CN116042508A - Kit and method for inducing differentiation of pluripotent stem cells into hepatocytes and application of kit and method - Google Patents

Abstract

The invention discloses a kit and a method for inducing pluripotent stem cells to differentiate into hepatocytes and application thereof. The kit contains low-concentration resveratrol, and the invention discovers that the albumin secretion of the liver cells obtained by adding the low-concentration resveratrol for induction is obviously increased, the expression level of liver function related genes such as drug metabolizing enzyme gene and polarized gene and the like are obviously increased, the use of high-concentration and expensive growth factors can be reduced, and the hepatic differentiation of the human multifunctional stem cells is further promoted; the method for differentiating the human multifunctional stem cells into the liver cells in a 3D suspension orientation is established by adopting the kit provided by the invention; greatly quickens the liver differentiation process of stem cells, reduces the differentiation cost, improves the maturity of the liver cells and the drug metabolism capability, and is more efficient and convenient.

Description

Kit, method and application for inducing pluripotent stem cells to differentiate into liver cells

technical field

The invention belongs to the technical field of biomedicine, and specifically relates to a kit, a method and an application for inducing pluripotent stem cells to differentiate into liver cells.

Background technique

Currently, the incidence of liver disease is increasing significantly worldwide, and orthotopic donor liver transplantation is the primary method for the treatment of end-stage liver disease. In the case of a shortage of donor liver organs, patients with end-stage liver disease and acute liver failure are clinically treated with hepatic cell transplantation or bioartificial liver. Each liver cell transplantation treatment or bioartificial liver treatment requires 10 ¹⁰ cubic meters Hundreds of millions of liver cells ^[1] . However, due to the same shortage of liver sources, the inability to obtain enough hepatocytes limits the clinical application of hepatocyte transplantation and bioartificial liver. 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 liver cells for drug metabolism and toxicology testing.

At present, there is still a certain gap in liver function between human pluripotent stem cell (hPSCs)-derived hepatocytes obtained by 3D differentiation method and freshly isolated primary human hepatocytes.

Freshly isolated primary human hepatocytes (PHHs) will rapidly lose their characteristics and functions when cultured in vitro. Therefore, in the clinical and biopharmaceutical fields, the cultivation of hepatocytes with functions comparable to PHHs has become an urgent problem to be solved ^{[2 ]} .

The directional differentiation of human pluripotent stem cells to hepatocytes is mainly divided into three stages: the first is the endoderm cell differentiation stage, that is, human pluripotent stem cells differentiate into endoderm cells; the second hepatocyte differentiation stage is that endoderm cells differentiate into hepatic progenitor cells and Hepatic precursor cell differentiation; the third stage is the maturation stage of hepatic cells, that 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] , which successfully induced the differentiation of human pluripotent stem cells cultured in two-dimensional (2D) adherent into hepatocytes.

However, 2D differentiation systems cannot mimic the 3D microenvironment of the human liver in vivo; second, increasing evidence shows that 3D culture systems can better mimic the in vivo microenvironment and promote the generation of liver lineages and hepatocyte maturation.

In summary, there are the following problems in the prior art: the differentiation route is time-consuming, the time span is long, and the time cost is high; there are many processes and many cytokines used, so expensive media and cytokines are consumed, and the cost of materials is high; The maturity and function of hepatocyte-like cells derived from pluripotent stem cell differentiation need to be improved. There is an urgent need for a method that can efficiently culture hepatocytes comparable to PHHs.

[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.

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

[3]Duan Y, Ma X, Zou W, et al.Differentiation and characterization of metabolically functioning hepatocytes from human embryonic stem cells[J].StemCells,2010,28(4):674-86.

[4]Ma X, Duan Y, Tschudy-Seney B, et al.Highly efficient differentiation of functional hepatocytes from human induced pluripotent stem cells[J].StemCells Transl Med,2013,2(6):409-19.

Contents of the invention

The purpose of the first aspect of the present invention is to provide a kit for inducing stem cells to differentiate into liver cells.

The purpose of the second aspect of the present invention is to provide the application of resveratrol and/or the kit of the first aspect of the present invention.

The object of the third aspect of the present invention is to provide a method for inducing stem cells into endoderm cells.

The purpose of the fourth aspect of the present invention is to provide a method for inducing stem cells to become hepatic progenitor cells.

The purpose of the fifth aspect of the present invention is to provide a method for inducing stem cells into liver cells.

The technical scheme that the present invention takes is:

The first aspect of the present invention provides a kit comprising at least one of (a1) to (a3);

a1 the first medium, the second medium and the third medium;

a2 fourth culture medium;

a3 fifth culture medium;

The first medium, the second medium, the third medium, the fourth medium, and the fifth medium contain 10nM～10μM Resveratrol;

Activin A is also included in the first culture medium, the second culture medium and the third culture medium;

BMP2 and BMP4 are also included in the fourth medium;

Oncostatin M is also included in the fifth medium.

Preferably, the first culture medium, the second culture medium, the third culture medium, the fourth culture medium and the fifth culture medium comprise 100nM～1μM Resveratrol;

More preferably, the first culture medium, the second culture medium, the third culture medium, the fourth culture medium and the fifth culture medium comprise 1 μM Resveratrol.

Preferably, the kit comprises a first medium, a second medium, a third medium, a fourth medium and a substrate medium.

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.

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

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

Preferably, the concentration of Oncostatin M in the fifth culture medium is 40-60 ng/mL.

Preferably, the first culture medium also includes a GSK3β inhibitor and a basal medium;

Preferably, the GSK3β inhibitor is CHIR99021.

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

Preferably, the second medium further comprises KSR and basal medium.

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

Preferably, the third medium further comprises KSR and basal medium.

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

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

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

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

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

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

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

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

Preferably, the concentration of dexamethasone in the fourth medium is 80-120 nM.

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

Preferably, the fifth medium further comprises HGF, dimethyl sulfoxide, dexamethasone, FGF-4, the first additive, and basal medium.

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

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

Preferably, the concentration of dexamethasone in the fifth medium is 80-120 nM.

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

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 Composed of seven components in the SingleQuots kit: 0.5mL ascorbic acid, 10.5mL BSA-FAF, 0.5mL hydrocortisone, 0.5mL transferrin, 0.5mL insulin, 0.5mL recombinant human epidermal growth factor and 0.5mL GA -1000, SingleQuots kit purchased from Lonza, the article number is CC-4182.

Preferably, the basal medium of the first medium is at least one of DMEM high sugar, DMEM-F12 and RPMI1640 medium.

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

Preferably, the basal medium of the third medium is at least one of DMEM high sugar, DMEM-F12 and RPMI1640 medium.

Preferably, the basal medium of the fourth medium is at least one of RPMI1640 and IMDM medium.

Preferably, the basal medium of the fifth medium is hepatocyte basal medium.

The second aspect of the present invention provides the application of resveratrol and/or the kit described in the first aspect of the present invention in at least one of b1-b6;

b1 preparation of endoderm cells;

b2 preparation of hepatic progenitor cells;

b3 preparation of liver cells;

b4 Preparation of products of endoderm cells;

b5 preparation of products of hepatic progenitor cells;

b6 Preparation of hepatocyte products.

The third aspect of the present invention provides a method for preparing endoderm cells. Stem cells are cultured in the first medium described in the first aspect of the present invention for 12 to 36 hours, and in the second culture described in the first aspect of the present invention medium for 12-36 hours; cultured in the third medium described in the first aspect of the present invention for 12-36 hours.

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

Preferably, the fourth medium is replaced every 16-32 hours during the cultivation process.

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

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

Preferably, the fifth medium is replaced every 16-32 hours during the cultivation process.

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

Preferably, the stem cells are human stem cells with multilineage differentiation potential.

Preferably, the human-derived stem cells with multilineage differentiation potential are human pluripotent stem cells, human parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, fat stem cells or umbilical cord blood stem cells.

Preferably, the stem cells are stem cell spheres.

Preferably, the preparation method of the stem cell sphere is as follows: mix the stem cells with the digestion solution, incubate, discard the digestion solution and add the mTeSR1 medium containing the Rock inhibitor to resuspend, and inoculate at a density of 250,000 to 1 million/mL to the medium containing the Rock inhibitor. culture medium for 12-36 hours.

Preferably, the stem cells are 4 to 5 days after subculture, the cell coverage rate reaches 70% to 80%, and the clones have regular edges and no differentiated cells.

Preferably, the Rock inhibitor is Y-27632.

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

The beneficial effects of the present invention are:

The present invention prepares a new induced pluripotent method by changing the culture method (from 2D culture to 3D culture), replacing some cytokines, and adding low-concentration resveratrol on the basis of the previous liver cell differentiation method in our laboratory. Kit for differentiation of stem cells into hepatocytes. The present invention finds for the first time that the hepatocytes obtained after adding resveratrol have significantly increased secretion of histone, gene expression levels of drug metabolizing enzymes, and expression levels of liver function-related genes such as polarization genes. Therefore, compared with the original differentiation scheme of our laboratory, the present invention replaces the use of high-concentration and expensive growth factors through the combination of the small molecule compound resveratrol and low-concentration growth factors, thereby reducing costs, Moreover, it can further promote the hepatic differentiation of human pluripotent stem cells, improve the maturity of hepatocytes and drug metabolism ability, and differentiate human pluripotent stem cells into more mature hepatocytes under 3D suspension culture differentiation conditions; shorten the differentiation time, improve the hepatocyte liver function.

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-saving method for 3D suspension-directed differentiation of human pluripotent stem cells into hepatocytes is established; the present invention The first stage of differentiation requires only 3 days of differentiation, the second stage only 6 days, and the third stage only 8 days. Compared with the traditional 2D differentiation method (differentiation takes 32 days in total), the present invention greatly speeds up the hepatic 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 final The differentiated hepatocytes have albumin secretion ability and drug metabolism function. Moreover, it has been verified that low concentration of resveratrol can promote the liver function level of hepatocytes and improve the maturity of hepatocytes under 3D suspension culture conditions.

Description of drawings

Figure 1 is a roadmap for the directed differentiation of human pluripotent stem cells to hepatocytes; Figure 1A is a roadmap for the directed differentiation of human pluripotent stem cells cultured in 2D adherent culture to hepatocytes, scale bar = 100 μm; Figure 1B is the roadmap for human pluripotent stem cells cultured in 3D suspension Roadmap of directional induction of pluripotent stem cells to hepatocytes, scale bar = 100 μm.

Figure 2 shows the directional induction and differentiation of human pluripotent stem cells to definitive endoderm. Figure 2A is a roadmap for the directed differentiation of human pluripotent stem cells to endoderm spheroids; Figure 2B is the flow cytometry diagram of SOX17 and FOXA2 in endoderm spheroids; Figure 2C is the directed differentiation of human pluripotent stem cells to endoderm spheroids Images of cell morphology changes in , scale bar = 100 μm. Figure 2D is the immunofluorescent staining of DAPI and FOXA2 of endoderm spheres. Figure 2E shows the endoderm gene expression of human pluripotent stem cell spheroids directed to induce qualitative endoderm spheroids under three different 3D conditions.

Fig. 3 is the directed differentiation of hepatic progenitor cell spheres cultured in 3D suspension to hepatocyte spheres under the condition of adding 1 μM resveratrol. Figure 3A is the route map of human pluripotent stem cells' directed differentiation to hepatocyte spheroids and the image of hepatocyte spheroids, scale bar = 100 μm; Figure 3B is the expression of ALB, AFP, and CYP3A4 in hepatocyte spheroids; Figure 3C is the expression of CPS1, Expression of FOXO1, BSEP, and NTCP; Figure 3D shows the expression of drug-metabolizing enzyme CYP3A4 in hepatocyte spheroids induced by 25 μM rifampicin; Figure 3E shows the secretion of hepatocyte globulin.

Fig. 4 is a diagram showing the results of screening the concentration of resveratrol using cell proliferation toxicity detection.

Detailed ways

The conception and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments, so as to fully understand the purpose, features and effects of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts belong to The protection scope of the present invention.

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

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

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

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

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

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

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

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

The fourth medium (HDM medium): IMDM media (Gibco, 31980030)+20% FBS (fetal bovine serum, VISTECH, SE100-B7953)+2mM L-glutamine (L-glutamine, Gibco, 25030081)+0.126 U/mLhuman insulin (recombinant human insulin, Sigma, 91077C-11MG)+0.3mM1-thioglycerol (1-thioglycerol, Sigma, M6145)+20ng/mL FGF-4 (fibroblast growth factor-4, Peprotech, 100 -18B-50)+20ng/mLHGF (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)+100nM dexamethasone (dexamethasone, Sigma, D4902)+1μM Resveratrol (Selleck, S3934);

The fifth medium (HCM medium): Hepatocyte basal medium (Lonza) (hepatocyte basal medium, Lonza, CC-3911) + Single Quots kit (Lonza) (Lonza, CC-4182, specifically including Ascorbicacid, BSA-FAF , Hydrocortisone, Transferrin, Insulin, rhEGF, seven additive components of GA-1000)+20ng/mL HGF (hepatocyte growth factor, Peprotech, 100-39)+50ng/mL oncostatin M (oncostatin M, Peprotech, 300-10)+0.5% DMSO (dimethylsulfoxide, MP Biomedical, 196055)+100nMdexamethasone (dexamethasone, Sigma, D4902)+20ng/mL FGF4 (fibroblast growth factor-4, Peprotech, 100-18B -50)+1 μM Resveratrol (Selleck, S3934).

This embodiment also provides a method for directional induction of differentiation of human pluripotent stem cells into hepatocytes in 3D suspension culture, and the roadmap is shown in FIG. 1 .

Embodiment 3 resveratrol concentration selection

A method for inducing cytotoxicity test, comprising the steps of:

S1. HepaRG recovery:

1) Select a tube of HepaRG in a good frozen state (provided by the laboratory of teacher Huang Lizhen of South China University of Technology), take the frozen tube out of the liquid nitrogen, and quickly put it into a 37°C water bath to melt;

2) Carefully open the bottle cap, transfer the cells into a centrifuge tube containing 5 mL of DMEM high glucose (Servicebio, G4512) medium with 10% FBS fetal bovine serum (VISTECH, SE100-B7953), centrifuge at 1500 rpm for 5 minutes; After completion, suck off the supernatant, resuspend with 10 mL of DMEM high glucose (Servicebio, G4512) medium with 10% FBS fetal bovine serum (VISTECH, SE100-B7953), inoculate the cell suspension into a 10 cm culture dish, Shake well.

S2.HepaRG passage:

1) For HepaRG cells (the cell coverage rate is about 70%-80%), add 1 mL of calcium and magnesium-free PBS to wash the cells after aspirating and discarding the medium, add 3 mL of 0.05% trypsin (Procell, PB180222) after aspirating and discarding, and put it back into the CO2 incubator Incubate for 2-4 minutes, then add 3 ml of medium and pipette the clones repeatedly to obtain a single-cell suspension;

2) Centrifuge at 1500rpm for 5 minutes; after centrifugation, suck off the supernatant, and resuspend the cells in 10 mL DMEM high glucose (Servicebio, G4512) medium with 10% FBS fetal bovine serum (VISTECH, SE100-B7953). The solution was inoculated into a 10 cm Petri dish and shaken crosswise.

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

1) Prepare the cell suspension, add 1mL calcium and magnesium-free PBS to wash the cells after aspirating HepaRG cells, add 3mL 0.05% trypsin (Procell, PB180222) after aspirating and discarding, put it back into the CO2 incubator and incubate for 2-4 minutes, Then add 3 ml of culture medium and pipette the clone repeatedly to obtain a single-cell suspension;

2) Centrifuge at 1500rpm for 5 minutes; after centrifugation, suck off the supernatant, resuspend with 10mL DMEM high glucose (Servicebio, G4512) medium with 10% FBS fetal bovine serum (VISTECH, SE100-B7953), and count.

3) Inoculate 5000 cells in a 96-well plate, about 100ul per well, and repeat 3 times.

4) Place the cells in an incubator for 24 hours. The concentrations of resveratrol in each well were 0nM, 10nM, 100nM, 1μM, 10μM, 100μM, 1mM.

4) Add 10u CCK-8 solution to each well, put the culture plate into the incubator and incubate for 1h.

5) Measure the absorbance (OD) at 450 nm with a microplate reader.

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

The difference between the kit in Comparative Example 1 and Example 2 is that Resveratrol is not added, and the others are the same as in Example 2.

Application Example 1

A high-efficiency method for directional induction of qualitative endoderm by 3D suspension of human pluripotent stem cells (hPSCs), specifically comprising the following steps (endoderm cell differentiation stage):

S1. Single-cell passaging hPSCs: select hPSCs in good condition: 4-5 days after hPSC passaging, the cell coverage rate reaches about 70%-80%, and the hPSCs with regular edges and no differentiated cells are cloned), discard the medium and add Wash the cells with 1mL calcium and magnesium-free PBS, add 1mL GCDR after aspiration and discard, put it back in the _CO2 incubator and incubate for 2-4 minutes, then aspirate and discard the GCDR in the well, add 1mL mTeSR1 medium containing 10μM Y-27632 (Rocki) , use a pipette gun to pipette the clone repeatedly, and finally obtain a single-cell suspension;

S2. Cell inoculation: draw 20 μl of single cell suspension, stain with trypan blue and count with a hemocytometer; use a Corning low-adhesion six-well culture plate, absorb 1 million single cell suspensions and inoculate them into the wells, and then supplement with 10 μM Y-27632 mTeSR1 medium until there is a total of 2 mL of liquid in the well, shake the well plate crosswise to suspend the cells evenly in the culture medium, which is recorded as day 0, and human pluripotent stem cells with a diameter of about 100 μm will be formed after 3-5 days ball;

S3. Cell replacement: After 3-5 days (the first day of differentiation), the human pluripotent stem cell spheres were washed with 1 mL of calcium and magnesium-free PBS in each well, and 2 mL of DE medium 1 was added after discarding; after 48 hours (the second day of differentiation) day) DE medium 2 was replaced, and DE medium 3 was replaced after 72 hours (differentiation day 3);

S4. Cell Harvesting: On the third day of culture, take pictures of the endoderm cell spheres, then collect the endoderm cell spheres, and settle for 1-2 minutes naturally. After discarding the supernatant, add 1 mL of PBS to wash the endoderm cell spheres, and naturally settle for 1-2 minutes. After 2 minutes, the supernatant was discarded, and RNA was extracted, and the expression levels of SOX17 and FOXA2 genes were detected by qPCR.

The experimental results are shown in Figure 2. Human pluripotent stem cells H9 in 3D suspension culture are induced to differentiate into endoderm spheres: Figure 2A is a roadmap for human pluripotent stem cells to differentiate into endoderm spheres; Figure 2B is endoderm spheres The flow cytometric detection of SOX17 and FOXA2 of human pluripotent stem cells, Figure 2C is the image of morphological changes of human pluripotent stem cells induced to differentiate into endoderm spheres, and Figure 2D is the immunofluorescence staining of DAPI and FOXA2 of endoderm spheres; it can be seen that using this The expression level of endoderm marker genes in the cell spheroid prepared by the kit in Example 1 was relatively high ( FIG. 2E ). Therefore, the kit in Example 1 can successfully differentiate human pluripotent stem cells into endoderm spheroids with high efficiency.

Application Example 2

A highly efficient method for 3D suspension of human pluripotent stem cells (hPSCs) to induce differentiation into hepatocytes, the roadmap is shown in Figure 3A; it specifically includes the following steps:

S1. Single-cell passaging hPSCs: select hPSCs in good condition (good condition: 4-5 days after hPSC passaging, the cell coverage rate reaches about 70%-80%, and the hPSCs with regular edges and no differentiated cells are cloned), discarded and cultured Add 1mL calcium and magnesium-free PBS to wash the cells after the base, add 1mL GCDR after aspiration and discard, put it back into the CO ₂ incubator and incubate for 2-4 minutes, then aspirate and discard the GCDR in the well, add 1mL containing 10μM Y-27632 (Rocki) For mTeSR1 medium, use a pipette gun to pipette clones repeatedly to obtain a single-cell suspension;

S2. Cell inoculation: draw 20 μl of single cell suspension, stain with trypan blue and count with a hemocytometer; use Corning low-adhesion six-well culture plate, absorb 500,000-2 million single cell suspension and inoculate into the wells again Supplement the mTeSR1 medium containing 10 μM Y-27632 until there is a total of 2 mL of liquid in the well, shake the well plate crosswise to suspend the cells evenly in the culture medium, which is recorded as day 0, and human cells with a diameter of 50-70 μm are formed after 3-5 days. Pluripotent stem cell spheres;

S3. Cell replacement: After 3-5 days (the first day of differentiation), the human pluripotent stem cell spheres were washed with 1 mL of calcium and magnesium-free PBS in each well, and 2 mL of DE medium 1 was added after discarding; after 48 hours (the second day of differentiation) day) DE medium 2 was replaced, and DE medium 3 was replaced after 72 hours (differentiation day 3);

S4. Cell replacement at the stage of directional induction of hepatic progenitor cells: wash the hepatic progenitor cell spheres with 1 mL of calcium and magnesium-free PBS in each well on the 6th to 8th day (the 4th day of differentiation), and add 2 mL of HDM medium after aspiration and discarding; Within 7-11 days (5-9 days of differentiation), replace 2 mL of HDM medium every day.

S5. Cell replacement at the stage of directional induction of hepatic cell maturation: within 12-14 days (the 10th day of differentiation), hepatic progenitor cell spheres were washed with 1 mL of calcium and magnesium-free PBS in each well, and 2 mL of HCM medium was added after discarding; Within 13-17 days (11-15 days of differentiation), replace 2mL of HCM medium every day.

S6. Cell harvesting: take pictures of the hepatocyte spheres every day, collect the hepatocyte spheres after the hepatocytes are mature, and settle naturally for 1-2 minutes, collect the medium supernatant, and use ELISA kit to detect the secretion of human albumin (ALB). Then add 1 mL of LPBS to wash the hepatocyte spheroids, settle for 1-2 minutes naturally, discard the supernatant and perform qPCR to detect the expression levels of human alpha-fetoprotein (AFP), ALB and CYP3A4 genes.

The experimental results are shown in Figure 3. In Figure 3, hepatic progenitor cell spheroids cultured in 3D suspension under the condition of adding 1uM resveratrol to mature and induce differentiation into hepatocyte spheroids: Figure 3A is a roadmap for the maturation of hepatic progenitor cell spheroids to hepatocyte spheroids and mature morphological images of hepatocyte spheroids; Figure 3B is the qPCR detection of functional genes ALB, AFP and CYP3A4 of hepatocyte spheroids, which can be drawn from the figure, compared with the control group (comparative example 1), the resveratrol group The alpha-fetoprotein gene was elevated, and the albumin gene and drug metabolism gene were also significantly elevated. Therefore, hepatic progenitor cells successfully matured into hepatocyte spheroids; Figure 3C shows the qPCR detection of CPS1, FOXO1, BSEP, and NTCP in hepatocyte spheroids. These four genes represent ammonia metabolism, autophagy, bottom membrane polarization, and apical membrane respectively. polarization. Compared with the control group, the expressions of CPS1, BSEP and NTCP in the resveratrol group were significantly increased, while FOXO1 was significantly decreased. The ammonia metabolism and polarization of hepatocytes in the resveratrol group are enhanced and autophagy is decreased; Figure 3D shows the expression of drug-metabolizing enzyme CYP3A4 in hepatocyte spheroids induced by 25 μM rifampicin. After 25 μM rifampicin induced hepatocyte spheroids for 48 hours, 1 mL of PBS was added to wash the hepatocyte spheroids, and the hepatocyte spheroids were naturally settled for 1-2 minutes. Inducer response is an important hallmark of mature hepatocytes. It can be seen that the addition of 1 μM Resveratrol significantly up-regulated the gene expression of metabolic enzymes compared with the control group, which shows that the differentiated hepatocyte spheroids in this example can respond to the induction and stimulation of drugs, and have good drug metabolism functions. Potential to become a cell model for drug screening; Figure 3E shows the ALB content in the medium supernatant detected by Elisa in the maturation stage of hepatocyte spheroids, and the secretion amounts are 102.8430ug/mL and 138.4630ug/mL respectively, indicating that the hepatocytes treated with resveratrol Balls have a stronger ability to secrete albumin.

The above-mentioned specific embodiments have described the present invention in detail, but the present invention is not limited to the above-mentioned embodiments, and various changes can be made within the knowledge of those of ordinary skill in the art without departing from the gist of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other if there is no conflict.

Claims (10)

1. A kit comprising at least one of (a 1) to (a 3);

a1, a first culture medium, a second culture medium and a third culture medium;

a2, a fourth culture medium;

a3, 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 comprise 10 nM-10 mu M Resveratrol;

the first culture medium, the second culture medium and the third culture medium also comprise Activin A;

the fourth culture medium also comprises BMP2 and BMP4;

the fifth culture medium also comprises Oncoinhibin M.

2. The kit according to claim 1, wherein the concentration of Activin a in the first medium, the second medium and the third medium is 80-120 ng/mL; 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 the Oncoinhibin M in the fifth culture medium is 40-60 ng/mL.

3. The kit according to claim 2, wherein,

the first medium further comprises a gsk3β inhibitor, a basal medium;

preferably, the second medium further comprises KSR, basal medium;

preferably, the third medium further comprises KSR, basal medium;

preferably, the fourth medium further comprises FBS, L-glutamine, insulin, 1-thioglycerol, FGF-4, HGF, dexamethasone, dimethyl sulfoxide, basal medium;

preferably, the fifth medium further comprises HGF, dimethyl sulfoxide, dexamethasone, FGF-4, a first additive, a basal medium;

preferably, the first additive comprises: ascorbic acid, BSA-FAF, hydrocortisone, transferrin, insulin, recombinant human epidermal growth factor and GA-1000.

4. The kit according to claim 3, wherein,

the basic culture medium of the first culture medium is at least one of DMEM high sugar, DMEM-F12 and RPMI1640 culture medium;

preferably, the basal medium of the second medium is at least one of DMEM high sugar, DMEM-F12 and RPMI1640 medium;

preferably, the basal medium of the third medium is at least one of DMEM high sugar, DMEM-F12 and RPMI1640 medium;

preferably, the basal medium of the fourth medium is at least one of RPMI1640 and IMDM medium;

preferably, the basal medium of the fifth medium is a hepatocyte basal medium.

5. Use of resveratrol and/or a kit according to any of claims 1 to 4 in at least one of b1 to b 6;

b1, preparing endoderm cells;

b2, preparing hepatic progenitors;

b3, preparing liver cells;

b4, preparing a product for inducing stem cells to differentiate into endoderm cells;

b5, preparing a product for inducing stem cells to differentiate into hepatic progenitor cells;

b6 preparing a product for inducing stem cells to differentiate into liver cells.

6. A method for preparing endoderm cells, comprising culturing stem cells in a first medium according to any one of claims 1 to 4 for 12 to 36 hours, in a second medium according to any one of claims 1 to 4 for 12 to 36 hours, and in a third medium according to any one of claims 1 to 4 for 12 to 36 hours.

7. A method for preparing hepatic progenitors comprising culturing endodermal cells in the fourth medium according to any one of claims 1 to 4 for 120 to 168 hours to obtain hepatic progenitors; preferably, the endoderm cells are prepared by the method of claim 6.

8. A method for preparing a hepatocyte, comprising culturing a hepatic progenitor in the fifth medium of any one of claims 1-4 for 120-168 hours to obtain a hepatocyte; preferably, the hepatic progenitors are prepared by the method of claim 7.

9. The method according to any one of claims 6 to 8, wherein: the stem cells are human stem cells with multidirectional differentiation potential;

preferably, the human stem cells having multipotential differentiation potential are human embryonic stem cells, human parthenogenesis stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells or cord blood stem cells.

10. The method of claim 9, wherein the stem cells are stem cell pellets;

preferably, the stem cell pellet is prepared by the following steps: mixing stem cells with digestive juice, incubating, discarding digestive juice, adding mTESR1 culture medium containing Rock inhibitor, re-suspending, inoculating into mTESR1 culture medium containing Rock inhibitor at 25-100 ten thousand/mL, and culturing for 12-36 h.

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