A kit, method and application of inducing pluripotent stem cells to differentiate into hepatocytes

Abstract

The present invention discloses a kit, method, and application for inducing pluripotent stem cell differentiation into hepatocytes. The kit contains a low concentration of resveratrol. The present invention, for the first time, finds that the addition of low-concentration resveratrol significantly increases albumin secretion, the expression levels of drug-metabolizing enzyme genes, and the expression levels of liver function-related genes, such as polarization genes, in the resulting hepatocytes. This method can also reduce the use of high-concentration and expensive growth factors, further promoting the hepatic differentiation of human pluripotent stem cells. By using the kit provided by the present invention, a method for directional 3D suspension differentiation of human pluripotent stem cells into hepatocytes has been established. This method significantly accelerates the hepatic differentiation process of stem cells, reduces differentiation costs, and improves the maturity and drug metabolism capacity of hepatocytes, making it more efficient and convenient.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a kit, a method and an application thereof for inducing pluripotent stem cells to differentiate into hepatocytes. Background Art

[0002] Currently, the incidence of liver disease is increasing significantly worldwide. Orthotopic liver transplantation is the primary method for treating end-stage liver disease. In the case of a shortage of donor liver organs, hepatocyte transplantation or bioartificial liver is used to treat patients with end-stage liver disease and acute liver failure. Each hepatocyte transplantation or bioartificial liver treatment requires 10 10 Tens of billions of liver cells [1] However, due to the same lack of liver sources, the inability to obtain sufficient hepatocytes limits the clinical application of hepatocyte transplantation and bioartificial livers. In addition, the liver is the main organ for drug metabolism in the human body. New drug development and drug screening also require a large number of functional hepatocytes for drug metabolism and toxicology testing.

[0003] Currently, there is still a certain gap between the liver function of human pluripotent stem cell (hPSCs)-derived hepatocytes obtained through 3D differentiation methods and freshly isolated human primary hepatocytes.

[0004] Freshly isolated primary human hepatocytes (PHHs) rapidly lose their properties and functions when cultured in vitro. Therefore, cultivating hepatocytes with functions comparable to PHHs has become an urgent problem to be solved in the clinical and biopharmaceutical fields. [2] .

[0005] The directed differentiation of human pluripotent stem cells into hepatocytes is mainly divided into three stages: the first is the endoderm differentiation stage, which is the differentiation of human pluripotent stem cells into endoderm cells; the second is the hepatocyte differentiation stage, which is the differentiation of endoderm cells into hepatic progenitor cells and hepatic precursor cells; the third is the hepatocyte maturation stage, which is the differentiation of hepatic progenitor cells and hepatic precursor cells into functional mature hepatocytes. In the previous work, our research group established a differentiation method [3,4] , successfully induced the differentiation of human pluripotent stem cells cultured in two-dimensional (2D) adherent culture into hepatocytes.

[0006] However, the 2D differentiation system cannot simulate the 3D microenvironment of the human liver; secondly, increasing evidence shows that the 3D culture system can better simulate the in vivo microenvironment and promote the generation of liver lineages and hepatocyte maturation.

[0007] In summary, existing technologies present the following challenges: The differentiation process is time-consuming, with a long time span and high time costs; the multiple steps and cytokines used result in high consumption of expensive culture media and cytokines, leading to high material costs; and the maturity and function of hepatocyte-like cells derived from human pluripotent stem cells need to be improved. A method for efficiently culturing hepatocytes comparable to PHHs is urgently needed.

[0008] [1]Chan C,Berthiaume F,Nath BD,et al.Hepatic tissue engineering for adjunct and temporary liver support:critical technologies[J].Liver Transpl,2004,10(11):1331-42.

[0009] [2] Baldari, S., Di Rocco, G., & Toietta, G. (2020). Current Biomedical Use of Copper Chelation Therapy. International journal of molecular sciences, 21(3),1069.

[0010] [3]Duan Y, Ma

[0011] [4]Ma Summary of the Invention

[0012] The first aspect of the present invention aims to provide a kit for inducing stem cells to differentiate into hepatocytes.

[0013] The second aspect of the present invention aims to provide applications of resveratrol and / or the kit of the first aspect of the present invention.

[0014] The third aspect of the present invention is to provide a method for inducing stem cells to become endoderm cells.

[0015] The fourth aspect of the present invention aims to provide a method for inducing stem cells to become hepatic progenitor cells.

[0016] The fifth aspect of the present invention is to provide a method for inducing stem cells to become hepatocytes.

[0017] The technical solution adopted by the present invention is:

[0018] In a first aspect of the present invention, a kit is provided, comprising at least one of (a1) to (a3);

[0019] a1 first culture medium, second culture medium and third culture medium;

[0020] a2 fourth culture medium;

[0021] a3 fifth culture medium;

[0022] The first culture medium, the second culture medium, the third culture medium, the fourth culture medium, and the fifth culture medium contain 10 nM to 10 μM Resveratrol;

[0023] The first culture medium, the second culture medium and the third culture medium further contain Activin A;

[0024] The fourth culture medium further comprises BMP2 and BMP4;

[0025] The fifth culture medium further comprises oncostatin M.

[0026] Preferably, the first culture medium, the second culture medium, the third culture medium, the fourth culture medium, and the fifth culture medium contain 100 nM to 1 μM Resveratrol;

[0027] More preferably, the first culture medium, the second culture medium, the third culture medium, the fourth culture medium, and the fifth culture medium contain 1 μM Resveratrol.

[0028] Preferably, the kit comprises a first culture medium, a second culture medium, a third culture medium, a fourth culture medium and a substrate culture medium.

[0029] Preferably, the concentration of Activin A in the first culture medium, the second culture medium and the third culture medium is 80-120 ng / mL.

[0030] Preferably, the concentration of BMP2 in the fourth culture medium is 5-15 ng / mL.

[0031] Preferably, the concentration of BMP4 in the fourth culture medium is 5-15 ng / mL.

[0032] Preferably, the concentration of oncostatin M in the fifth culture medium is 40-60 ng / mL.

[0033] Preferably, the first culture medium further comprises a GSK3β inhibitor and a basal culture medium;

[0034] Preferably, the GSK3β inhibitor is CHIR99021.

[0035] Preferably, the concentration of the GSK3β inhibitor is 2-4 μM.

[0036] Preferably, the second culture medium further comprises KSR and basal culture medium.

[0037] Preferably, the concentration of KSR in the second culture medium is 0.6-1 w / w%.

[0038] Preferably, the third culture medium further comprises KSR and basal culture medium.

[0039] Preferably, the concentration of KSR in the third culture medium is 6-10 w / w%.

[0040] Preferably, the fourth culture medium further comprises FBS, L-glutamine, insulin, 1-thioglycerol, FGF-4, HGF, dexamethasone, dimethyl sulfoxide, and basal culture medium.

[0041] Preferably, the FBS concentration in the fourth culture medium is 10-30 w / w%.

[0042] Preferably, the concentration of L-glutamine in the fourth culture medium is 1-3 mM.

[0043] Preferably, the concentration of insulin in the fourth culture medium is 0.1-0.2 U / mL.

[0044] Preferably, the concentration of 1-thioglycerol in the fourth culture medium is 0.2-0.4 mM.

[0045] Preferably, the concentration of FGF-4 in the fourth culture medium is 10-30 ng / mL.

[0046] Preferably, the concentration of HGF in the fourth culture medium is 10-30 ng / mL.

[0047] Preferably, the concentration of dexamethasone in the fourth culture medium is 80-120 nM.

[0048] Preferably, the concentration of dimethyl sulfoxide in the fourth culture medium is 0.4-0.6 w / w%.

[0049] Preferably, the fifth culture medium further comprises HGF, dimethyl sulfoxide, dexamethasone, FGF-4, a first additive, and a basal culture medium.

[0050] Preferably, the concentration of HGF in the fifth culture medium is 10-30 ng / mL.

[0051] Preferably, the concentration of dimethyl sulfoxide in the fifth culture medium is 0.4-0.6 w / w%.

[0052] Preferably, the concentration of dexamethasone in the fifth culture medium is 80-120 nM.

[0053] Preferably, the concentration of FGF-4 in the fifth culture medium is 10-30 ng / mL.

[0054] Preferably, the first additive comprises the following components: ascorbic acid, BSA-FAF, hydrocortisone, transferrin, insulin, recombinant human epidermal growth factor and GA-1000; further preferably, the first additive consists of seven components in the SingleQuots kit: 0.5 mL ascorbic acid, 10.5 mL BSA-FAF, 0.5 mL hydrocortisone, 0.5 mL transferrin, 0.5 mL insulin, 0.5 mL recombinant human epidermal growth factor and 0.5 mL GA-1000, and the SingleQuots kit is purchased from Lonza with the product number CC-4182.

[0055] Preferably, the basal culture medium of the first culture medium is at least one of DMEM high glucose, DMEM-F12 and RPMI1640 culture medium.

[0056] Preferably, the basal culture medium of the second culture medium is at least one of DMEM high glucose, DMEM-F12 and RPMI1640 culture medium.

[0057] Preferably, the basal culture medium of the third culture medium is at least one of DMEM high glucose, DMEM-F12 and RPMI1640 culture medium.

[0058] Preferably, the basal culture medium of the fourth culture medium is at least one of RPMI1640 and IMDM culture medium.

[0059] Preferably, the basal medium of the fifth culture medium is a hepatocyte basal medium.

[0060] The second aspect of the present invention provides use of resveratrol and / or the kit described in the first aspect of the present invention in at least one of items b1 to b6;

[0061] b1 Preparation of endoderm cells;

[0062] b2 Preparation of hepatic progenitor cells;

[0063] b3 Preparation of liver cells;

[0064] b4 Preparation of endoderm cell products;

[0065] b5 Product for preparing hepatic progenitor cells;

[0066] b6 preparation of liver cell products.

[0067] The third aspect of the present invention provides a method for preparing endoderm cells, comprising culturing stem cells in the first culture medium described in the first aspect of the present invention for 12 to 36 hours, culturing them in the second culture medium described in the first aspect of the present invention for 12 to 36 hours, and culturing them in the third culture medium described in the first aspect of the present invention for 12 to 36 hours.

[0068] A fourth aspect of the present invention provides a method for preparing hepatic progenitor cells, comprising culturing endoderm cells in the fourth culture medium described in the first aspect of the present invention for 120 to 168 hours to obtain hepatic progenitor cells.

[0069] Preferably, the fourth culture medium is replaced every 16 to 32 hours during the culture process.

[0070] Preferably, the endoderm cells are prepared by the method described in the third aspect of the present invention.

[0071] A fifth aspect of the present invention provides a method for preparing hepatocytes, comprising culturing hepatic progenitor cells in the fifth culture medium described in the first aspect of the present invention for 120 to 168 hours to obtain hepatocytes.

[0072] Preferably, the fifth culture medium is replaced every 16 to 32 hours during the culture process.

[0073] Preferably, the hepatic progenitor cells are prepared by the method described in the fourth aspect of the present invention.

[0074] Preferably, the stem cells are human stem cells with multidirectional differentiation potential.

[0075] Preferably, the human stem cells with multidirectional differentiation potential are human pluripotent stem cells, human parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells or umbilical cord blood stem cells.

[0076] Preferably, the stem cells are stem cell spheres.

[0077] Preferably, the preparation method of the stem cell spheres is as follows: mixing stem cells with digestion fluid, incubating, discarding the digestion fluid, adding mTeSR1 culture medium containing Rock inhibitor and resuspending, seeding into mTeSR1 culture medium containing Rock inhibitor at a density of 250,000 to 1,000,000 cells / mL, and culturing for 12 to 36 hours.

[0078] Preferably, the stem cells are stem cells with a cell coverage rate of 70% to 80% 4 to 5 days after passage, regular clone edges and no differentiated cells.

[0079] Preferably, the Rock inhibitor is Y-27632.

[0080] Preferably, the final concentration of the Rock inhibitor in mTeSR1 culture medium is 8-12 μM.

[0081] The beneficial effects of the present invention are:

[0082] Based on the previous hepatocyte differentiation method of this laboratory, the present invention prepares a new kit for inducing pluripotent stem cell differentiation into hepatocytes by changing the culture method (from 2D culture to 3D culture), replacing some cytokines, and adding low-concentration resveratrol. The present invention discovered for the first time that the albumin secretion, drug metabolizing enzyme gene expression levels, and liver function-related gene expression levels of the hepatocytes obtained after adding resveratrol were significantly increased. Therefore, compared with the existing differentiation scheme of this laboratory, the present invention replaces the use of high-concentration and expensive growth factors by combining the small molecule compound resveratrol and low-concentration growth factors, reducing costs, and can further promote the hepatic differentiation of human pluripotent stem cells, improve the maturity and drug metabolism ability of hepatocytes, and differentiate human pluripotent stem cells into more mature hepatocytes under 3D suspension culture differentiation conditions; shorten the differentiation time and improve the liver function of hepatocytes.

[0083] The present invention also provides a method for inducing stem cells to differentiate into stem cells. By using the kit provided by the present invention, a more efficient and cost-effective method for directional differentiation of human pluripotent stem cells into hepatocytes in 3D suspension is established; the first stage of differentiation of the present invention only requires 3 days of differentiation, the second stage only requires 6 days, and the third stage only requires 8 days. Compared with the traditional 2D differentiation method (differentiation requires a total of 32 days), the present invention greatly accelerates the liver differentiation process of stem cells and also reduces the differentiation cost; the differentiation efficiency is also tested at each stage of differentiation, and it is proved that the liver cells finally differentiated have albumin secretion capacity and drug metabolism function. It is also verified that low concentrations of resveratrol can promote the liver function level of hepatocytes and improve the maturity of hepatocytes under 3D suspension culture conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 A roadmap for the directional differentiation of human pluripotent stem cells into hepatocytes; Figure 1 A is a roadmap for the directional differentiation of human pluripotent stem cells into hepatocytes in 2D adherent culture, scale bar = 100 μm; Figure 1 B is a roadmap for the directional differentiation of human pluripotent stem cells cultured in 3D suspension into hepatocytes. Scale bar = 100 μm.

[0085] Figure 2 Directed differentiation of human pluripotent stem cells into definitive endoderm. Figure 2 A is a roadmap for the directional differentiation of human pluripotent stem cells into endoderm cell spheres; Figure 2 B is the flow cytometry analysis of SOX17 and FOXA2 in endoderm cell spheres; Figure 2 C is an image showing the cell morphological changes during the directional differentiation of human pluripotent stem cells into endoderm cell spheres. Scale bar = 100 μm. Figure 2 D is the immunofluorescence staining of DAPI and FOXA2 in endoderm cell spheres. Figure 2 E shows the expression of endoderm genes in human pluripotent stem cell spheres induced into definitive endoderm cell spheres under three different 3D conditions.

[0086] Figure 3 The 3D suspension cultured hepatic progenitor cell spheres were induced to differentiate into hepatocyte spheres under the condition of adding 1 μM resveratrol. Figure 3 A is a roadmap of the directional differentiation of human pluripotent stem cells into hepatocyte spheres and an image of hepatocyte spheres. Scale bar = 100 μm. Figure 3 B is the expression of ALB, AFP, and CYP3A4 in hepatospheres; Figure 3 C shows the expression of CPS1, FOXO1, BSEP, and NTCP in hepatospheres; Figure 3 D is the expression of drug metabolizing enzyme CYP3A4 induced by 25 μM rifampicin in hepatospheres; Figure 3 E is the amount of globulin secreted by hepatocytes.

[0087] Figure 4 A graph showing the concentrations of resveratrol used in a cell proliferation toxicity assay. DETAILED DESCRIPTION

[0088] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0089] Example 1 A kit for inducing stem cells to differentiate into endoderm cells

[0090] The first culture medium (DE medium 1): RPMI1640 (Gibco, 61870036) + 100 ng / mL Activin A (recombinant human activin-A, Peprotech, 120-14) + 3 μM CHIR99021 (Selleck, CT99021) + 1 μM Resveratrol (Selleck, S3934);

[0091] Second culture medium (DE medium 2): RPMI1640 (Gibco, 61870036) + 100 ng / mL Activin A (recombinant human activin-A, Peprotech, 120-14) + 0.8% KSR (Thermo, 10828028) + 1 μM Resveratrol (Selleck, S3934);

[0092] The third culture medium (DE medium 3): RPMI1640 (Gibco, 61870036) + 100 ng / mL Activin A (recombinant human activin-A, Peprotech, 120-14) + 8% KSR (Thermo, 10828028) + 1 μM Resveratrol (Selleck, S3934).

[0093] Example 2 A kit for inducing stem cells to differentiate into hepatocytes

[0094] The first culture medium (DE medium 1): RPMI1640 (Gibco, 61870036) + 100 ng / mL Activin A (recombinant human activin-A, Peprotech, 120-14) + 3 μM CHIR99021 (Selleck, CT99021) + 1 μM Resveratrol (Selleck, S3934);

[0095] Second culture medium (DE medium 2): RPMI1640 (Gibco, 61870036) + 100 ng / mL Activin A (recombinant human activin-A, Peprotech, 120-14) + 0.8% KSR (Thermo, 10828028) + 1 μM Resveratrol (Selleck, S3934);

[0096] The third culture medium (DE medium 3): RPMI1640 (Gibco, 61870036) + 100 ng / mL Activin A (recombinant human activin-A, Peprotech, 120-14) + 8% KSR (Thermo, 10828028) + 1 μM Resveratrol (Selleck, S3934);

[0097] The fourth culture medium (HDM medium): IMDM media (Gibco, 31980030) + 20% FBS (fetal bovine serum, VISTECH, SE100-B7953) + 2mM L-glutamine (L-glutamine, Gibco, 25030081) + 0.126U / mL human insulin (recombinant human insulin, Sigma, 91077C-11MG) + 0.3mM 1-thioglycerol (1-thioglycerol, Sigma, M6145) + 20ng / mL FGF-4 (fibroblast growth factor-4, Peprotech, 100-18B-50) + 20ng / mL HGF (hepatocyte growth factor, Peprotech, 100-39) + 10ng / mL BMP2 (bone morphogenetic protein 2, Peprotech, 120-02) + 10ng / mL BMP4 (bone morphogenetic protein 4, Peprotech, 120-05) + 0.5% DMSO (dimethyl sulfoxide, MP Biomedical, 196055) + 100 nM dexamethasone (dexamethasone, Sigma, D4902) + 1 μM Resveratrol (Selleck, S3934);

[0098] The fifth culture medium (HCM medium): Hepatocyte basal medium (Lonza) (hepatocyte basal medium, Lonza, CC-3911) + Single Quots kit (Lonza) (Lonza, CC-4182, specifically including seven supplementary components: Ascorbic acid, BSA-FAF, Hydrocortisone, Transferrin, Insulin, rhEGF, and GA-1000) + 20 ng / mL HGF (hepatocyte growth factor, Peprotech, 100-39) + 50 ng / mL oncostatin M (oncostatin M, Peprotech, 300-10) + 0.5% DMSO (dimethyl sulfoxide, MP Biomedical, 196055) + 100 nM dexamethasone (dexamethasone, Sigma, D4902) + 20 ng / mL FGF4 (Fibroblast Growth Factor-4, Peprotech, 100-18B-50) + 1 μM Resveratrol (Selleck, S3934).

[0099] This embodiment also provides a method for inducing the differentiation of human pluripotent stem cells cultured in 3D suspension into hepatocytes. The roadmap is as follows: Figure 1 shown.

[0100] Example 3 Resveratrol concentration selection

[0101] A method for inducing cytotoxicity testing comprises the following steps:

[0102] S1. HepaRG Resuscitation:

[0103] 1) Select a well-preserved tube of HepaRG (provided by Professor Huang Lizhen's laboratory at South China University of Technology), remove the tube from liquid nitrogen, and quickly thaw it in a 37°C water bath.

[0104] 2) Carefully open the bottle cap and transfer the cells into a centrifuge tube containing 5 mL of DMEM high glucose (Servicebio, G4512) supplemented with 10% FBS fetal bovine serum (VISTECH, SE100-B7953). Centrifuge at 1500 rpm for 5 minutes. After centrifugation, aspirate the supernatant and resuspend the cells in 10 mL of DMEM high glucose (Servicebio, G4512) supplemented with 10% FBS fetal bovine serum (VISTECH, SE100-B7953). Inoculate the cell suspension into a 10-cm culture dish and shake crosswise to mix.

[0105] S2. HepaRG passage:

[0106] 1) After aspirating the culture medium of HepaRG cells (cell confluence of approximately 70%-80%), wash the cells with 1 mL of calcium- and magnesium-free PBS. After aspirating, add 3 mL of 0.05% trypsin (Procell, PB180222) and incubate the cells in a CO2 incubator for 2-4 minutes. Then, add 3 mL of culture medium and pipette repeatedly to obtain single-cell suspensions.

[0107] 2) Centrifuge at 1500 rpm for 5 minutes. After centrifugation, aspirate the supernatant and resuspend the cell suspension in 10 mL of DMEM high glucose (Servicebio, G4512) supplemented with 10% FBS fetal bovine serum (VISTECH, SE100-B7953). Inoculate the cell suspension into a 10-cm culture dish and shake crosswise to mix well.

[0108] S3. Cytotoxicity test Cell Counting Kit-8 (CCK-8 kit, (Dongren, CK04):

[0109] 1) Prepare a cell suspension. After aspirating the culture medium from HepaRG cells, wash the cells with 1 mL of calcium- and magnesium-free PBS. After aspirating, add 3 mL of 0.05% trypsin (Procell, PB180222) and incubate in a CO2 incubator for 2-4 minutes. Then, add 3 mL of culture medium and pipette repeatedly to obtain single-cell suspension.

[0110] 2) Centrifuge at 1500 rpm for 5 minutes. After centrifugation, remove the supernatant and resuspend in 10 mL of DMEM high glucose (Servicebio, G4512) medium supplemented with 10% FBS fetal bovine serum (VISTECH, SE100-B7953) and count.

[0111] 3) Seed 5000 cells in a 96-well plate, approximately 100 μl per well, and replicate three times.

[0112] 4) Place the cells in an incubator and culture for 24 hours. The concentrations of resveratrol in each well are 0nM, 10nM, 100nM, 1μM, 10μM, 100μM, and 1mM.

[0113] 4) Add 10 μL of CCK-8 solution to each well and place the culture plate in an incubator for 1 hour.

[0114] 5) Measure the absorbance (OD) at 450 nm using a microplate reader.

[0115] According to the comparison of cytotoxicity test results ( Figure 4 ), and finally 1 μM resveratrol was selected as the final added concentration. Comparative Example 1 A kit for inducing stem cells to differentiate into hepatocytes

[0116] The difference between the kit in Comparative Example 1 and that in Example 2 is that Resveratrol is not added, and the rest is the same as in Example 2.

[0117] Application Example 1

[0118] A highly efficient method for inducing definitive endoderm formation from human pluripotent stem cells (hPSCs) in 3D suspension culture, comprising the following steps (endodermal cell differentiation stage):

[0119] Single-cell passaging of hPSCs: Select hPSCs in good condition (4-5 days after passage, with a cell confluence of approximately 70%-80%, regular colony edges, and no differentiated cells). Aspirate the culture medium and wash the cells with 1 mL of calcium- and magnesium-free PBS. After aspirating the culture medium, add 1 mL of GCDR and return the cells to the CO2 incubator for 2-4 minutes. Then, aspirate the GCDR and add 1 mL of mTeSR1 medium containing 10 μM Y-27632 (Rocki). Use a pipette to repeatedly pipette the colonies to obtain a single-cell suspension.

[0120] S2. Cell Seeding: Aspirate 20 μl of single-cell suspension, stain with trypan blue, and count using a hemocytometer. Using a Corning low-adhesion six-well culture plate, aspirate 1 million single-cell suspension and inoculate into the wells. Add mTeSR1 medium containing 10 μM Y-27632 until the wells contain 2 mL of liquid. Shake the plate crosswise to evenly suspend the cells in the culture medium. This is marked as day 0. After 3-5 days, human pluripotent stem cell spheres with a diameter of approximately 100 μm will form.

[0121] S3. Cell medium replacement: After 3-5 days (differentiation day 1), wash each well of the human pluripotent stem cell spheres with 1 mL of calcium- and magnesium-free PBS, aspirate, and add 2 mL of DE medium 1. After 48 hours (differentiation day 2), replace with DE medium 2, and after 72 hours (differentiation day 3), replace with DE medium 3.

[0122] S4. Cell Harvest: On the third day of culture, take photos of the endoderm spheres, collect them, allow them to settle naturally for 1-2 minutes, discard the supernatant, and add 1 mL of PBS to wash the endoderm spheres. Allow them to settle naturally for 1-2 minutes, discard the supernatant, and extract RNA. qPCR is then used to detect the expression levels of SOX17 and FOXA2 genes.

[0123] The experimental results are shown in Figure 2 , 3D suspension culture of human pluripotent stem cells H9 directed differentiation into endoderm cell spheres: Figure 2 A is a roadmap for the directional differentiation of human pluripotent stem cells into endoderm cell spheres; Figure 2B is the flow cytometry detection of SOX17 and FOXA2 in endoderm cell spheres. Figure 2 C is an image of the morphological changes of human pluripotent stem cells induced to differentiate into endoderm cell spheres. Figure 2 D is the immunofluorescence staining of DAPI and FOXA2 of the endoderm cell sphere; it can be seen that the expression level of endoderm marker genes in the cell sphere prepared by the kit in Example 1 is higher ( Figure 2 E). Therefore, the kit in Example 1 can successfully and efficiently differentiate human pluripotent stem cells into endoderm cell spheres.

[0124] Application Example 2

[0125] A method for efficient 3D suspension-directed differentiation of human pluripotent stem cells (hPSCs) into hepatocytes, with a roadmap such as Figure 3 As shown in A; specifically including the following steps:

[0126] Single-cell passaging of hPSCs: Select hPSCs in good condition (good condition: 4-5 days after hPSC passaging, cell confluence reaches approximately 70%-80%, with regular colony edges and no differentiated cells). Aspirate the culture medium and wash the cells with 1 mL of calcium- and magnesium-free PBS. After aspirating the culture medium, add 1 mL of GCDR and return the cells to the CO2 incubator for incubation for 2-4 minutes. Then, aspirate the GCDR and add 1 mL of mTeSR1 medium containing 10 μM Y-27632 (Rocki). Use a pipette to repeatedly pipette the colonies to obtain a single-cell suspension.

[0127] S2. Cell Seeding: Pipette 20 μl of single-cell suspension, stain with trypan blue, and count using a hemocytometer. Using a Corning low-adhesion six-well plate, pipette 500,000-2,000,000 single-cell suspensions into the wells. Add mTeSR1 medium containing 10 μM Y-27632 until the wells contain 2 mL of liquid. Shake the plate crosswise to evenly suspend the cells in the medium. This is marked as day 0. After 3-5 days, human pluripotent stem cell spheres with a diameter of 50-70 μm will form.

[0128] S3. Cell medium replacement: After 3-5 days (differentiation day 1), wash each well of the human pluripotent stem cell spheres with 1 mL of calcium- and magnesium-free PBS, aspirate, and add 2 mL of DE medium 1. After 48 hours (differentiation day 2), replace with DE medium 2, and after 72 hours (differentiation day 3), replace with DE medium 3.

[0129] S4. Cell culture medium change during the directed induction of hepatic progenitor cells: on days 6-8 (differentiation day 4), wash the hepatic progenitor cell spheres with 1 mL of calcium- and magnesium-free PBS per well, discard the PBS, and add 2 mL of HDM culture medium; on days 7-11 (differentiation day 5-9), replace 2 mL of HDM culture medium every day.

[0130] S5. Cell culture medium replacement during the directed induction of hepatocyte maturation: On days 12-14 (differentiation day 10), wash the hepatic progenitor cell spheres with 1 mL of calcium- and magnesium-free PBS per well, discard the PBS, and add 2 mL of HCM culture medium. On days 13-17 (differentiation day 11-15), replace 2 mL of HCM culture medium every day.

[0131] S6. Cell harvesting: The hepatocyte spheres were photographed daily. After the hepatocytes matured, the hepatocyte spheres were collected and allowed to settle naturally for 1-2 minutes. The culture supernatant was collected and the secretion of human albumin (ALB) was detected using an ELISA kit. The hepatocyte spheres were then washed with 1 mL of PBS and allowed to settle naturally for 1-2 minutes. The supernatant was discarded and the expression levels of human alpha-fetoprotein (AFP), ALB, and CYP3A4 genes were detected by qPCR.

[0132] The experimental results are shown in Figure 3 ,exist Figure 3 In the experiment, 1uM resveratrol was added to induce the differentiation of hepatocyte progenitor cell spheres cultured in 3D suspension into mature hepatocyte spheres: Figure 3 A is the roadmap of the maturation of hepatic progenitor cell spheres into hepatocyte spheres and the morphological image of the maturation of hepatocyte spheres; Figure 3 Figure B shows the qPCR detection of the functional genes ALB, AFP, and CYP3A4 in the hepatospheres. As can be seen from the figure, compared with the control group (Comparative Example 1), the resveratrol group had an increase in the alpha-fetoprotein gene, as well as the albumin gene and drug metabolism genes. Therefore, the hepatic progenitor cells successfully matured into hepatospheres; Figure 3 C shows qPCR analysis of CPS1, FOXO1, BSEP, and NTCP in hepatocyte spheroids. These four genes represent ammonia metabolism, autophagy, basal membrane polarization, and apical membrane polarization, respectively. Compared to the control group, the expression of CPS1, BSEP, and NTCP in the resveratrol group was significantly increased, while FOXO1 was significantly decreased. This indicates that ammonia metabolism and polarization of hepatocytes in the resveratrol group were enhanced, and autophagy was decreased. Figure 3 D is the expression of drug metabolizing enzyme CYP3A4 in hepatocyte spheres induced by 25 μM rifampicin. After 48 hours of induction of hepatocyte spheres by 25 μM rifampicin, 1 mL of PBS was added to wash the hepatocyte spheres, and the spheres were naturally settled for 1-2 minutes. The supernatant was discarded and qPCR detection was performed. Drug metabolizing enzyme induction experiments were performed using differentiated hepatocyte spheres. Responding to inducers is an important sign of mature hepatocytes. It can be seen that the addition of 1 μM Resveratrol significantly increased the expression of metabolic enzyme genes compared with the control group. This shows that the hepatocyte spheres differentiated in this example can respond to drug induction and stimulation, have good drug metabolism function, and have the potential to become a drug screening cell model. Figure 3E is the ALB content in the culture medium supernatant detected by Elisa at the mature stage of hepatocyte spheres. The secretion amounts were 102.8430ug / mL and 138.4630ug / mL, respectively, indicating that the hepatocyte spheres had stronger albumin secretion ability after resveratrol treatment.

[0133] The above specific embodiments provide a detailed description of the present invention. However, the present invention is not limited to the above embodiments. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with each other unless there is a conflict.

Claims

1. A kit comprising a first culture medium, a second culture medium, a third culture medium, a fourth culture medium, and a fifth culture medium; The first culture medium, the second culture medium, the third culture medium, the fourth culture medium, and the fifth culture medium contain 10 nM to 10 μM Resveratrol; The first culture medium, the second culture medium and the third culture medium further contain Activin A; The concentration of Activin A in the first culture medium, the second culture medium, and the third culture medium is 80 to 120 ng / mL; The second culture medium and the third culture medium further comprise KSR and basal culture medium; The concentration of KSR in the second culture medium is 0.6-1 w / w%; The KSR concentration in the third culture medium is 6-10 w / w%; The fourth culture medium further comprises BMP2 and BMP4; The fifth culture medium further comprises oncostatin M; The concentration of BMP2 in the fourth culture medium is 5-15 ng / mL, and the concentration of BMP4 is 5-15 ng / mL; the concentration of oncostatin M in the fifth culture medium is 40-60 ng / mL; the first culture medium further comprises a GSK3β inhibitor and a basal culture medium; the fourth culture medium further comprises FBS, L-glutamine, insulin, 1-thioglycerol, FGF-4, HGF, dexamethasone, dimethyl sulfoxide, and a basal culture medium; the fifth culture medium further comprises HGF, dimethyl sulfoxide, dexamethasone, FGF-4, a first additive, and a basal culture medium; the first additive comprises ascorbic acid, BSA-FAF, hydrocortisone, transferrin, insulin, recombinant human epidermal growth factor, and GA-1000; The basal culture medium of the first culture medium is at least one of DMEM high glucose, DMEM-F12 and RPMI1640 culture medium; the basal culture medium of the second culture medium is at least one of DMEM high glucose, DMEM-F12 and RPMI1640 culture medium; the basal culture medium of the third culture medium is at least one of DMEM high glucose, DMEM-F12 and RPMI1640 culture medium; the basal culture medium of the fourth culture medium is at least one of RPMI1640 and IMDM culture medium; and the basal culture medium of the fifth culture medium is hepatocyte basal culture medium.

2. Use of the kit according to claim 1 in preparing a product for inducing stem cells to differentiate into hepatocytes.

3. A method for preparing hepatocytes, characterized in that: The stem cells are cultured in the first culture medium of claim 1 for 12 to 36 hours, in the second culture medium of claim 1 for 12 to 36 hours, and in the third culture medium of claim 1 for 12 to 36 hours to obtain endoderm cells; the endoderm cells are cultured in the fourth culture medium of claim 1 for 120 to 168 hours to obtain hepatic progenitor cells; and the hepatic progenitor cells are cultured in the fifth culture medium of claim 1 for 120 to 168 hours to obtain hepatocytes.

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