Methods of inducing mesenchymal stem cells to differentiate into islet-like cell clusters

Abstract

The application discloses a method for inducing mesenchymal stem cells to differentiate into islet-like cell clusters, which comprises the following steps: (1) taking mesenchymal stem cells, inoculating into a culture plate, adding a stem cell basic culture medium, and culturing for 1 day; (2) entering a differentiation stage 1, replacing the culture solution with a DMEM / F12 culture medium containing 10% fetal bovine serum, and culturing for 3 days, wherein liquid replacement is performed once during the culturing process; (3) entering a differentiation stage 2, replacing the culture solution with a nerve cell conditional culture medium, and culturing for 5 days, wherein liquid replacement is performed once during the culturing process; (4) entering a differentiation stage 3, replacing the culture solution with a differentiation culture medium, and culturing for 9 days, wherein liquid replacement is performed once every 2 days during the culturing process; and the islet-like cell clusters are obtained. The method for inducing mesenchymal stem cells to differentiate into islet-like cell clusters has only three differentiation stages, can be completed in 17 days, only needs one differentiation culture medium, has a remarkable effect, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to a method for inducing mesenchymal stem cells to differentiate into pancreatic islet-like cell clusters, belonging to the field of embryonic development and tissue regeneration medicine technology. Background Technology

[0002] Diabetes mellitus is a systemic metabolic disease that can lead to multi-organ complications, including diabetic nephropathy, retinopathy, and diabetic foot. It is the disease with the most known complications, severely impacting patients' quality of life. Poor blood sugar control in diabetic patients can induce ketoacidosis and hyperosmolar hyperglycemia, threatening their lives. Type 1 diabetes is caused by insufficient insulin secretion due to pancreatic β-cell dysfunction, while type 2 diabetes is caused by relative insulin deficiency due to insulin resistance. The chronic management of diabetes is lengthy and complex, involving long-term lifestyle interventions, blood glucose monitoring, oral medication, and continuous insulin replacement therapy. This complex process reduces patient adherence. Therefore, in recent years, many studies have explored the feasibility of cell replacement therapy.

[0003] Direct islet transplantation is the earliest cell replacement therapy. Researchers abroad obtain pancreatic samples from patients who have just died of brain disease, inject collagenase into the main pancreatic duct for digestion, and then sort the islets under a microscope for transplantation into diabetic patients. However, this method has significant drawbacks: First, suitable brain-dead donors are extremely scarce, resulting in a shortage of islets; second, the timing of collagenase digestion of the pancreas is difficult to control, and manually separating the islets under a microscope is challenging and requires a high level of skill; third, once the donor's islets are implanted in the diabetic patient, they are attacked by the host's immune system, leading to poor survival rates due to immune rejection.

[0004] The abundance of stem cell sources has opened up new avenues for cell replacement therapy in diabetes. Currently, there are hundreds of registered clinical trials worldwide for stem cell therapy in diabetes. Some researchers inject stem cells directly into the blood vessels or livers of diabetic patients, while others induce stem cells to differentiate into islet-like cell clusters with insulin-secreting function in vitro before transplanting them into diabetic patients via microencapsulation. In 2006, Kevin et al. established a method for inducing ESCs into islet-like cell clusters. This study was the first to successfully induce stem cell differentiation in vitro and obtain cell products capable of secreting insulin, glucagon, and somatostatin, marking a significant milestone. In 2014, Douglas et al. established a method for inducing iPSCs into islet-like cell clusters and demonstrated that these cell clusters, after being transplanted into the subcapsular region of mice, could secrete insulin into the bloodstream. On March 10, 2023, the U.S. Food and Drug Administration (FDA) approved VX-264 to begin clinical trials. VX-264 is a stem cell-derived islet encased in an immune-protective device that has the potential to functionally cure type 1 diabetes without the need for immunosuppressants.

[0005] However, both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) present safety concerns, namely potential tumorigenicity. In March 2022, researchers at Yunnan Cancer Hospital reported a case of an immature teratoma developing after iPSC-derived cell therapy, and this teratoma was more aggressive than typical teratomas. Stem cells have received attention and importance due to their multipotent differentiation potential, but they have also been questioned due to their uncertainty in differentiation. In 2013, Wang Ling et al. proposed that ESCs are a heterogeneous group of cells, and residual phenomena during the differentiation process may be an inherent characteristic. iPSCs, on the other hand, activate "oncogenes," such as c-Myc, during the cell preparation process. Furthermore, the source of seed cells in the preparation of ESC cells may also involve ethical issues.

[0006] Ideal stem cell-derived islets should meet the following four conditions: 1. Stem cells, serving as the starting point of differentiation, should have an abundant source, be easy to prepare, and not involve ethical issues; 2. Islet-like cell clusters, serving as the endpoint of differentiation, should have sufficient safety guarantees and should not cause tumorigenesis after transplantation; 3. The differentiation process should be simple, highly operable, and the culture media for each differentiation stage should be easy to prepare; 4. The differentiation effect should be stable, with sufficient evidence to show that the islet-like cell clusters have the function of secreting insulin. However, current methods for preparing stem cell-derived islets mostly use iPSCs or ESCs, which have shortcomings such as high differentiation uncertainty, difficulty in targeted differentiation, high risk of tumorigenesis, numerous and cumbersome differentiation steps, and low differentiation efficiency.

[0007] Chinese invention patent application CN 110423720 A discloses a method for inducing differentiation of human amniotic epithelial stem cells into functional pancreatic β cells, comprising the following steps: 1) isolation, culture, expansion, and identification of amniotic epithelial stem cells; 2) in vitro induction of differentiation: amniotic epithelial stem cells of the 2nd to 3rd generation are selected and induced to differentiate using a culture medium containing nicotinamide. After 14 days of in vitro differentiation, insulin-secreting cells expressing pancreatic β cell-specific markers and possessing normal pancreatic β cell function are obtained; 3) in vivo transplantation: the induced insulin-secreting cells are transplanted into type 1 diabetic mice, which can significantly alleviate the hyperglycemic state in mice. Two induction culture media were used for the differentiation. Summary of the Invention

[0008] In view of the above-mentioned prior art, the present invention provides a method for inducing mesenchymal stem cells to differentiate into pancreatic islet-like cell clusters.

[0009] This invention is achieved through the following technical solution: A method for inducing mesenchymal stem cells to differentiate into pancreatic islet-like cell clusters includes the following steps: (1) Take mesenchymal stem cells, seed them into a culture plate, add stem cell basal culture medium, and culture at 37°C and 5% CO2 for 1 day; (2) After 1 day of culture, the cells adhered to the wall and entered the differentiation stage 1. The culture medium was replaced with DMEM / F12 medium containing 10% (volume percentage) fetal bovine serum (FBS). The cells were cultured at 37°C and 5% CO2 for 3 days, with one medium change during the culture process. (3) After co-culturing for 3 days, the cells enter the differentiation stage 2. The medium is changed to neural-conditioned medium (NCM). The cells are co-cultured at 37°C and 5% CO2 for 5 days, with one medium change during the culture process. (4) After culturing for 5 days, the cells enter the differentiation stage 3. The medium is changed to differentiation medium and cultured for 9 days at 37°C and 5% CO2. The medium is changed every 2 days during the culture process, and differentiation medium is added a total of 5 times; thus, pancreatic islet cell clusters are obtained. The differentiation medium consisted of the following components: 15–20 mM glucose, 8–12 mM nicotinamide, 8–12 nM exendin-4 (exenatide), 8–12 nM pentagastrin, 80–120 pM hepatocyte growth factor, 8–12 μM 5-hydroxytryptamine (5-HT), 8–12 μM butyramide, 0.08–0.12 mg / ml TPO (thrombopoietin), 1%–5% (v / v) of serum-free B27 additive, and the remainder being DMEM / F12 medium.

[0010] Furthermore, in step (1), the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0011] Furthermore, in step (1), the inoculation density is 2 × 10⁻⁶. 5 Cells / pores.

[0012] Furthermore, in step (1), 2 ml of culture medium is added to each well.

[0013] Furthermore, in step (1), the stem cell basal culture medium is a commercially available culture medium in the prior art, which can be selected from Daco's Dayou mesenchymal stem cell basal culture medium (product number: 6114011).

[0014] Further, in step (3), the conditioned medium for nerve cells is prepared by the following method: rat brain is taken and placed in DMEM / F12 medium containing 10% FBS. The rat brain is homogenized and thoroughly ground. The cell suspension is filtered through a 50μm cell filter to obtain a cell suspension. The cell suspension is cultured at 37℃ and 5% CO2 for 20-28 hours. The supernatant of the culture is taken out, and DMEM / F12 medium containing 10% FBS is added to the supernatant. The culture is then cultured at 37℃ and 5% CO2 for 4 days. The supernatant of the culture is collected, centrifuged, and filtered through a 0.22μm cell filter to obtain the conditioned medium for nerve cells.

[0015] Further, in step (4), the differentiation medium consists of the following components: 17.5 mM glucose, 10 mM nicotinamide, 10 nM exendin-4, 10 nM pentagastrin, 100 pM hepatocyte growth factor, 10 μM 5-hydroxytryptamine, 10 μM butyramide, 0.1 mg / ml TPO, 2% B27 serum-free additive, and the balance being DMEM / F12 medium.

[0016] This invention provides a method for inducing mesenchymal stem cell differentiation into islet-like cell clusters. It utilizes readily available and abundant umbilical cord mesenchymal stem cells (UC-MSCs) to establish a stable system for MSC isolation and differentiation. Starting with MSCs, the method induces their differentiation into islet-like cell clusters. The differentiation of MSCs exhibits relatively low uncertainty, making clinical application safer, and the extraction process does not violate ethical principles. Although researchers have concerns about the tumorigenic risk of MSCs, years of clinical application have shown no such incidents. Previous procedures for inducing ESC or iPSC differentiation typically involved 4-6 differentiation stages, lasting approximately one month, with each stage requiring the preparation of different differentiation media, a cumbersome process. In contrast, this invention's method involves only 3 differentiation stages, completed in 17 days, significantly reducing time and requiring only one type of differentiation media, making the operation simple. This method for inducing MSC differentiation into islet-like cell clusters demonstrates significant efficacy and promising application prospects, holding important significance for the development of cell replacement therapy for diabetes.

[0017] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0018] Figure 1 Schematic diagram of flow cytometry results for mesenchymal stem cell identification, where A: CD34-; B: CD45-; C: CD73+; D: CD90+.

[0019] Figure 2 Schematic diagram of dithizone staining results for islet-like cell clusters.

[0020] Figure 3 Schematic diagram of neural nestin staining results in differentiation stage 2 cells.

[0021] Figure 4 Schematic diagram of insulin staining results for three types of pancreatic islet cell clusters during the differentiation stage.

[0022] Figure 5 Schematic diagram of immunofluorescence staining results of three types of pancreatic islet cell clusters during the differentiation stage. Implementation

[0023] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0024] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0025] Example 1: Collection of Umbilical Cord Mesenchymal Stem Cells The steps are as follows: (1) Wash the umbilical cord with 0.9% saline solution and then place it in a 10 cm culture dish. Add 75% ethanol solution to the culture dish to submerge the entire umbilical cord and soak for 5 minutes. Transfer the umbilical cord to a new culture dish, add saline solution to wash, and repeat the washing until the blood stains and alcohol are removed.

[0026] (2) Place the cleaned umbilical cord into a new petri dish, cut off the ligated ends and discard them, and cut the remaining umbilical cord into several small segments of 2-3 cm in length. Add physiological saline to clean the cut umbilical cord segments, repeat the cleaning until the blood stains are removed and the washing solution is clear.

[0027] (3) Locate the umbilical vein and cut the small section of umbilical cord along the spiral direction of the vein, removing the arteries and veins from the umbilical cord. Use tissue forceps to peel off the Wharton's jelly and transfer the peeled Wharton's jelly to a 50 ml centrifuge tube. Add an appropriate amount of physiological saline to the centrifuge tube beforehand to prevent the Wharton's jelly from drying out. Cut the Wharton's jelly into small pieces of 1–8 mm in the centrifuge tube. 3 Small pieces.

[0028] (4) Add physiological saline to the centrifuge tube to 45 ml, centrifuge at 750 g for 10 min, and discard the supernatant.

[0029] (5) Add physiological saline to the centrifuge tube to 45 ml, centrifuge at 750 g for 10 min, and discard the supernatant.

[0030] (6) Wharton's jelly was seeded into T75 culture flasks, with 1 g of Wharton's jelly seeded into each flask, and 4 ml of complete umbilical cord cell culture medium was added. Culture conditions: temperature 37℃, CO2 concentration 5%, saturated humidity. Mesenchymal stem cells could be observed crawling out of the tissue.

[0031] (7) The obtained mesenchymal stem cells were cultured in MSCBM medium.

[0032] Example 2: Identification of mesenchymal stem cells Cell surface markers were detected using flow cytometry. Mesenchymal stem cell surface markers included CD73+, CD90+, CD34-, and CD45-. The steps are as follows: (1) Transfer 1 ml of cell suspension to a 1.5 ml centrifuge tube. Centrifuge at 300 g for 5 minutes at 4°C, carefully aspirate and discard the supernatant.

[0033] (2) Wash the cells with an appropriate amount of PBS buffer, centrifuge at 300 g for 5 minutes at 4°C, carefully aspirate and discard the supernatant.

[0034] (3) Resuspend the cells in pre-cooled PBS buffer and adjust the final cell concentration to 1×10⁻⁶. 7 cells / ml, gently pipette to mix.

[0035] (4) Take 100 μl of cell suspension as blank control group, take 100 μl of cell suspension as isotype control group, add isotype control antibody, take 200 μl as experimental group and add detection antibody, incubate at 4℃ for 30 minutes.

[0036] (5) Add an appropriate amount of PBS buffer to wash the cells, centrifuge at 300 g for 5 minutes at 4°C, carefully aspirate and discard the supernatant.

[0037] (6) Resuspend cells in 500 μl PBS buffer and perform instrumental analysis.

[0038] The results are as follows Figure 1 As shown, the biomarkers for mesenchymal stem cells are: CD73+ 99.98% and CD90+ 99.26%. The results indicate that the cells cultured in Example 1 are mesenchymal stem cells, highly expressing CD73+ and CD90+, and low expressing CD34- and CD45-.

[0039] Example 3: Preparation of conditioned medium for rat brain nerve cells The steps are as follows: (1) Sacrifice seven-day-old rats, remove the rat brain and place it in a 10 cm culture dish, add PBS buffer to thoroughly wash away blood stains.

[0040] (2) Transfer the rat brain to a new 10 cm culture dish and add DMEM / F12 medium containing 10% FBS. Cut the rat brain into small pieces with ophthalmic scissors and transfer them to a 25 ml glass tissue homogenizer. Add 5 ml of DMEM / F12 medium containing 10% FBS. After thoroughly grinding the rat brain, add 10 ml of DMEM / F12 medium containing 10% FBS to the glass homogenizer.

[0041] (3) Take a new 10 cm culture dish and place a cell filter with a 50 μm pore size on it. Use a dropper to draw up the cell suspension obtained in the glass homogenizer and add it to the filter to obtain the cell suspension. Place the sample in a temperature of 37℃ and a CO2 concentration of 5% for 24 hours.

[0042] (4) After 24 hours of culture, the culture was observed. The supernatant contained nerve cells, while oligodendrocytes had adhered to the culture vessel. Therefore, the supernatant was transferred to a new 10 cm culture dish, and 15 ml of DMEM / F12 medium containing 10% FBS was added. The culture was continued for four days.

[0043] (5) Collect the supernatant into a 50 ml centrifuge tube and centrifuge at 1000 g for 15 minutes. Use a 20 ml syringe to extract the supernatant obtained by centrifugation in several portions, pass it through a 0.22 μm cell filter, and inject it into a new 50 ml centrifuge tube to obtain NCM.

[0044] Example 4: Induction of mesenchymal stem cell differentiation The steps are as follows: (1) The umbilical cord mesenchymal stem cells obtained in Example 1 were seeded in a six-well plate at a seeding density of 2 × 10⁻⁶. 5 Cells / well: Add 2 ml of stem cell basal culture medium (Dakeway's Dayou mesenchymal stem cell basal culture medium, catalog number: 6114011) to each well and culture at 37°C and 5% CO2 concentration.

[0045] (2) After the cells adhere to the wall on the second day, they enter the differentiation stage 1. The medium is changed to DMEM / F12 medium containing 10% FBS. The cells are cultured for 3 days, with one medium change in between.

[0046] (3) Enter the differentiation stage 2. The medium is changed to the medium prepared in Example 3. The culture is carried out for 5 days, with one medium change in between.

[0047] (4) Enter the differentiation stage 3, change the medium to differentiation medium, and culture for a total of 9 days. Change the medium once every 2 days in between, and add differentiation medium a total of 5 times.

[0048] The differentiation medium consisted of the following components: 17.5 mM glucose, 10 mM nicotinamide, 10 nM exendin-4, 10 nM pentagastrin, 100 pM hepatocyte growth factor, 10 μM 5-hydroxytryptamine (5-HT), 10 μM butyramide, 0.1 mg / ml TPO (thrombopoietin), 2% B27 serum-free additive, and the balance being DMEM / F12 medium.

[0049] (5) Gently pick up the cell clusters in the six-well plate with the tip of a needle; these are pancreatic islet cell clusters.

[0050] Example 5: Observation of in vitro differentiation effect When mesenchymal stem cells are initially seeded, they are a flat monolayer of cells; however, after entering differentiation stage 3, they spontaneously aggregate to form islet-like cell clusters with a diameter of 300–2000 μm. These cell clusters can be stained to detect the effectiveness of in vitro differentiation.

[0051] (a) Dithizone staining Dithizone staining is the most common and simplest method for identifying pancreatic islets. Zinc ions contained in pancreatic β cells can chelate with dithizone to form a cherry-red complex.

[0052] (1) Dissolve 10 mg of dithizone in 1 ml of DMSO, and then dilute with PBS buffer at a ratio of 1:100 to obtain dithizone staining working solution.

[0053] (2) Add dithizone staining working solution to the 6-well plate, 2 ml / well, and incubate at 37°C for 30 minutes.

[0054] (3) Wash three times with PBS buffer, then add glycerol for coating, and observe under a light microscope.

[0055] The results are as follows Figure 2 As shown, the differentiated islet-like cell clusters were positive for dithizone staining.

[0056] (ii) Immunohistochemical staining (1) Pick the differentiated cell products, add 4% paraformaldehyde solution, and fix for 60 min. Embed the fixed cell products in paraffin, and then perform serial sectioning. The thickness of each section is 4 μm, and the sections are attached to a glass slide.

[0057] (2) The paraffin sections were sequentially placed in xylene I for 10 min, xylene II for 10 min, xylene III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 90% ethanol solution for 5 min, 80% ethanol solution for 5 min, 70% ethanol solution for 5 min, and 50% ethanol solution for 5 min for dewaxing.

[0058] (3) Use 0.5% Triton X-100 to permeate the perforation hole for 10 minutes.

[0059] (4) Wash the slices three times with PBS buffer, and then soak the slices in 3% hydrogen peroxide solution (diluted with methanol) for 15 min to block endogenous peroxidase.

[0060] (5) Immerse the slides in 10 mM citric acid solution and heat to 100°C for 10 min to repair the antigen. Then place the slides and citric acid solution at room temperature to cool slowly and thoroughly.

[0061] (6) Wash the slides three times with PBS buffer, then transfer the slides to a humidified chamber, add 10% bovine serum albumin (BSA) solution to the surface, and block at room temperature for one hour.

[0062] (7) Remove the blocking solution and blot away any water. For cells in differentiation stage 2, add neurotrophic protein antibody (CST, 10959S, diluted 1:200 in PBS buffer); for cell clusters in differentiation stage 3, add insulin antibody (CST, 3014S, diluted 1:1000 in PBS buffer). Incubate overnight at 4°C.

[0063] (8) After the sections were warmed to room temperature for one hour, they were washed three times with PBS buffer, and then GTVision was used to analyze them. TM The Detection System / Mo&Rb (Genetech, GK800511) was used for color development and incubated at room temperature for 10 minutes.

[0064] (9) Wash away the DAB staining solution with PBS buffer, then add hematoxylin and counterstain for 90 seconds.

[0065] (10) Dehydration was carried out using a gradient passing method, and then the film was sealed with neutral resin.

[0066] (11) Observe and photograph under a light microscope.

[0067] The results are as follows Figure 3 , Figure 4 As shown, cells obtained in differentiation stage 2 are positive for nestin staining. Islet-like cell clusters obtained in differentiation stage 3 are positive for insulin staining.

[0068] Results analysis: By the end of differentiation stage 2, mesenchymal stem cells had differentiated into neuroendocrine cells. By the end of differentiation stage 3, mesenchymal stem cells had differentiated into cells with insulin-producing function.

[0069] (III) Immunofluorescence staining (1) The sample fixation, embedding, sectioning, dewaxing, punching, catalase blocking, heat repair, and blocking process are the same as above.

[0070] (2) Cell clusters in differentiation stage 3 were divided into two groups. One group was given insulin antibody (CST, 3014S, diluted 1:1000 in PBS buffer), and the other group was given C-peptide antibody (CST, 4593S, diluted 1:100 in PBS buffer). They were incubated overnight at 4°C.

[0071] (3) After the sections were warmed to room temperature for one hour, they were washed three times with PBS buffer, and then fluorescent secondary antibodies (Immunoway, RS3211, RS3811) were added and incubated at room temperature in the dark for one hour.

[0072] (4) Wash the sections three times with PBS buffer, then add DAPI staining solution and incubate at room temperature in the dark for 10 min.

[0073] (5) Wash the slides three times with PBS buffer, then add anti-quenching mounting medium and mount.

[0074] (6) Observe and photograph under a confocal microscope.

[0075] The results are as follows Figure 5 As shown, the islet-like cell clusters obtained in differentiation stage 3 are positive for insulin and C-peptide staining.

[0076] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for inducing mesenchymal stem cells to differentiate into pancreatic islet-like cell clusters, characterized in that, Includes the following steps: (1) Take mesenchymal stem cells, seed them into a culture plate, add stem cell basal culture medium, and culture at 37°C and 5% CO2 for 1 day; (2) After 1 day of culture, the cells adhered to the wall and entered the differentiation stage 1. The culture medium was replaced with DMEM / F12 medium containing 10% fetal bovine serum and cultured for 3 days at 37°C and 5% CO2. The medium was changed once during the culture process. (3) After co-culturing for 3 days, the cells enter the differentiation stage 2. The medium is changed to neural cell conditioned medium and co-cultured for 5 days at 37°C and 5% CO2. The medium is changed once during the culture process. (4) After culturing for 5 days, the culture enters the differentiation stage 3. The medium is changed to differentiation medium and cultured for 9 days at 37℃ and 5% CO2. The medium is changed once every 2 days during the culture process, and differentiation medium is added a total of 5 times. This yields islet-like cell clusters; The differentiation medium consists of the following components: 15–20 mM glucose, 8–12 mM nicotinamide, 8–12 nM exendin-4, 8–12 nM pentagastrin, 80–120 pM hepatocyte growth factor, 8–12 μM 5-hydroxytryptamine, 8–12 μM butyramide, 0.08–0.12 mg / ml TPO, 1%–5% B27 serum-free additive, and the balance being DMEM / F12 medium.

2. The method for inducing mesenchymal stem cells to differentiate into pancreatic islet-like cell clusters according to claim 1, characterized in that: In step (1), the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

3. The method for inducing mesenchymal stem cells to differentiate into pancreatic islet-like cell clusters according to claim 1, characterized in that: In step (1), the inoculation density is 2×10⁻⁶. 5 Cells / pores.

4. The method for inducing mesenchymal stem cells to differentiate into pancreatic islet-like cell clusters according to claim 1, characterized in that: In step (1), 2 ml of culture medium is added to each well.

5. The method for inducing mesenchymal stem cells to differentiate into pancreatic islet-like cell clusters according to claim 1, characterized in that: In step (1), the stem cell basal culture medium is Dayou mesenchymal stem cell basal culture medium.

6. The method for inducing mesenchymal stem cells to differentiate into pancreatic islet-like cell clusters according to claim 1, characterized in that: In step (3), the conditioned medium for nerve cells is prepared by the following method: rat brain is taken and placed in DMEM / F12 medium containing 10% FBS. The rat brain is homogenized and thoroughly ground. The cell suspension is filtered through a 50μm cell filter to obtain a cell suspension. The cell suspension is cultured at 37℃ and 5% CO2 for 20-28 hours. The supernatant of the culture is taken out, and DMEM / F12 medium containing 10% FBS is added to the supernatant. The culture is then cultured at 37℃ and 5% CO2 for 4 days. The supernatant of the culture is collected, centrifuged, and filtered through a 0.22μm cell filter to obtain the conditioned medium for nerve cells.

7. The method for inducing mesenchymal stem cells to differentiate into pancreatic islet-like cell clusters according to claim 1, characterized in that: In step (4), the differentiation medium consists of the following components: 17.5 mM glucose, 10 mM nicotinamide, 10 nM exendin-4, 10 nM pentagastrin, 100 pM hepatocyte growth factor, 10 μM 5-hydroxytryptamine, 10 μM butyramide, 0.1 mg / ml TPO, 2% B27 serum-free additive, balance DMEM / F12 medium.

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