impact of animal viruses on safety of application. Even though some researches adopt some nutritional ingredients to replace animal-derived ingredients, the survival rate and purity of hepatic progenitor cells are still not ideal enough, and the stability of different batches is not good enough, so that the increasingly urgent clinical application cannot be met. Moreover, absent specific methods of hepatic progenitor digestion, it is difficult to obtain hepatic progenitors with high viability rates.

Disclosure of Invention

In view of the above, the present invention provides a culture medium and method for differentiating human pluripotent stem cells into hepatic progenitor cells. The culture medium can efficiently differentiate human pluripotent stem cells into hepatic progenitor cells, and has high purity and survival rate.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a culture medium for differentiating human pluripotent stem cells into hepatic progenitor cells, which comprises the following components: a first culture medium and a second culture medium for differentiating the human pluripotent stem cells into endoderm cells, and a third culture medium for inducing the differentiation of the endoderm cells into hepatic progenitor cells;

the first culture medium takes RPMI1640 or DMEM/F12 as a basic culture medium and comprises the following components: activin A, PI3K inhibitors, GSK-3 inhibitors, L-ascorbic acid and carnitine;

the second culture medium takes RPMI1640 or DMEM/F12 as a basic culture medium and comprises the following components: activin a, PI3K inhibitor, L-ascorbic acid, and carnitine;

the third culture medium takes DMEM/F12 or IMDM as a basic culture medium and comprises the following components: l-ascorbic acid, carnitine, ALK5 inhibitors, insulin and transferrin.

In some embodiments, the first medium is a basal medium, RPMI1640 or DMEM/F12, comprising the following components:

50-150 ng/mL of Activin A, 5-20 mu M, GSK-3 inhibitor of PI3K, 2-20 mu M, L-ascorbic acid, 20-100 mg/mL of carnitine and 1-10 mu g/mL of carnitine;

the second culture medium takes RPMI1640 or DMEM/F12 as a basic culture medium and comprises the following components:

50-150 ng/mL of Activin A, 5-20 mu M, L of PI3K inhibitor-20 mg/mL of ascorbic acid and 1-10 mu g/mL of carnitine;

the third culture medium takes DMEM/F12 or IMDM as a basic culture medium and comprises the following components: 20-100 mg/mL of L-ascorbic acid, 1-10 mu g/mL of carnitine, 5-20 mu M of ALK5 inhibitor, 5-20 mu g/mL of insulin and 5-20 mu g/mL of transferrin.

In some embodiments, the first medium is a basal medium based on RPMI1640 or DMEM/F12, and comprises the following components:

activin A50 ng/mL, PI3K inhibitor 5 μ M, GSK-3 inhibitor 2 μ M, L-ascorbic acid 20mg/mL and carnitine 1 μ g/mL;

activin A50 ng/mL, PI3K inhibitor 5. mu. M, L-ascorbic acid 20mg/mL and carnitine 1. mu.g/mL.

The third culture medium takes DMEM/F12 or IMDM as a basic culture medium and comprises the following components: 20mg/mL of L-ascorbic acid, 1. mu.g/mL of carnitine, 5. mu.M of ALK5 inhibitor, 5. mu.g/mL of insulin and 5. mu.g/mL of transferrin.

activin A150 ng/mL, PI3K inhibitor 20 μ M, GSK-3 inhibitor 20 μ M, L-ascorbic acid 100mg/mL, and carnitine 10 μ g/mL;

activin A150 ng/mL, PI3K inhibitor 20 μ M, L-ascorbic acid 100mg/mL, and carnitine 10 μ g/mL;

the third culture medium takes DMEM/F12 or IMDM as a basic culture medium and comprises the following components: 100mg/mL of L-ascorbic acid, 10. mu.g/mL of carnitine, 20. mu.M of ALK5 inhibitor, 20. mu.g/mL of insulin and 20. mu.g/mL of transferrin.

activin A100 ng/mL, PI3K inhibitor 10 μ M, GSK-3 inhibitor 10 μ M, L-ascorbic acid 50mg/mL, and carnitine 5 μ g/mL;

activin A100 ng/mL, PI3K inhibitor 10 μ M, L-ascorbic acid 50mg/mL, and carnitine 5 μ g/mL;

the third culture medium takes DMEM/F12 or IMDM as a basic culture medium and comprises the following components: l-ascorbic acid 50mg/mL, carnitine 5. mu.g/mL, ALK5 inhibitor 10. mu.M, insulin 10. mu.g/mL, and transferrin 10. mu.g/mL.

In some embodiments, the PI3K inhibitor is LY294002, the GSK-3 inhibitor is CHIR99021, and the ALK5 inhibitor is SB 431542.

Aiming at different stages of differentiation from pluripotent stem cells to endoderm cells and differentiation from endoderm cells to hepatic progenitor cells, the invention reasonably matches various nutrient components, and the obtained three culture media mutually promote and act together in the process of differentiating the human pluripotent stem cells into the hepatic progenitor cells. Experiments show that the culture medium provided by the invention can induce and differentiate the pluripotent stem cells into the hepatic progenitor cells, the differentiation efficiency and the survival rate of the hepatic progenitor cells are both high, the differentiation efficiency reaches more than 84%, and the survival rate reaches more than 90%. Based on the above, the invention also provides the application of the culture medium in the differentiation of the human pluripotent stem cells into hepatic progenitor cells and the application of the culture medium in the preparation of a preparation for the differentiation of the human pluripotent stem cells into hepatic progenitor cells.

The present invention also provides a method for differentiating human pluripotent stem cells into hepatic progenitor cells, comprising:

step 1: culturing the pluripotent stem cells by using the first culture medium in the culture medium of the invention on day 0-1;

step 2: continuing culturing with a second culture medium on days 2-3;

and step 3: and continuing culturing with a third culture medium on days 4-10.

Further, the method for differentiating human pluripotent stem cells into hepatic progenitors according to the present invention further comprises a step of digesting the cells in stages after the culturing in step 3, wherein the stage digestion is: incubating the cells for 3 times, 5min each time, with 0.1-0.5 mM EDTA; accutase was digested for 5 min.

In some embodiments, the method for differentiating human pluripotent stem cells into hepatic progenitors according to the present invention further comprises the step of culturing the pluripotent stem cells using one of Matrigel or Vitronectin matrix, Essential 8 or TeSR E8 culture solution before the step 1.

Wherein in the step 1-3, the culture conditions are 37 ℃, 95% relative humidity and 5% CO₂。

In the present invention, the pluripotent stem cells preferably include embryonic stem cells and induced pluripotent stem cells.

The culture medium provided by the invention can induce and differentiate the pluripotent stem cells into the hepatic progenitor cells, and the differentiation efficiency and the survival rate of the hepatic progenitor cells are both high. Experiments show that the differentiation efficiency of the hepatic progenitor cells reaches more than 84 percent, the survival rate reaches more than 90 percent, and the differentiation efficiency and the cell survival rate are both obviously higher than those of a control. Meanwhile, the culture medium and the culture method of the invention at least have one of the following beneficial effects:

(1) at the level of the culture system: the raw material level is high, the culture medium is serum-free and animal-derived component-free, the components are clear, and the selection principle of the raw materials of the cell therapy product is met;

(2) at the level of raw material and product quality control: the batch is stable and the application is safe;

(3) in terms of production cost of the product: compared with the commercial product StemXVivo specificity Kit, the cost is reduced by more than 50 percent;

(4) the survival rate of hepatic progenitor cells obtained by the segmented digestion method is obviously higher than that of a control group.

Drawings

FIG. 1 shows the results of flow cytometry for the efficiency of endoderm differentiation using SOX17 as a marker after induction of differentiation in the media of example 1 and comparative example 1 (i.e., control), 1-a to 1-c being the results of the uninduced group, experimental group and comparative example 1 (i.e., control), respectively;

FIG. 2 shows the results of flow cytometry for the detection of the differentiation efficiency of endoderm cells using FOXA2 as a marker after the induction of differentiation in the culture media of example 1 and comparative example 1 (i.e., control group), and 2-a to 2-c are the results of the uninduced group, the experimental group, and the comparative example 1 (i.e., control group), respectively;

FIG. 3 shows the results of flow cytometry for the differentiation efficiency of hepatic progenitors using HNF4 α as a marker after induction of differentiation in the culture media of example 1 and comparative example 1 (i.e., control group), and 3-a to 3-c are the results of the uninduced group, experimental group, and comparative example 1 (i.e., control group), respectively;

FIG. 4 shows the results of flow cytometry for detecting the differentiation efficiency of hepatic progenitors using AFP as a marker after induction of differentiation in the media of example 1 and comparative example 1 (i.e., control group), 4-a to 4-c being the results of the uninduced group, experimental group and comparative example 1 (i.e., control group), respectively;

FIG. 5 shows that after differentiation was induced in the medium of example 2, 5-a represents an uninduced group, and 5-b to 5-e respectively measure the differentiation efficiency of hepatic progenitors using a flow cytometer using SOX17, FOXA2, HNF4 alpha and AFP as markers;

FIG. 6 shows that 6-a represents an uninduced group and 6-b-6-e detects differentiation efficiency of hepatic progenitors by flow cytometry using SOX17, FOXA2, HNF4 alpha and AFP as markers, respectively, after differentiation was induced in the medium of example 3;

FIG. 7 shows that 7-a represents an uninduced group and 7-b to 7-e detect the differentiation efficiency of hepatic progenitors by flow cytometry using SOX17, FOXA2, HNF4 alpha and AFP as markers, respectively, after induction of differentiation in the medium of comparative example 2;

FIG. 8 shows the differentiation efficiency of different stem cell lines into hepatic progenitors measured by flow cytometry using the medium of example 1 to induce differentiation, 8-a for uninduced group, 8-b to 8-f for HNF4 α marker;

FIG. 9 shows the effect of different digestion methods on hepatic progenitor cell viability.

Detailed Description

The invention provides a culture medium and a method for differentiating human pluripotent stem cells into hepatic progenitor cells. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market. Specific sources of the antibodies used are shown in table 1.

TABLE 1

Name (R)	Species of species	Manufacturer of the product	Goods number
				Anti-SOX17 antibody	goat	R&D System	AF1924
Anti-FOXA2 antibody	goat	R&D System	AF2400
				Anti-HNF4α antibody	mouse	R&D System	MAB4605
Anti-AFP antibody	rabbit	Abcam	Ab169552

The invention is further illustrated by the following examples:

example 1

A first medium: basal medium RPMI1640 or DMEM/F12, cytokine Activin A50 ng/mL, small molecule LY 2940025 mu M, CHIR 990212 mu M, L-ascorbic acid 20mg/mL, carnitine 1 mu g/mL.

A second medium: basal medium RPMI1640 or DMEM/F12, cytokine Activin A50 ng/mL, small molecule LY 2940025 mu M, L-ascorbic acid 20mg/mL, carnitine 1 mu g/mL.

A third medium: basal medium DMEM/F12 or IMDM, adding small molecule L-ascorbic acid 20mg/mL, carnitine 1. mu.g/mL, SB 4315425. mu.M, protein Insulin 5. mu.g/mL, apo-Transferrin 5. mu.g/mL.

Example 2

A first medium: basal medium RPMI1640 or DMEM/F12, cytokine Activin A150 ng/mL, small molecule LY 29400220 mu M, CHIR 9902120 mu M, L-ascorbic acid 100mg/mL, and carnitine 10 mu g/mL.

A second medium: basal medium RPMI1640 or DMEM/F12, cytokine Activin A150 ng/mL, small molecule LY 29400220 mu M, L-ascorbic acid 100mg/mL, and carnitine 10 mu g/mL.

A third medium: basal medium DMEM/F12 or IMDM, adding small molecule L-ascorbic acid 100mg/mL, carnitine 10. mu.g/mL, SB 43154220. mu.M, protein Insulin 20. mu.g/mL, apo-Transferrin 20. mu.g/mL.

Example 3

A first medium: basal medium RPMI1640 or DMEM/F12, cytokine Activin A100 ng/mL, small molecule LY 29400210 mu M, CHIR 9902110 mu M, L-ascorbic acid 50mg/mL, carnitine 5 mu g/mL.

A second medium: basal medium RPMI1640 or DMEM/F12, cytokine Activin A100 ng/mL, small molecule LY 29400210 mu M, L-ascorbic acid 50mg/mL, carnitine 5 mu g/mL.

A third medium: basal medium DMEM/F12 or IMDM, adding small molecule L-ascorbic acid 50mg/mL, carnitine 5. mu.g/mL, SB 43154210. mu.M, protein Insulin 10. mu.g/mL, Transferrin 10. mu.g/mL.

Example 4 method for the differentiation of pluripotent Stem cells of the present inventors into hepatic progenitors

The medium of example 1 is used as an example to illustrate:

(1) culturing the pluripotent stem cells: using Matrigel or Vitronectin matrix, Essential 8 or TeSR E8 culture solution;

(2) differentiation of pluripotent stem cells into endodermal cells:

day0~ 1: culturing the pluripotent stem cells by using a first culture medium;

day 2-3: replacing the culture medium with a second culture medium, and continuing to culture;

the culture conditions are as follows: 37 ℃ 95% relative humidity, 5% CO₂Changing the liquid every day;

(3) endoderm cells to hepatic progenitor differentiation:

day4~ 10: culturing the cells by using a third culture medium;

the culture conditions are as follows: 37 ℃ 95% relative humidity, 5% CO₂Changing the liquid every other day

(4) Obtaining hepatic progenitor cells by a segmented digestion method:

the first stage is as follows: incubating the hepatic progenitor cells induced and differentiated in the step (3) with 0.1-0.5 mM EDTA for 3 times, 5min each time;

and a second stage: accutase was digested for 5 min.

Comparative example 1

Compared with the culture medium of example 3, the first culture medium, the second culture medium and the third culture medium lack ascorbic acid and carnitine, and the specific composition is as follows:

a first medium: basal medium RPMI1640 or DMEM/F12, cytokine Activin A100 ng/mL, small molecule LY 29400210 μ M, CHIR 9902110 μ M.

D1-D3: basal medium RPMI1640 or DMEM/F12, cytokine Activin A100 ng/mL, small molecule LY 29400210. mu.M.

D4-D10: basal medium DMEM/F12 or IMDM, small molecule SB 43154210. mu.M, protein Insulin 10. mu.g/mL, apo-Transferrin 10. mu.g/mL.

Comparative example 2

D0-D1: basal medium RPMI1640 or DMEM/F12, cytokine Activin A100 ng/mL, small molecule CHIR 9902110. mu.M.

D1-D3: basal medium RPMI1640 or DMEM/F12, cytokine Activin A100 ng/mL, and nutritional supplement B27.

D4-D10: basal medium DMEM/F12 or IMDM, 20% Knockout Serum Replacement.

Test example 1

Pluripotent stem cells were induced to differentiate in the same manner as in example 4 using the media of examples 1 to 3 and comparative examples 1 to 2.

The differentiation efficiency of pluripotent stem cells into endoderm cells during the culture in step (2) (endoderm cell stage) was examined by a flow cytometer by the following method:

1) cells were collected 3 days after differentiation: discarding the culture supernatant, washing with PBS once, adding an appropriate amount (0.5 mL per well, taking a 6-well plate as an example) of Accutase, digesting at 37 ℃ for 3 min, adding 1 mL of DMEM/F12, blowing off the cells from the bottom of the dish by using a 1 mL pipette, collecting the cells in a 15 mL centrifuge tube, and centrifuging at 1000 rpm for 3 min;

2) discarding the supernatant, adding 1 mL of 4% PFA, and fixing at room temperature for 30 min;

3) centrifuging at 1000 rpm for 3 min, discarding the supernatant, adding 1 mL PBS, and repeating the step three times;

4) adding 1 mL of 0.5% Triton, and performing permeation treatment at room temperature for 20 min;

5) centrifuging at 1000 rpm for 3 min, discarding the supernatant, adding 1 mL PBS +5% BSA blocking solution, and blocking at room temperature for 1 h;

6) centrifuging at 1000 rpm for 3 min, discarding the supernatant, adding primary anti-stain solution (PBS +2% BSA +1:200 anti-SOX17 anti/anti-FOXA 2 anti), and incubating at room temperature for 1 h;

7) centrifuging at 1000 rpm for 3 min, discarding the supernatant, adding 1 mL PBS, and repeating the step three times;

8) centrifuging at 1000 rpm for 3 min, discarding the supernatant, adding corresponding secondary dye solution PBS +2% BSA +1:200 donkey-anti-goat secondary antibody (donkey-anti-goat-AF 488, Abcam, ab 150129), and incubating at room temperature for 1 h;

9) centrifuging at 1000 rpm for 3 min, discarding the supernatant, adding 1 mL PBS, and repeating the step three times;

10) and analyzing by a flow type computer, and obtaining results shown in the figures 1-2.

And (3) detecting the differentiation efficiency of the endoderm cells to the hepatic progenitor cells in the culture process of the step (3) (the hepatic progenitor cell stage) by using a flow cytometer, wherein the detection method comprises the following steps:

1) cells were collected 10 days after differentiation: the digestion method is as described in the technical scheme part, and centrifugation is carried out for 3 min at 1000 rpm;

6) centrifuging at 1000 rpm for 3 min, discarding the supernatant, adding primary anti-stain solution (PBS +2% BSA +1:200 anti-HNF4 alpha antibody/anti-AFP antibody), and incubating at room temperature for 1 h;

8) centrifuging at 1000 rpm for 3 min, discarding the supernatant, adding corresponding secondary antibody solution PBS +2% BSA +1:200 of a high-anti-mouse secondary antibody/high-anti-mouse secondary antibody (high-anti-mouse-AF 488, Abcam, ab 150113; goat-anti-rabbitaf 488, Abcam, ab 150077), incubating for 1 h at room temperature;

10) and (4) analyzing on a flow type computer. The results are shown in Table 2, wherein the results of example 1 and comparative example 1 are shown in FIGS. 3 to 4, and the results of examples 2 to 3 and comparative example 2 are shown in FIGS. 5 to 7, respectively.

TABLE 2

As shown in Table 2 and FIGS. 1 to 9, the differentiation efficiency of the culture medium of the present invention into endoderm cells was 91.7% to 97.6% and the differentiation efficiency of the comparative examples 1 to 2 was 57% to 77.6% using SOX17 and FOXA2 as markers, which are very different from each other (see that: (A) the differentiation efficiency of the culture medium of the present invention into endoderm cells is very different from each otherp< 0.01). By using HNF4 alpha and AFP as markers, the differentiation efficiency of hepatic progenitor cells of the culture medium is 81.3% -87.0%, the differentiation efficiency of comparative examples 1-2 is 43.9% -57.6%, and the differentiation efficiency have very significant difference (2)p＜0.01）。

Test example 2 determination of differentiation efficiency of hepatic progenitors in different cell lines

Four pluripotent stem cell lines of H1, H9, IPSC1, IPSC 2 and IPSC 3 were induced to differentiate, respectively, according to the method of example 4 using the medium of example 1. The differentiation efficiency of endoderm cells into hepatic progenitor cells during the culture in step (3) (hepatic progenitor stage) was examined by the flow cytometer in the same manner as in test example 1, and the results are shown in FIG. 8.

The result shows that the culture medium can induce different pluripotent stem cell lines to differentiate into hepatic progenitor cells, and the differentiation efficiency reaches over 84.5 percent.

Test example 3 cell viability assay

Experimental groups: pluripotent stem cells were induced to differentiate according to the method of example 4 using the medium of example 1.

Control group: induced differentiation was performed on pluripotent stem cells according to the steps (1) to (3) of example 4 using the medium of example 1, and the step (4) was performed by a general digestion method, specifically:

and (4):

the survival rate of the hepatic progenitors obtained in step (4) of the experimental and control groups was measured by AO/PI staining using a CountStar apparatus, and the results are shown in FIG. 9.

The results show that the survival rate of the hepatic progenitor cells obtained by the sectional digestion method is more than 90 percent, and the survival rate of the common digestion method is about 80 percent. Compared with the common digestion method, the segmented digestion method can further improve the survival rate of the hepatic progenitor cells.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

1. A medium for differentiation of human pluripotent stem cells into hepatic progenitor cells comprising: a first culture medium and a second culture medium for differentiating the human pluripotent stem cells into endoderm cells, and a third culture medium for inducing the differentiation of the endoderm cells into hepatic progenitor cells;

2. The culture medium according to claim 1, wherein the first culture medium is a basal medium based on RPMI1640 or DMEM/F12, comprising the following components:

3. The culture medium according to claim 1, wherein the first culture medium is a basal medium based on RPMI1640 or DMEM/F12, comprising the following components:

activin A50 ng/mL, PI3K inhibitor 5 μ M, L-ascorbic acid 20mg/mL and carnitine 1 μ g/mL;

4. The culture medium according to any one of claims 1 to 3, wherein the PI3K inhibitor is LY294002, the GSK-3 inhibitor is CHIR99021, and the ALK5 inhibitor is SB 431542.

5. Use of a culture medium according to any one of claims 1 to 4 in the preparation of functional cells derived from the downstream of hepatic progenitors, said functional cells derived from the downstream being hepatocytes or cholangiocytes.

6. Use of a culture medium according to any one of claims 1 to 4 in the preparation of hepatic progenitors or in the preparation of a product for inducing differentiation of human pluripotent stem cells into hepatic progenitors, said product being a reagent or kit.

7. A method of inducing differentiation of human pluripotent stem cells into hepatic progenitors comprising:

step 1: on days 0 to 1, culturing pluripotent stem cells in the first medium of the medium according to any one of claims 1 to 4;

step 2: continuously culturing with a second culture medium on days 2-3 to obtain endoderm cells;

and step 3: and continuously culturing with a third culture medium on days 4-10 to obtain hepatic progenitor cells.

8. The method of claim 7, further comprising the step of performing a step of stepwise digestion of the cells after the culturing of step 3, the stepwise digestion being: incubating the cells for 3 times, 5min each time, with 0.1-0.5 mM EDTA; accutase was digested for 5 min.

9. The method according to claim 7, wherein the culturing conditions in steps 1 to 3 are 37 ℃ and 95% relative humidity and 5% CO₂。

10. The method of claim 7, wherein the human pluripotent stem cells comprise embryonic stem cells and induced pluripotent stem cells; the human pluripotent stem cells at least express one or more of the following characteristic markers: OCT4, NANOG, SOX2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81.

11. The method of claim 7, wherein the endoderm cells express one or both of SOX17, FOXA2 as characteristic markers;

the hepatic progenitors express one or both of the characteristic markers HNF4 α, AFP.