Method for differentiating pluripotent stem cells into insulin-secreting cells

Abstract

The invention relates to a method for differentiating pluripotent stem cells into insulin-secreting cells, and relates to the technical field of stem cell biology, and the method comprises the following steps: culturing pluripotent stem cells in a culture medium of a phytohormone combination comprising curcumin, sophocarpidine, apigenin, artemisinin, codonopsis pilosula lactone and duranol angelica acid ester, therefore, the insulin-secreting cells are differentiated. The application has the effects of reducing cost, improving safety and promoting maturation of insulin-secreting cells, provides powerful support for cell therapy of diabetes, and has a good clinical application prospect.

Description

Technical Field

[0001] This application relates to the field of stem cell biology, and in particular to a method for differentiating pluripotent stem cells into insulin-secreting cells, insulin-secreting cells obtained by the method, and compositions comprising such cells. Background Art

[0002] Human islet transplantation can effectively restore endogenous insulin secretion in patients, thereby reversing type 1 diabetes (T1DM). However, due to the lack of easily accessible sources of human islets, the widespread application of islet transplantation has been severely hindered. Pluripotent stem cells have the ability of unlimited self-renewal and differentiation into multiple cell types, and are ideal "seed cells" in regenerative medicine. In recent years, studies have found that pluripotent stem cells can be induced to differentiate into insulin-secreting cells, providing new hope for cell replacement therapy of T1DM.

[0003] Currently, a variety of methods have been developed for inducing pluripotent stem cells to differentiate into insulin-secreting cells. For example, using a combination of small molecule compounds or growth factors, pluripotent stem cells can be efficiently induced to differentiate into insulin-secreting cells. Although certain progress has been made, existing methods still have some challenges. For example, due to reasons such as the lack of glucose-stimulated insulin secretion (GSIS) function, cell therapy for diabetes has been greatly restricted; relying on cytokines or small molecule compounds derived from animal sources or recombinant proteins, these factors or / and compounds have problems such as high cost, potential immune responses, and safety issues.

[0004] Therefore, there is still an urgent need in the art to develop a method for differentiating pluripotent stem cells into insulin-secreting cells with lower cost, higher safety, and mature functions. Summary of the Invention

[0005] The object of this application is to provide a method for differentiating pluripotent stem cells into insulin-secreting cells, which has the effects of reducing cost, improving safety, and promoting the maturation of insulin-secreting cells, provides strong support for cell therapy of type 1 diabetes, and has good clinical application prospects.

[0006] In the first aspect, this application provides a method for differentiating pluripotent stem cells into insulin-secreting cells, the method comprising: (a) Culturing pluripotent stem cells in medium A in differentiation stage one, thereby differentiating into definitive endoderm cells; (b) Culturing the cells obtained in step (a) in medium B in differentiation stage two, thereby differentiating into primitive gut tube cells; (c) Culturing the cells obtained in step (b) in medium C in differentiation stage three, thereby differentiating into posterior foregut cells; (d) Culturing the cells obtained in step (c) in medium D at the fourth differentiation stage, so as to differentiate into pancreatic progenitor cells; (e) Culturing the cells obtained in step (d) in medium E at the fifth differentiation stage, so as to differentiate into pancreatic endocrine progenitor cells; (f) Culturing the cells obtained in step (e) in medium F at the sixth differentiation stage, so as to differentiate into insulin-secreting cells.

[0007] Optionally, the medium A contains curcumin, matrine and activin A; the medium B contains artemisinin, apigenin, FGF-7 and a Wnt inhibitor.

[0008] Optionally, the medium C contains lobetyolin, retinoic acid, an SHH inhibitor and a Wnt inhibitor.

[0009] Optionally, the medium D contains decursinol angelate, EGF, nicotinamide and an SHH inhibitor.

[0010] Optionally, the medium E contains lobetyolin, an ALK5 inhibitor, T3, ISX9, heparin, a Wnt inhibitor and a γ-secretase inhibitor.

[0011] Optionally, the medium F contains an ALK5 inhibitor, T3, an Axl inhibitor, heparin, an adenylate cyclase activator, zinc sulfate and an IκB kinase inhibitor.

[0012] In a second aspect, the present application provides a cell population, comprising a cell population containing insulin-secreting cells obtained by any of the methods in the first aspect.

[0013] In a third aspect, the present application provides a pharmaceutical composition, comprising the cell population described in the second aspect.

[0014] In a fourth aspect, the present application provides the use of a phytohormone composition in promoting the differentiation of pluripotent stem cells into insulin-secreting cells, wherein the phytohormone composition includes curcumin, matrine, apigenin, artemisinin, lobetyolin and decursinol angelate.

[0015] The present application has the following remarkable advantages and effects compared with the prior art: 1. The method can significantly improve the induction and differentiation efficiency of pluripotent stem cells, promote the maturation of insulin-secreting cells in vitro, and can significantly increase the positive rate of insulin + / C-peptide + / PDX-1 + / MAFA + cells; 2. Replacing cytokines derived from animals or recombinant proteins with phytohormones can significantly reduce costs and improve the safety of clinical medications; 3. After transplantation of the obtained insulin-secreting cells into a diabetic animal model, endogenous insulin secretion was effectively restored and blood glucose control was improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a graph of cell viability after the action of various phytohormones; Figure 2 It is the immunohistochemical result of the insulin-secreting cells prepared in this application; Figure 3 It is the result of insulin secretion after different glucose stimulations of the insulin-secreting cells prepared in this application; Figure 4 It is the body weight change curve after transplantation of the insulin-secreting cells prepared in this application into a mouse diabetes model; Figure 5 It is the blood glucose concentration change curve after transplantation of the insulin-secreting cells prepared in this application into a mouse diabetes model; Figure 6 It is the mRNA expression result of cells at each differentiation stage prepared in this application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] As used herein, "insulin-secreting cells" mainly contain β cells, including C-peptide + , PDX-1 + and MAFA + cells. Therefore, the insulin-secreting cells of the present disclosure can be used to treat, alleviate or reverse diseases or disorders caused by or associated with the dysfunction or deficiency of the Islets of Langerhans, such as T1DM.

[0018] As used herein, "pluripotent stem cells" (PSC) refer to undifferentiated cells with the potential for differentiation and proliferation (especially self-renewal ability) and maintaining the potential for differentiation, which can be cultured in vitro and have the potential to differentiate into cell types of the three germ layers (endoderm, ectoderm, mesoderm). Examples of PSC include embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), etc.

[0019] As used herein, "basal medium" refers to the basic component or matrix of a medium (such as a differentiation medium, induction medium or expansion medium), which is relative to the supplements or additives of the medium. The basal medium in the medium can act as a source of nutrients, hormones and / or factors that contribute to cell proliferation, maintenance or differentiation. The basal medium usually accounts for about 95 to 99% by volume of the medium.

[0020] As used herein, "supplement" or "additive" refers to an added or supplementary component of a culture medium (such as a differentiation medium, an induction medium, or an expansion medium), which is relative to its basal medium. The supplemented substances can include commercially available premixes or can be formulated as needed. Exemplary supplements include, but are not limited to, B27, glucose, BSA, serum replacement, N2, and GlutaMAX.

[0021] As used herein, "ROCK inhibitor" refers to a substance that inhibits the Rho kinase (ROCK) signaling pathway, such as: Y27632, HA100, HA1152, and Blebbistatin. In some embodiments, during the process of differentiating insulin-secreting cells from PSCs, a ROCK inhibitor is preferably added to the induction medium used after cell dissociation to affect the expression of cell cycle- and apoptosis-related proteins to regulate cell proliferation and maintain cell viability. The use concentration of the ROCK inhibitor can be any suitable concentration. In one or more embodiments, the ROCK inhibitor is Y27632, and the concentration of Y27632 in the medium can be, for example, 1 - 50 μM, but is not limited thereto, and is preferably 10 μM.

[0022] As used herein, "Wnt inhibitor" refers to a substance that inhibits the Wnt signaling pathway, such as: Wnt-C59. In some embodiments, during the process of differentiating definitive endoderm cells into posterior foregut cells, adding Wnt-C59 to inhibit the Wnt signaling pathway helps maintain and establish correct cell polarity, promotes the formation of a functional intestinal tube structure, prevents excessive cell proliferation, and at the same time, inhibiting the Wnt signaling pathway helps activate the Notch and FGF signaling pathways, which play key roles in the formation of intestinal tube cell characteristics. In some embodiments, during the process of differentiating pancreatic progenitor cells into pancreatic endocrine progenitor cells, adding Wnt-C59 to inhibit the Wnt signaling pathway promotes differentiation in the endocrine direction.

[0023] As used herein, "SHH inhibitor" refers to a substance that inhibits the Sonic hedgehog signaling pathway, such as: Sant1. In some embodiments, during the process of differentiating primitive gut tube cells into posterior foregut cells, adding Sant1 to inhibit the Sonic hedgehog signaling pathway thus affects the differentiation direction of intestinal cells. In some embodiments, during the process of differentiating posterior foregut cells into pancreatic progenitor cells, adding Sant1 to inhibit the Sonic hedgehog signaling pathway affects the proliferation and differentiation of posterior foregut cells and promotes the development of pancreatic progenitor cells.

[0024] As used herein, "ALK5 inhibitor" refers to a substance that inhibits TGF-β signaling, such as: ALK5 inhibitor Ⅱ. In some embodiments, during the differentiation of pancreatic progenitor cells into insulin-secreting cells, an ALK5 inhibitor is added. By inhibiting TGF-β signaling, the inhibitory effect on the differentiation process is reduced, thereby promoting the differentiation of pancreatic progenitor cells and inducing the differentiation of pancreatic progenitor cells into insulin-producing cells.

[0025] As used herein, "γ-secretase inhibitor" refers to a substance that inhibits NOTCH signaling, such as: Xxi. In some embodiments, during the differentiation of pancreatic progenitor cells into pancreatic endocrine progenitor cells, γ-secretase inhibitor Xxi is added to inhibit the NOTCH signaling pathway and reduce the interference with the differentiation of pancreatic progenitor cells.

[0026] As used herein, "Axl inhibitor" refers to a substance that inhibits PI3K / Akt signaling, such as: R428. In some embodiments, during the differentiation of pancreatic endocrine progenitor cells into insulin-secreting cells, the Axl inhibitor R428 is added to affect the signaling pathway related to islet cell differentiation, thereby promoting or inhibiting the differentiation of islet cells.

[0027] As used herein, "adenylate cyclase activator" refers to a substance that increases the intracellular cyclic adenosine monophosphate (cAMP) level, such as: Forskolin. In some embodiments, during the differentiation of pancreatic endocrine progenitor cells into insulin-secreting cells, Forskolin is added to significantly increase the intracellular cyclic adenosine monophosphate (cAMP) level, thereby promoting the differentiation of insulin-secreting cells.

[0028] As used herein, "IκB kinase inhibitor" refers to a substance that blocks NF-κB activity, such as: N-acetylcysteine or its analogs. In some embodiments, during the differentiation of pancreatic endocrine progenitor cells into insulin-secreting cells, N-acetylcysteine is added to block the conduction of its downstream signaling pathway, thereby regulating the differentiation state and function of islet cells and increasing the sensitivity and responsiveness of islet cells to insulin. Examples

[0029] This application is described in detail by the following examples. However, this application is not limited to the embodiments shown in these examples, and those skilled in the art can make various modifications.

[0030] Experimental materials Curcumin, matrine, apigenin, artemisinin, and decursinol angelate were all purchased from Chem Faces, and ligustilide was purchased from Shanghai Chunyou Biotechnology Co., Ltd. The experimental materials used in this application are all commercially available products unless otherwise specified. For those not indicating specific techniques or conditions in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the corresponding product instructions. For those reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0031] The pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells, and the invention purpose can be achieved by using this application for both of them. Human pluripotent stem cells (hPSC) are preferred.

[0032] Example 1 Screening of the safety concentration range of phytohormones for inducing differentiation In this example, the safety concentration ranges of curcumin, matrine, apigenin, artemisinin, ligustilide, and decursinol angelate for inducing differentiation were screened. The specific method is as follows: The safety investigation condition was pluripotent stem cells, which were inoculated into a 96-well plate at a density of 5×10 5 cells / mL, and after culturing overnight, they were replaced with various phytohormones at 1 - 20 μM. After acting for 24 h, 72 h, and 144 h, the CCK 8 kit was used for detection, and the safe concentrations were selected as the induction concentrations for standby.

[0033] The finally screened safe concentrations were as follows: curcumin: 0.5 μM - 5 μM, matrine: 0.5 μM - 2 μM, apigenin: 0.5 μM - 5 μM, artemisinin: 0.5 μM - 15 μM, ligustilide: 0.5 μM - 5 μM, decursinol angelate: 0.5 μM - 2 μM.

[0034] Example 2 Preparation of insulin-secreting cells In this example, the basal medium of Medium A and Medium B used MCDB131 (Gibco). Due to its low protein and serum-free characteristics, it is suitable for the culture of various cell types. The additives were 4.5 mM glucose, 1% GlutaMAX, 1% B27, and 0.25 mM vitamin C.

[0035] In this example, the basal medium of Medium C, Medium D, Medium E, and Medium F used DMEM (basic). Therefore, no additional glucose supplement was required, and 1% GlutaMAX, 1% B27, and 0.25 mM vitamin C needed to be added.

[0036] Pre-culture stage: Culture of pluripotent stem cells Coat Matrigel on a 6-well plate, and after 2 h at 37 °C, inoculate well-differentiated pluripotent stem cells in good condition.

[0037] Differentiation stage 1: Differentiation of pluripotent stem cells into definitive endoderm cells (culture time is 4 - 6 days) When the confluence of the cultured pluripotent stem cells reaches 90%, digest with TrypLE and resuspend in medium A containing the Rock inhibitor Y27632 at a concentration of 10 μM. Inoculate the cell suspension at a density of 2×10 4 / cm 2 onto a Matrigel-coated culture plate and culture in an incubator at 37ºC and 5% CO 2 with saturated humidity. Medium A is supplemented with artemisinin, matrine, and activin A in addition to the basal medium and additives. Among them, the concentration of curcumin is 0.5 μM - 5 μM, preferably 1.0 μM - 2.0 μM, and 1.5 μM is the optimal; the concentration of matrine is 0.5 μM - 2 μM, preferably 1.0 μM - 2.0 μM, and 1.5 μM is the optimal; the concentration of activin A is 100 ng / mL.

[0038] Differentiation stage 2: Differentiation of definitive endoderm cells into primitive gut tube cells (culture time is 2 - 4 days) Remove the old medium and replace it with fresh medium B. Medium B is supplemented with curcumin, apigenin, Wnt-C59, and FGF-7 in addition to the basal medium and additives. Among them, the concentration of artemisinin is 0.5 μM - 15 μM, preferably 2 μM - 5 μM, and 5 μM is the optimal; the concentration of apigenin is 0.5 μM - 5 μM, preferably 2 μM - 5 μM, and 5 μM is the optimal; the concentration of Wnt-C59 is 100 nM, and the concentration of FGF-7 is 50 nM.

[0039] Differentiation stage 3: Differentiation of primitive gut tube cells into posterior foregut cells (culture time is 4 - 6 days) Remove the old medium and replace it with fresh medium C. Medium C is supplemented with tangshenolide, retinoic acid, Sant1, and Wnt-C59 in addition to the basal medium and additives. Among them, the concentration of tangshenolide is 0.5 μM - 5 μM, preferably 1 - 2 μM, and 2 μM is the optimal; the concentration of retinoic acid is 2 μM, the concentration of Sant1 is 0.25 μM, and the concentration of Wnt-C59 is 100 nM.

[0040] Differentiation stage 4: Differentiation of posterior foregut cells into pancreatic progenitor cells (culture time is 6 - 8 days) Digest the cells obtained from the culture in stage 3 with TrypLE, and resuspend the cell suspension in medium D containing the Rock inhibitor Y27632 at a preferred concentration of 10 μM. At a density of 1×10 6 / cm 2Inoculate the cell suspension with the density into a new Matrigel-coated culture plate and place it in an incubator at 37°C and 5% CO 2 Incubate in an incubator with saturated humidity. Medium D is supplemented with imperatorin angelate, EGF, nicotinamide, and Sant1 in addition to the basal medium and additives. Among them, the concentration of imperatorin angelate is 0.5 μM to 2 μM, preferably 1 μM to 2 μM, and 2 μM is the optimal; the concentration of EGF is 100 ng / mL, the concentration of nicotinamide is 10 mM, and the concentration of Sant1 is 0.25 μM.

[0041] Differentiation stage five: Differentiation of pancreatic progenitor cells into pancreatic endocrine progenitor cells (culture time is 6 - 8 d) Digest the cells obtained from the culture in stage four using TrypLE, and resuspend the cell suspension in medium E containing the Rock inhibitor Y27632 with a preferred concentration of 10 μM. At a density of 2×10 6 / cm 2 Inoculate the cell suspension into a new Matrigel-coated culture plate and place it in an incubator at 37°C and 5% CO 2 Incubate in an incubator with saturated humidity. Medium E is supplemented with lactucin, ALK5 inhibitor Ⅱ, T3, ISX9, heparin, Wnt-C59, and γ-secretase inhibitor Xxi in addition to the basal medium and additives. Among them, the concentration of lactucin is 0.5 μM - 5 μM, preferably 1 - 2 μM, and 2 μM is the optimal; the concentration of ALK5 inhibitor Ⅱ is 10 μM, the concentration of T3 is 10 μM, the concentration of ISX9 is 10 μM, the concentration of heparin is 10 μg / mL, the added concentration of γ-secretase inhibitor Xxi is 0.1 μM, and the added concentration of Wnt-C59 is 100 nM.

[0042] Differentiation stage six: Differentiation of pancreatic endocrine progenitor cells into insulin-secreting cells (culture time is 4 - 6 d) Remove the old medium and replace it with fresh medium F. Medium F is supplemented with ALK5 inhibitor Ⅱ, T3, R428, heparin, forskolin, zinc sulfate, and N-acetylcysteine in addition to the basal medium and additives. Among them, the concentration of ALK5 inhibitor Ⅱ is 10 μM, the concentration of T3 is 1 μM, the concentration of R428 is 0.5 uM, the concentration of heparin is 10 μg / mL, the concentration of forskolin is 10 μM, the concentration of zinc sulfate is 10 μM, and the concentration of N-acetylcysteine is 2 mM.

[0043] In vitro maturation of insulin-secreting cells The cells completed in Stage 6 were digested into single cells with trypsin and seeded into AggreWell TM -400 (STEMCELL) six-well plates. After centrifugation, they were cultured overnight at 37 °C in an incubator. Then they were taken out and transferred to a low-attachment six-well plate. They were shaken at 80 rpm on a shaker and cultured in the incubator for 48 h to complete the process.

[0044] Example 3 Identification of the prepared insulin-secreting cells The cell aggregates or tissues were rinsed with PBS and then fixed with 4% PFA at 4 °C for 2 h (cell aggregates) or 24 h (tissues). The samples were washed 3 times with PBS and dehydrated overnight in 30% sucrose solution at 4 °C. The samples were covered with OCT solution, frozen with liquid nitrogen and stored at -80 °C. Sections of 10 μm were cut with a cryomicrotome and placed on glass slides. The glass slides were rinsed with PBS and permeabilized with PBST solution at room temperature for 1 h. The primary antibodies (anti-insulin antibody / anti-C peptide antibody / anti-PDX-1 antibody / anti-MAFA antibody) diluted with PBST solution were incubated with the glass slides overnight at 4 °C. After rinsing three times with PBS, the glass slides were incubated with conjugated secondary antibody in PBST solution at 1:1000 for 1 h and stained with DAPI for 5 min at room temperature. Images were acquired using a confocal microscope.

[0045] The results are as Figure 2 shown, demonstrating that the insulin-secreting cells prepared in this application can highly express insulin / C peptide / PDX-1 / MAFA simultaneously.

[0046] Example 4 Evaluation of the function of insulin-secreting cells 4.1 Glucose-stimulated insulin secretion (GSIS) The cultured insulin-secreting cells were transferred to a 50 mL sterile centrifuge tube at a density of 4.5×10 6 . 1.5×10 6 cells were added to the cell culture chambers of a 24-well plate. 2.8 mM D-glucose solution and 28 mM D-glucose solution were added respectively, and they were placed in an incubator at 37 °C with 5% CO 2 for 1 h. The chambers and insulin-secreting cells were taken out with forceps. The bottom of the chamber was gently touched to the corresponding culture well in the well plate to allow the insulin solution in the chamber to flow into the culture well as much as possible. The chamber was discarded. According to the manufacturer's instructions, the detection liquid in the culture well was placed in an automated chemiluminescent immunoassay analyzer (New Hope, MAGLUMI X8) using a sugar stimulation kit (purchased from Yikang Cell Transplantation) for detection.

[0047] The results are as Figure 3 shown, indicating that the insulin-secreting cells prepared in this application show a strong response to glucose stimulation and their function is mature.

[0048] 4.2 In Vivo Experiments on C57 / B6 Mice Establishment of model mice: C57 / B6 mice were fasted for one day, and then streptozotocin (STZ) at a dose of 180 mg / g was intraperitoneally injected the next day. Blood glucose was measured on the 3rd day, and if the blood glucose was higher than 20 mmol / L for three consecutive days, the mice were considered to have successfully modeled.

[0049] Experimental grouping: blank control group (i.e., normal mouse group), diabetes model group, IPC administration model group (abbreviated as IPC group in the attached figure), n = 6. Transplantation was performed in the peritoneal cavity of mice. The number of transplanted IPC cells was 2×10 6 / mouse. From the next day, blood glucose and body weight were measured daily for 14 consecutive days.

[0050] The results are as Figure 4 and Figure 5 shown. In the IPC administration group transplanted with the insulin-secreting cells prepared in this application, both body weight and blood glucose concentration were well controlled.

[0051] Example 5 qRT-PCR Detection of Cells from Differentiation Stage 1 to Differentiation Stage 5 The molecular markers of cells at each differentiation stage in Example 2 were detected by qRT-PCR, using hPSC as a negative control. The molecular markers of cells at each specific differentiation stage are shown in Table 1.

[0052] The results are as Figure 6 shown. The molecular markers of cells at each detected differentiation stage were all positive, indicating that the corresponding cells were obtained at each differentiation stage and the differentiation direction was correct.

[0053] Table 1

[0054] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for differentiating pluripotent stem cells into insulin-secreting cells, characterized in that: The method comprises: (a) culturing pluripotent stem cells in medium A at differentiation stage 1, thereby differentiating into definitive endoderm cells; (b) culturing the cells obtained in step (a) in medium B of differentiation stage 2, thereby differentiating into primitive intestinal tube cells; (c) culturing the cells obtained in step (b) in medium C of differentiation stage three, thereby differentiating into posterior foregut cells; (d) culturing the cells obtained in step (c) in medium D of differentiation stage 4, thereby differentiating the cells into pancreatic progenitor cells; (e) culturing the cells obtained in step (d) in medium E of differentiation stage five, thereby differentiating into pancreatic endocrine progenitor cells; (f) Culturing the cells obtained in step (e) in medium F of differentiation stage VI to differentiate into insulin-secreting cells.

2. The method for differentiating pluripotent stem cells into insulin-secreting cells according to claim 1, characterized in that: The culture medium A contains curcumin, matrine and activin A; the culture medium B contains artemisinin, apigenin, FGF-7 and a Wnt inhibitor.

3. The method for differentiating pluripotent stem cells into insulin-secreting cells according to claim 1, characterized in that: The culture medium C contains codonopsis pilosula lactone, retinoic acid, a SHH inhibitor and a Wnt inhibitor.

4. The method for differentiating pluripotent stem cells into insulin-secreting cells according to claim 1, characterized in that: The culture medium D contains purpurogenol angelate, EGF, niacinamide and SHH inhibitor.

5. The method for differentiating pluripotent stem cells into insulin-secreting cells according to claim 1, characterized in that: The culture medium E contains codonopsis pilosula lactone, ALK5 inhibitor, T3, ISX9, heparin, Wnt inhibitor and γ-secretase inhibitor.

6. The method for differentiating pluripotent stem cells into insulin-secreting cells according to claim 1, characterized in that: The medium F contains ALK5 inhibitor, T3, Axl inhibitor, heparin, adenylate cyclase activator, zinc sulfate and IκB kinase inhibitor.

7. The method for differentiating pluripotent stem cells into insulin-secreting cells according to claim 1, characterized in that: The culture medium A, culture medium D and culture medium E further comprise a ROCK inhibitor.

8. A cell population, characterized in that A cell population comprising insulin-secreting cells obtained by the method of any one of claims 1 to 7.

9. A pharmaceutical composition, characterized in that Comprising the cell population of claim 8.

10. Use of a phytochemical composition in promoting differentiation of pluripotent stem cells into insulin-secreting cells, characterized in that: The phytochemical composition comprises curcumin, matrine, apigenin, artemisinin, codonopsis pilosula lactone and purpurogenol angelate.

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