Culture medium composition for inducing pluripotent stem cells to be differentiated into insulin-secreting cells and application of culture medium composition

Abstract

The invention discloses a culture medium composition for inducing pluripotent stem cells to be differentiated into insulin-secreting cells and application of the culture medium composition, and belongs to the technical field of cell differentiation. The culture medium combination comprises an induction culture medium 1 and an induction culture medium 2, wherein the induction culture medium 1 comprises 80-90 vol% of a DMEM high glucose culture medium, 10-20 vol% of FBS, 1.5-2.5 mmol / L of glutamine, 1.5-2.5 mmol / L of non-essential amino acid, 8-12 mmol / L of nicotinamide, 8-12 [mu] g / L of activin A, 8-12 [mu] g / L of hepatocyte growth factors and 8-12 [mu] g / L of beta-cytokine, and the pH value of the induction culture medium 1 is 7.2-7.4; the induction culture medium 2 comprises 94 to 98 vol% of a DMEM / F-12 culture medium, 2 to 6 vol% of FBS, 1.5 to 2.5 mmol / L of glutamine, 1.5 to 2.5 mmol / L of non-essential amino acid, 8 to 12 mmol / L of nicotinamide, 20 to 30 [mu] g / mL of insulin, 0.8 to 1.2 [mu] g / mL of laminin and 8 to 12 [mu] g / L of activin A, and the pH value of the induction culture medium 2 is 7.2 to 7.4. The method for inducing the pluripotent stem cells to be differentiated into the insulin-secreting cells by using the culture medium combination is short in time consumption, only needs 8 days, and is simple in process and lower in cost.

Description

Technical Field

[0001] This invention belongs to the field of cell differentiation technology, and in particular relates to a culture medium combination for inducing pluripotent stem cells to differentiate into insulin-secreting cells and its application. Background Technology

[0002] With the increasing incidence of diabetes year by year, finding effective treatments for diabetes has become a hot topic in medical research. Embryonic stem cells (ESCs) have shown great potential in regenerative medicine and cell therapy due to their multi-directional differentiation potential. Embryonic stem cells (ESCs) have the potential to differentiate into various tissue cells, and under suitable conditions, they can differentiate into various target cells needed for the treatment of certain diseases. Compared with gene therapy, using functional cells for transplantation can avoid problems such as gene vector safety, gene expression instability, and gene product processing maturation, and may be subject to in vivo metabolic regulation, making their function closer to the physiological state. Inducing embryonic stem cells into insulin-producing cells (IPCs) for the treatment of diabetes has been a hot research topic both domestically and internationally in recent years. Lumelsky et al. (Science, 2001) induced mouse ES cells into insulin-secreting pancreatic β-like cells through five stages of culture induction totaling approximately 28-32 days. After transplantation of these IPCs into diabetic mice, blood glucose levels decreased, and growth and development improved, showing promising application prospects. Similar reports have increased in recent years, but current research in this area shares some common problems: ① Low yield: During the induction of ES cells into IPCs, the conversion rate of IPCs is low, with most cells dying during the induction process. Only a limited number of IPCs can be obtained from a single culture, which is insufficient for large-scale clinical application. ② Poor IPC function: IPCs differentiated from ES cells have only about 1 / 50th the insulin secretion function of normal β cells, failing to produce enough insulin to meet therapeutic requirements. ③ Long IPC induction cycle: Inducing ES cells to differentiate into IPCs involves multiple induction stages, with an experimental cycle typically lasting about a month. Such a lengthy experimental cycle does not meet the requirements for clinical application. ④ Complex procedures: Due to the high requirements for ES cell culture and expansion, the differentiation and induction process of IPCs is complex, difficult to standardize, and hinders quality control. Summary of the Invention

[0003] To address the above technical problems, this invention provides a culture medium combination for inducing pluripotent stem cells to differentiate into insulin-secreting cells, the culture medium combination comprising induction medium 1 and induction medium 2, wherein: The induction medium 1 comprises 80-90 vol% DMEM high glucose medium, 10-20 vol% FBS, 1.5-2.5 mmol / L glutamine, 1.5-2.5 mmol / L non-essential amino acids, 8-12 mmol / L nicotinamide, 8-12 μg / L activin A, 8-12 μg / L hepatocyte growth factor and 8-12 μg / L β-cytokinin, pH 7.2-7.4; The induction medium 2 comprises 94-98 vol% DMEM / F-12 medium, 2-6 vol% FBS, 1.5-2.5 mmol / L glutamine, 1.5-2.5 mmol / L non-essential amino acids, 8-12 mmol / L nicotinamide, 20-30 μg / mL insulin, 0.8-1.2 μg / mL laminin and 8-12 μg / L activin A, pH 7.2-7.4.

[0004] In one embodiment of the present invention, the induction medium 1 comprises 85 vol% DMEM high glucose medium, 15 vol% FBS, 2 mmol / L glutamine, 2 mmol / L non-essential amino acids, 10 mmol / L nicotinamide, 10 μg / L activin A, 10 μg / L hepatocyte growth factor and 10 μg / L β-cytokinin, pH 7.2-7.4.

[0005] In one embodiment of the present invention, the induction medium 2 comprises 96 vol% DMEM / F-12 medium, 4 vol% FBS, 2 mmol / L glutamine, 2 mmol / L non-essential amino acids, 10 mmol / L nicotinamide, 25 μg / mL insulin, 1 μg / mL laminin and 10 μg / L activin A, pH 7.2-7.4.

[0006] This invention also provides the application of the above-mentioned culture medium combination in the induction of pluripotent stem cell differentiation.

[0007] The present invention also provides a method for inducing pluripotent stem cells to differentiate into insulin-secreting cells, comprising the following steps: (1) Resuspend pluripotent stem cells in the above-mentioned induction culture medium 1, and then transfer them to a 6-well plate coated with 45-55 μg / mL type I rat tail collagen for further culture. Replace the culture medium every 24 hours and culture for 3-5 days. (2) Replace with the above-mentioned induction medium 2 for culture, replace with new medium every 24 hours, and culture for 3-5 days.

[0008] In one embodiment of the present invention, the concentration of type I rat tail collagen in step (1) is 50 μg / mL.

[0009] In one embodiment of the present invention, the culture time in step (1) is 4 days.

[0010] In one embodiment of the present invention, the culture time in step (2) is 4 days.

[0011] This invention also provides the application of the above method in the induction of pluripotent stem cell differentiation.

[0012] Compared with the prior art, the present invention has the following beneficial effects: Existing techniques for inducing embryonic stem cells into insulin-secreting cells mostly employ stage-specific growth factor combinations, but these methods are complex, time-consuming, and yield limited numbers and functions of insulin-secreting cells, failing to meet clinical application needs. Current technologies suffer from low induction efficiency, low numbers of differentiated insulin-secreting cells, complex procedures, long cycles, and high costs. In contrast, the induction method of this invention has a short induction time of only 8 days, a simpler process, and lower costs. Attached Figure Description

[0013] Figure 1 The results of DTZ staining identification in Example 1 are shown.

[0014] Figure 2 The results of DTZ staining identification in Comparative Example 1 are shown. Detailed Implementation

[0015] The non-essential amino acids used in this invention can be any non-essential amino acids known to those skilled in the art, and there are no special restrictions. The non-essential amino acids used in the examples are finished products from Solarbio.

[0016] Example 1 1. Preparation of ES-D3 cell feeder layer MEF cell culture medium: 90% DMEM high glucose medium + 9% FBS + 1% penicillin and antibiotic-resistant medium, pH 7.2-7.4; MEF cell cryopreservation solution: 70% DMEM high glucose medium + 20% FBS + 10% DMSO.

[0017] (1) Take a clean culture flask, add 2 mL of 0.1% gelatin aqueous solution, place it in an incubator for 30 min, aspirate the gelatin, and let the culture flask air dry before use. (2) Take the MEF cells that have been passaged to the 4th generation and have covered the bottom of the culture flask out of the incubator, add 1 mL of prepared mitomycin C solution to the culture flask, and continue to culture for 1.5 h. (3) Aspirate the culture medium, rinse repeatedly with PBS buffer 5 times to wash away mitomycin C residue and eliminate its influence on cell growth. (4) Add 1 mL of 0.25% trypsin digestion solution containing EDTA, and digest in an incubator for 2 min. (5) Add 3 mL of MEF cell culture medium to stop digestion, transfer the digestion solution in the culture flask to a centrifuge tube, and centrifuge at 1000 rpm for 5 min. (6) After centrifugation, remove the centrifuge tube, aspirate the supernatant, resuspend the cells in the prepared ES-D3 cell culture medium, gently pipette them into a single-cell suspension, and transfer them to a culture flask coated with gelatin as a feeder layer for ES-D3 cell culture.

[0018] 2. ES-D3 cell resuscitation and culture ES-D3 cell culture medium: 85% DMEM high glucose medium + 15% FBS + 2 mmol / L non-essential amino acids + 1000 IU / L LIF + 0.1 mmol / L β-mercaptoethanol + 2 mmol / L glutamine.

[0019] (1) Remove the frozen ES-D3 cells from the liquid nitrogen tank and thaw them in a water bath at 37°C.

[0020] (2) Take the lysed cells to a pre-sterilized ultraviolet laminar flow hood, wipe the cryovial with 75% ethanol, transfer the cells to a 15mL centrifuge tube, add 3mL of complete culture medium, and centrifuge at 1000rpm for 5min.

[0021] (3) After centrifugation, remove the centrifuge tube, discard the supernatant, add 2 mL of ES-D3 cell culture medium to the centrifuge tube, pipette evenly, and resuspend the cells.

[0022] (4) Transfer the cells to a 25T culture flask containing a feeder layer and place it in an incubator with conditions set in advance at 37°C, 5% CO2 and 95% humidity. Change the medium every other day and observe under a microscope every day.

[0023] 3. Induction of ES-D3 cells into insulin-secreting cells Induction medium 1: 85% DMEM high glucose medium (without LIF) + 15% FBS + 2 mmol / L glutamine + 2 mmol / L non-essential amino acids + 10 mmol / L nicotinamide + 10 μg / L activin A + 10 μg / L hepatocyte growth factor + 10 μg / L β-cytokinin, pH 7.2-7.4.

[0024] Induction medium 2: 96% DMEM / F-12 medium + 4% FBS + 2 mmol / L glutamine + 2 mmol / L non-essential amino acids + 10 mmol / L nicotinamide + 25 μg / mL insulin + 1 μg / mL laminin + 10 μg / L activin A, pH 7.2-7.4.

[0025] Glutamine: Functions: A fundamental nutrient in cell culture, providing energy (through the tricarboxylic acid cycle) and nitrogen for rapidly proliferating and differentiating cells. It is a precursor for the synthesis of nucleotides, amino acids (such as other non-essential amino acids), and the antioxidant glutathione.

[0026] Its significance in differentiation: It ensures that cells have sufficient energy and biosynthetic raw materials during the energy-intensive differentiation process, maintains cell viability and normal metabolism, and is a fundamental component of any cell culture (including the differentiation process).

[0027] Non-essential amino acids (NEAAs): Function: Provides various amino acids required for cell protein synthesis. Although cells can synthesize these amino acids themselves, exogenous addition can reduce the cellular synthetic burden and optimize cell growth and function under culture conditions with high metabolic demands.

[0028] Its significance in differentiation: It supports the synthesis of a large number of new proteins during differentiation, ensuring cell health and the smooth progress of the differentiation process. It is a standard additive in basal culture media.

[0029] Nicotinamide: Functions: The amide form of vitamin B3 is a precursor to coenzymes NAD⁺ / NADH and NADP⁺ / NADPH, participating in energy metabolism, redox reactions, and DNA repair. More importantly, it is a key factor in the differentiation and survival of pancreatic endocrine cells (especially β cells).

[0030] The significance of differentiation: Promotes endocrine fate: Inhibits the differentiation of non-endocrine pancreatic cells (such as ductal cells) and specifically guides pluripotent stem cell-derived precursor cells to differentiate into insulin-positive endocrine cells (including β cells).

[0031] Enhanced cell survival: Improved survival of newly generated endocrine / β-like cells by regulating energy metabolism and reducing oxidative stress.

[0032] Improved function: It helps differentiated cells acquire the ability to secrete insulin stimulated by glucose.

[0033] Inhibition of terminal differentiation / dedifferentiation: helps maintain the mature state of β cells at certain stages.

[0034] Activin A: Function: It belongs to the TGF-β superfamily and is a key developmental signaling molecule.

[0035] Significance in differentiation: Usually used in the earliest stages of differentiation.

[0036] Induction of stable endoderm: Activin A (usually used in combination with Wnt signaling pathway activators such as CHIR99021) is the gold standard signal driving the efficient differentiation of pluripotent stem cells into stable endoderm. Stable endoderm is the common tissue of origin for organs such as the pancreas (including β cells), liver, lungs, and thyroid gland. It is an absolutely necessary starting point for subsequent differentiation of pancreas and β cells.

[0037] Hepatocyte growth factor (HGF): Function: A pleiotropic growth factor that activates multiple signaling pathways (such as PI3K / Akt, MAPK) after binding to its receptor c-Met.

[0038] Significance in differentiation: It is usually added in the middle and late stages of differentiation (after the formation of endocrine precursors or during the maturation of β cells).

[0039] Promotes proliferation and survival: Stimulates the proliferation of pancreatic endocrine precursor cells and new β-like cells, and inhibits their apoptosis, increasing the number of cells ultimately produced.

[0040] Promoting maturation and function: Studies have shown that HGF can promote the expression of β-cell maturation markers (such as MafA) and enhance glucose-stimulated insulin secretion. It may help cells acquire a more mature phenotype.

[0041] β-cytokinin (BTC): Function: It belongs to the epidermal growth factor family and binds to the EGFR / ErbB receptor.

[0042] Its significance in differentiation: It is mainly used in the later stages of differentiation (differentiation of endocrine precursors into mature β cells or the maturation period).

[0043] Potent β-cell differentiation factor: BTC has been shown to effectively induce pancreatic ductal cells or endocrine precursor cells to differentiate into insulin-positive cells.

[0044] Promotes proliferation: Stimulates the proliferation of β cells (or precursor cells).

[0045] Enhanced function: It can increase the insulin content and glucose responsiveness of differentiated β-like cells.

[0046] Synergistic with EGF receptor signaling: It often works synergistically with EGF or other EGF family members in differentiation protocols to optimize β cell differentiation and functional maturation.

[0047] Induction of ES to IPC differentiation (1) ES-D3 cell culture and expansion are the same as ES-D3 cell resuscitation and culture (2) IPC pre-induction: A. After expanding and culturing ES-D3 cells for 3-4 days, add 1 mL of 0.25% trypsin digestion solution containing EDTA and digest in an incubator for 2 minutes to digest into single cells.

[0048] B. Add 3 mL of ES-D3 cell culture medium to stop digestion, transfer the digestion solution in the culture flask to a centrifuge tube, and centrifuge at 1000 rpm for 5 min.

[0049] C. After centrifugation, remove the centrifuge tube, discard the supernatant, resuspend the cells in prepared ES-D3 cell induction culture medium 1, gently pipette to form a single-cell suspension, and transfer to a 6-well plate coated with 50 μg / mL type I rat tail collagen. Culture in induction medium 1, changing the medium every 24 hours for 4 days. Detect the mRNA expression of differentiation markers SOX17, Foxa2, PDX-1, Ngn3, and Nkx6.1 every 2 days.

[0050] (3) IPC induction: After IPC pre-induction for 4 days, the medium was replaced with induction medium 2 for further culture. The medium was replaced with fresh medium every 24 hours for 4 days.

[0051] 4. Identification (1) Cell morphology observation Microscopic observation of cell growth status and morphological changes during cell induction, and DTZ staining to identify the maturation of insulin-secreting cells.

[0052] Weigh 50 mg of DTZ powder and dissolve it in 5 mL of DMSO. Shake well until completely dissolved to prepare the DTZ staining solution. Filter through a 0.22 μM filter for sterilization. After inducing maturation of insulin-secreting cells, add DTZ staining solution to the induction medium at a ratio of 100:1, incubate for 20 min, observe under a microscope and photograph the results. See [link to results]. Figure 1 .

[0053] (2) mRNA expression of cell differentiation markers A. Total RNA extraction: Wash cells twice with 2 mL PBS buffer in a culture flask, remove the PBS, add 1 mL RNA extraction buffer, and pipette to lyse the cells. Transfer the liquid to a 1.5 mL sterile EP tube, add 250 μL chloroform, mix thoroughly, let stand for 3 min, and centrifuge at 12000 rpm for 10 min at 4 °C. Take 400 μL of the supernatant and add it to a new EP tube, add 0.8 times the volume of isopropanol, mix thoroughly, incubate at -20 °C for 15 min, and centrifuge again under the same conditions. The white precipitate at the bottom is the total RNA. B. RNA Concentration Determination: Aspirate the supernatant, add 1.5 mL of 75% ethanol to the precipitate for washing, centrifuge at 12000 rpm for 10 min at 4°C, aspirate the supernatant, dry the centrifuge tube, add 15 μL of RNase-free pure water to dissolve the RNA, incubate at 55°C for 5 min, and detect the RNA absorbance value using a microplate reader. Dilute any excessively high concentrations of RNA again to achieve a final RNA concentration of 100-500 ng / L. C. Reverse Transcription Reaction System Preparation (20 μL reaction system): Prepare the reaction system in a PCR tube according to the instructions, mix thoroughly by pipetting, incubate at 25°C for 5 min, then at 42°C for 30 min, and finally incubate at 85°C for 5 min to inactivate reverse transcriptase. D. PCR quantification: The reaction system consisted of 7.5 μL of 2×qPCR Mix, 1.5 μL of 2.5 μM gene primers, 2.0 μL of reverse transcription product, and 4.0 μL of ddH2O. PCR amplification conditions were: pre-denaturation at 95℃ for 10 min, followed by 40 cycles of 95℃ for 15 s and 60℃ for 60 s; melting curve was prepared at 60℃-95℃, with a temperature increase of 0.3℃ every 15 s. Results were expressed as 2... -△△CTMethods: PCR primer synthesis was performed by Wuhan Saiweier Biotechnology Co., Ltd., and the synthesized primers are shown in Table 1 below. On day 2 of cell induction, there was no significant difference in the relative expression levels of mRNAs of SOX17, Foxa2, PDX-1, Ngn3, and Nkx6.1. On day 4 of induction, the relative expression level of Ngn3, a marker of pancreatic endocrine progenitor cells, was higher than that of SOX17, Foxa2, PDX-1, and Nkx6.1, consistent with the Ngn3 expression status in the pancreatic endocrine progenitor cell phase reported in the literature, indicating that the cells were in the pancreatic endocrine progenitor cell phase on day 4 of induction. On days 6 and 8 of cell induction, the relative expression level of Nkx6.1, a marker of insulin-secreting cells, was the highest, consistent with the expression trend in the insulin-secreting cell phase reported in the literature, indicating that the induced cells were in the insulin-secreting cell phase at this time.

[0054] Table 1 Primer sequences

[0055] Table 2. Relative mRNA Expression Levels

[0056] (3) Expression of insulin and C-peptide under stimulation with 18.0 g / L glucose IPCs were cultured in induction medium 2 for 4 days, and then glucose at a concentration of 10 mmol / L was added to the culture medium. After 24 hours, the culture medium was replaced with fresh induction medium containing 20 mmol / L glucose, and induction continued for another 24 hours before IPC identification. The expression of insulin and C-peptide was detected by ELISA, following the kit instructions. The results are shown in Table 3.

[0057] Table 3

[0058] In summary, the results, combined with DTZ staining, insulin and C-peptide mRNA expression markers, demonstrate that ES-D3 successfully induced insulin-secreting cells (IPCs).

[0059] Comparative Example 1 Induction of ES to IPC differentiation ES culture medium DMEM high glucose medium contains 15% FCS, 1000U / ml LIF, 0.1mmol / L β-ME (β-mercaptoethanol), 2.0g / L each of NaHCO3 and HEPES, pH 7.2-7.4.

[0060] The EB culture medium is identical to the ES culture medium except that it does not contain LIF.

[0061] NPC selective medium DMEM / F12, serum-free, containing 1% N2 supplement, bFGF 50 ng / ml, and fibronectin 50 ng / ml.

[0062] IPC selective medium: DMEM / F12 medium, serum-free, containing nicotinamide 10 mmol / L, bFGF 50 ng / ml, activin A (ATA) 10 ng / ml, β-cytokinin (BCL) 10 ng / ml, hepatocyte growth factor (HGF) 10 ng / ml, and 1% B27 supplement.

[0063] (a) Phased induction 1. First stage: ES cell culture and expansion for 4 days. 2. Formation of the second-stage embryonic body (EB) ES cells are digested using the hanging drop method, pipetted into single cells, centrifuged, and then the ES cell density is adjusted to 1×10⁻⁶ cells using EB culture medium. 3 Add 100 μl / well in droplets onto the lid of a culture dish or into a 96-well cell culture plate. Invert the culture plate and add PBS to the culture dish (or the lid of the 96-well culture plate) to maintain humidity. Incubate at 37°C with 5% CO2 for 4 days.

[0064] 3. The induction of EB to NPC differentiation in the third stage (1) Coating with 50 μg / ml type I collagen (pH 4.0) at 37°C for 12 h, removing the fibronectin solution, washing twice with PBS, and storing aseptically for later use.

[0065] (2) Inoculation with EB: Collect embryos cultured on day 4 into a 10ml centrifuge tube and allow them to settle naturally at room temperature for 30min. After the EB settles, remove some of the supernatant, centrifuge at 1000rpm for 10min, suspend the precipitated EB in EB culture medium, and inoculate into coated culture plates or culture flasks at an appropriate density.

[0066] (3) Selective induction of NPC: After culturing in EB medium for 24 hours, most of the EB adhered to the wall. The non-adhered EB was removed, and the culture medium was replaced with NPC selective medium. The culture was continued, and the fresh culture medium was replaced every 2 days for 4 days of induction culture.

[0067] 4. Induction of NPC to IPC differentiation in the fourth stage: After 4 days of NPC induction culture, the medium was replaced with IPC induction medium. Fresh medium was replaced every 2 days. On the 4th day, glucose at a concentration of 10 mmol / L was added to the culture medium. After 24 hours, the medium was replaced with fresh induction medium containing 20 mmol / L glucose, and induction continued for another 24 hours.

[0068] 5. Identification Cell morphology observation: Microscopic observation of cell growth status and morphological changes during cell induction, and DTZ staining to identify the maturation of insulin-secreting cells.

[0069] Weigh 50 mg of DTZ powder and dissolve it in 5 mL of DMSO. Shake well until completely dissolved to prepare the DTZ staining solution. Filter through a 0.22 μM filter for sterilization. After inducing insulin-secreting cells to mature, add DTZ staining solution to the induction medium at a ratio of 100:1, incubate for 20 min, observe under a microscope and photograph the results. Figure 2 As shown.

[0070] Compared with Comparative Example 1, Example 1, after the induction solution was improved and optimized, reduced the difficult step of embryoid preparation in the induction of ES-D3 cells into insulin-secreting cells, greatly reduced the difficulty of induction and shortened the induction time, making it more efficient, lower in cost and more reproducible.

[0071] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A culture medium composition for inducing pluripotent stem cells to differentiate into insulin-secreting cells, characterized in that, The culture medium combination includes induction medium 1 and induction medium 2, wherein: The induction medium 1 comprises 80-90 vol% DMEM high glucose medium, 10-20 vol% FBS, 1.5-2.5 mmol / L glutamine, 1.5-2.5 mmol / L non-essential amino acids, 8-12 mmol / L nicotinamide, 8-12 μg / L activin A, 8-12 μg / L hepatocyte growth factor and 8-12 μg / L β-cytokinin, pH 7.2-7.4; The induction medium 2 comprises 94-98 vol% DMEM / F-12 medium, 2-6 vol% FBS, 1.5-2.5 mmol / L glutamine, 1.5-2.5 mmol / L non-essential amino acids, 8-12 mmol / L nicotinamide, 20-30 μg / mL insulin, 0.8-1.2 μg / mL laminin and 8-12 μg / L activin A, pH 7.2-7.

4.

2. The culture medium combination according to claim 1, characterized in that, The induction medium 1 comprises 85 vol% DMEM high glucose medium, 15 vol% FBS, 2 mmol / L glutamine, 2 mmol / L non-essential amino acids, 10 mmol / L nicotinamide, 10 μg / L activin A, 10 μg / L hepatocyte growth factor and 10 μg / L β-cytokinin, pH 7.2-7.

4.

3. The culture medium combination according to claim 2, characterized in that, The induction medium 2 comprises 96 vol% DMEM / F-12 medium, 4 vol% FBS, 2 mmol / L glutamine, 2 mmol / L non-essential amino acids, 10 mmol / L nicotinamide, 25 μg / mL insulin, 1 μg / mL laminin and 10 μg / L activin A, pH 7.2-7.

4.

4. The use of the culture medium combination according to any one of claims 1-3 in the induction of pluripotent stem cell differentiation.

5. A method for inducing pluripotent stem cells to differentiate into insulin-secreting cells, characterized in that, Includes the following steps: (1) Resuspend pluripotent stem cells in the induction culture medium according to any one of claims 1-3, and then transfer them to a 6-well plate coated with 45-55 μg / mL type I rat tail collagen for further culture. Replace the culture medium every 24 hours and culture for 3-5 days. (2) Replace the induction medium 2 as described in any one of claims 1-3 for culturing, replace the medium with a new one every 24 hours, and culture for 3-5 days.

6. The method according to claim 5, characterized in that, The concentration of type I rat tail collagen in step (1) is 50 μg / mL.

7. The method according to claim 6, characterized in that, The culture time in step (1) is 4 days.

8. The method according to claim 7, characterized in that, The culture time in step (2) is 4 days.

9. The application of the method of claim 5 in inducing pluripotent stem cell differentiation.

10. The application of the method according to any one of claims 6-8 in the induction of pluripotent stem cell differentiation.

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