Induction method and application of human amniotic epithelial stem cells

Abstract

The embodiment of the invention discloses an induction method and application of human amniotic epithelial stem cells (hAESCs). According to the invention, hAESCs are successfully induced and differentiated into insulin secreting cells (IPCs) through combination of a small molecule compound and a basic induction liquid, meanwhile, METTL3 gene expression is found to present dynamic change in the induction process, and IPCs with better functions are obtained through overexpression of the METTL3 gene expression. According to the invention, hAESCs can be differentiated to endoderm, pancreatic progenitor cells and IPCs, maturation of beta-like cells is realized, the efficiency of induced differentiation of hAESCs into IPCs is increased, and the obtained IPCs have ideal functions and have potential application value in treatment of diabetes.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of stem cell technology, and specifically to a method for inducing human amniotic epithelial stem cells and applications thereof. Background Art

[0002] In recent years, the global incidence of diabetes has risen rapidly, which has brought huge economic pressure to patients. As a pluripotent cell, stem cells have the potential to differentiate into multiple germ layers, including pancreatic β-like cells in the endoderm.

[0003] Human amniotic epithelial stem cells (hAESCs) are considered ideal candidate cells for induced differentiation due to their convenient sources, low immunogenicity, and strong proliferation ability. In stem cell induced differentiation technology, researchers have used genetic engineering, such as introducing pancreatic development-related genes into stem cells through lentivirus, adenovirus, retrovirus, or plasmid vectors, and have achieved good results. However, the efficiency of induction and the function of the resulting insulin-secreting cells (IPCs) are still not ideal. Therefore, in-depth research on changes in gene expression during the induction process and possible mechanisms will be of great significance for obtaining IPCs with better functions, and is also crucial for seeking safer and more effective methods. Summary of the invention

[0004] To this end, an embodiment of the present invention provides a method for inducing human amniotic epithelial stem cells and its application, which can increase the efficiency of inducing hAESCs to differentiate into IPCs, and the obtained IPCs have ideal functions and have potential application value in the treatment of diabetes.

[0005] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions:

[0006] According to a first aspect of an embodiment of the present invention, the present invention provides a method for inducing human amniotic epithelial stem cells, the method comprising:

[0007] hAESCs cells were plated in a six-well plate, and when the cells filled 70%-80% of the six-well plate, the first stage induction solution, the second stage induction solution and the third stage induction solution were used in sequence for culture; wherein, the first stage induction solution included basic induction solution, 80-120 ng / mL activinA and 1-5 μM CHIR99021, the second stage induction solution included basic induction solution, 0.5-1.5 μM dorsomorphin, 1-4 μM retinoic acid and 5-15 μM SB431542, and the third stage induction solution included basic induction solution, 8-12 μM forskolin, 6-10 μM dexamethasone, 3-6 μM Alk5 inhibitor II and 10-12 mM nicotinamide.

[0008] Among them, activinA: a multifunctional cytokine that promotes cell differentiation; CHIR99021: an activator of the Wnt / β-catenin signaling pathway that protects cells from apoptosis. In the first stage, the use of the above small molecule compounds can make stem cells differentiate into endoderm cells.

[0009] Dorsomorphin: AMPK inhibitor reduces autophagy; retinoic acid: a metabolite of vitamin A, plays an important role in cell growth, differentiation and organogenesis; SB431542: a TGF-β receptor kinase inhibitor, which has the effect of inhibiting cell apoptosis. In the second stage, the cells can be differentiated into pancreatic progenitor cells by using the above small molecule compounds.

[0010] Forskolin: adenylate cyclase activator, increases intracellular cAMP levels; dexamethasone: a glucocorticoid receptor agonist, can inhibit inflammation levels; Alk5 inhibitor II: can inhibit the activity of TGF-β type II receptor and activin type II receptor; nicotinamide: a form of vitamin B3 or nicotinic acid, can inhibit the activity of SIRT2 and inhibit cell differentiation towards tumors. In the third stage, the use of the above small molecule compounds can allow cells to differentiate into insulin-secreting cells (IPCs).

[0011] Further, the composition of the basic induction solution is: knockout-DMEM, containing 1-3% knockout-serum, 0.5-1.5mM β-mercaptoethanol, 0.5-1% non-essential amino acids, 1-2% B27, 0.5-2% GlutaMax, 1.5-2.5% N-2, 10-30ng / ml basic fibroblast growth factor (bFGF), 15-35ng / ml epidermal growth factor (EGF), 1% penicillin-streptomycin. These ingredients can enhance the induction and differentiation effect of the above-mentioned small molecule compounds on stem cells.

[0012] Furthermore, the preparation method of the hAESCs cells includes: culturing human amniotic tissue using LG-DMEM culture medium containing 10% (v / v) fetal bovine serum (FBS), 1% (v / v) L-GlutaMAX, 1% (v / v) non-essential amino acids and 10 ng / mL EGF, and when the cell fusion rate reaches 70%-80%, subculturing is carried out, and when the second generation is reached, primary cultured hAESCs cells are obtained.

[0013] Furthermore, when the first-stage induction solution is used for culturing, the culture conditions are: culturing in a 5% CO2 cell culture incubator at 37°C for 3 days.

[0014] Furthermore, when the second-stage induction solution is used for culturing, the culture conditions are: culturing in a 5% CO2 cell culture incubator at 37°C for 7 days, and replacing half of the induction solution every 3 days.

[0015] Furthermore, when the third stage induction solution is used for culture, the culture conditions are: culture in a 5% CO2 cell culture incubator at 37°C for 11 days, and replace half of the induction solution every 3 days.

[0016] Furthermore, the hAESCs cells are hAESCs cells overexpressing the METTL3 gene.

[0017] According to a second aspect of an embodiment of the present invention, the present invention provides use of the method for inducing human amniotic epithelial stem cells as described in any one of the above items in the preparation of a product for treating diabetes.

[0018] The embodiments of the present invention have the following advantages:

[0019] The present invention successfully induces hAESCs to differentiate into insulin-secreting cells (IPCs) through a combination of small molecule compounds and a basic induction solution. During the induction process, it is found that the expression of the METTL3 gene shows dynamic changes, and IPCs with better functions are obtained by overexpressing it. The present invention can differentiate hAESCs into endoderm, pancreatic progenitor cells and IPCs, achieve the maturation of β-like cells, increase the efficiency of inducing differentiation of hAESCs into IPCs, and the obtained IPCs have ideal functions and have potential application value in the treatment of diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0021] Figure 1 These are the cell morphology diagrams of hAESCs; A. P0 hAESCs on the 7th day; B. P1 hAESCs on the 9th day; C. P2 hAESCs on the 13th day; D. P3 hAESCs on the 16th day; all pictures were magnified 200x.

[0022] Figure 2 The cell morphology at each stage of hAESCs induction; A. hAESCs cell morphology without induced differentiation (S0); B. cell morphology at the first stage of induced differentiation (S1); C. cell morphology at the second stage of induced differentiation (S2); D. cell morphology at the third stage of induced differentiation (S3); all pictures were magnified 200x.

[0023] Figure 3 qRT-PCR was used to detect the mRNA expression levels of marker genes at each stage of induced differentiation; A. Foxa2 mRNA expression level at each stage of induced differentiation; B. Sox17 mRNA expression level at each stage of induced differentiation; C. Pdx1 mRNA expression level at each stage of induced differentiation; D. Nkx6.1 mRNA expression level at each stage of induced differentiation; E. OCT4 mRNA expression level at each stage of induced differentiation; F. Glut2 mRNA expression level at each stage of induced differentiation; G. MAFA mRNA expression level at each stage of induced differentiation; H. Insulin mRNA expression level at each stage of induced differentiation, *, ** indicate p<0.05 and p<0.01, respectively, compared with S0, the differences are statistically significant.

[0024] Figure 4 To show the immunofluorescence expression of markers at each stage of induction; A. Sox17 protein expression at S1 stage; B. Pdx1 protein expression at S2 stage; C. Insulin protein expression at S3 stage; all magnifications are 100x.

[0025] Figure 5 WB was used to detect the expression of Glut2, Pdx1, MAFA, Insulin and METTL3 proteins at each stage of induction; * indicates P<0.05, and the difference is statistically significant.

[0026] Figure 6 A is the protein expression of METTL3 in normal stem cell induction (WT) and induction after overexpression of METTL3 gene (METTL3-OE); Figure 6 B: The two groups of cells were collected separately, and the mRNA level of METTL3 was detected by real-time fluorescence quantitative PCR reaction; * indicates that compared with WT, P<0.05, the difference is statistically significant.

[0027] Figure 7 A shows the islet spheroidization results and insulin immunofluorescence expression of normal induction (WT) and induction after overexpression of METTL3 gene (METTL3-OE); Figure 7 B is the supernatant of cells in the two groups at each stage S0, S1, S2, and S3, and the C-peptide content; Figure 7 C shows the in vitro glucose-stimulated release experiment of the two groups; * indicates that compared with WT, P<0.05, the difference is statistically significant. DETAILED DESCRIPTION

[0028] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the examples are regarded as conventional methods; the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0030] knockout-DMEM (Thermo Fisher Scientific, Cat. No. 12660012)

[0031] knockout-serum (Thermo Fisher Scientific, Cat. No. 10828028)

[0032] β-Mercaptoethanol (Thermo Fisher Scientific, Catalog No. 21985023)

[0033] Non-essential amino acids (Procell, Cat. No. PB180424)

[0034] B27 (Thermo Fisher Scientific, Cat. No. 17504044)

[0035] GlutaMax (Procell, Cat. No. PB180419)

[0036] N-2 (Thermo Fisher Scientific, Cat. No. 17502048)

[0037] Basic fibroblast growth factor (bFGF) (PeproTech, Cat. No. AF-100-18B)

[0038] Epidermal Growth Factor (EGF) (PeproTech, Cat. No. AF-100-15)

[0039] Penicillin-Streptomycin (Procell, Catalog No. PB180120)

[0040] activinA (Medchemexpress, Catalog No. HY-P70311)

[0041] CHIR99021 (Medchemexpress, Catalog No. HY-10182)

[0042] Dorsomorphin (Medchemexpress, Catalog No. HY-13418A)

[0043] Retinoic acid (Medchemexpress, Catalog No. HY-14649)

[0044] SB431542 (Medchemexpress, Cat. No. HY-10431)

[0045] Forskolin (Medchemexpress, Catalog No. HY-15371)

[0046] Dexamethasone (Medchemexpress, product number HY-14648)

[0047] Alk5 inhibitorII (sigma, catalog number 616452)

[0048] Nicotinamide (Medchemexpress, product number HY-B0150)

[0049] Isolation, culture, passaging and cryopreservation of hAESC

[0050] Isolation and culture: The human amniotic membrane tissue retrieved from the hospital was stored in D-hanks buffer with penicillin-streptomycin, and placed in a sterile culture dish for isolation. The amniotic membrane was washed with buffer until the surface was clean and blood-free, then transferred to a clean culture dish, cut into small pieces with scissors, and then transferred to a 50ml centrifuge tube. It was digested in a buffer containing 0.05% trypsin at 37°C for 40 minutes, and the undigested tissue was removed and continued to be digested in a new 50ml centrifuge tube for 40 minutes. The liquid from the two times was centrifuged at 1200rpm for 10 minutes, and the obtained cells were plated on T25cm 2 The culture flask contained the culture medium including LG-DMEM and 10% (v / v) fetal bovine serum (FBS), 1% (v / v) L-GlutaMAX, 1% (v / v) non-essential amino acids and 10 ng / mL EGF, and was cultured in a 37°C incubator. Half of the culture medium was replaced every 3 days after the cells adhered to the wall and grew.

[0051] Passaging and freezing: When the cell fusion rate reaches 70%-80%, discard the culture medium, wash twice with PBS, add 2ml trypsin to the culture medium, digest for 5-6min, and stop digestion with culture medium when the cell morphology changes from long spindle to round under the microscope. Transfer to a centrifuge tube, centrifuge for 5min at 1200rpm, and continue to culture by inoculating in 1:3 bottles. When it reaches the second generation, wash some cells with PBS twice, centrifuge, and store 5x10^6-1x10^7 cells in each cryopreservation tube in 1ml FBS:DMSO=9:1 (volume ratio). Some cells are used for induction experiments.

[0052] The results of cell morphology observation are shown in Figure 1 The primary cultured hAESCs were obtained. After subsequent expansion culture, the cells all grew in a spindle shape, which was consistent with the cell morphology of hAESCs.

[0053] In the following content, the composition of the basic induction solution is as follows:

[0054] The basic culture medium is knockout-DMEM, containing knockout-serum 2%, β-mercaptoethanol 1mM, non-essential amino acids 1%, B27 1%, GlutaMax 1%, N-22%, EGF 20ng / ml, bFGF 20ng / ml, and penicillin-streptomycin 1%.

[0055] Example 1

[0056] This embodiment provides a method for inducing hAESCs, comprising the following steps:

[0057] (1) After the primary hAESCs were subcultured, P2 cells were subjected to induction experiments. After cell digestion, 2x10^5 cells / well were plated in a six-well plate. When the cells were 70%-80% full of the 6-well plate, the first stage induction solution was added: basic induction solution, 100 ng / mL activinA and 3 μM CHIR99021, and cultured in a cell culture incubator at 37°C and 5% CO2 for 3 days;

[0058] (2) On the 4th day, the cells were replaced with the second-stage induction medium: basal induction medium, 1 μM dorsomorphin, 2 μM retinoic acid, and 10 μM SB431542. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 7 days, with half of the induction medium replaced every 3 days.

[0059] (3) On the 11th day, the cells were replaced with the third-stage induction medium: basal induction medium, 10 μM forskolin, 10 μM dexamethasone, 5 μM Alk5 inhibitor II, and 10 mM nicotinamide. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 11 days. Half of the induction medium was replaced every 3 days to terminate the induction.

[0060] Example 2

[0061] This example provides a method for inducing hAESCs, which differs from Example 1 only in that the induction solution in the first stage includes: basic induction solution, 80 ng / mL activinA and 5 μM CHIR99021.

[0062] Example 3

[0063] This example provides a method for inducing hAESCs, which differs from Example 1 only in that the second stage induction solution includes: basic induction solution, 0.5 μM dorsomorphin, 1 μM retinoic acid and 15 μM SB431542.

[0064] Example 4

[0065] This example provides an induction method for hAESCs, which differs from Example 1 only in that the third stage induction solution includes: basic induction solution, 12 μM forskolin, 6 μM dexamethasone, 6 μM Alk5 inhibitor II and 12 mM nicotinamide.

[0066] Comparative Example 1

[0067] This comparative example provides a method for inducing hAESCs, which differs from Example 1 only in that the induction solution used in the three stages of steps (1) to (3) is only the basic induction solution.

[0068] Comparative Example 2

[0069] This comparative example provides a method for inducing hAESCs, which differs from Example 1 only in that the composition of the induction solution used in the first stage of step (1) is only the basic induction solution, and the other steps are the same as Example 1.

[0070] Comparative Example 3

[0071] This comparative example provides a method for inducing hAESCs, which differs from Example 1 only in that the composition of the induction solution used in the second stage of step (2) is only the basic induction solution, and the other steps are the same as Example 1.

[0072] Comparative Example 4

[0073] This comparative example provides a method for inducing hAESCs, which differs from Example 1 only in that the composition of the induction solution used in the third stage of step (3) is only the basic induction solution, and the other steps are the same as Example 1.

[0074] Comparative Example 5

[0075] The difference between this comparative example and Example 1 is that the method of Example 1 is used to induce differentiation of umbilical cord mesenchymal stem cells, wherein the preparation method of umbilical cord mesenchymal stem cells is a conventional method in the art, and a-MEM complete culture medium containing 10% FBS is used as the culture medium.

[0076] Test Example 1

[0077] 1. Cell morphology observation

[0078] Figure 2 The morphological changes in each stage of Example 1. hAESCs were induced in vitro. The cells were added with CHIR99021 and ActicinA in the first stage. The cell morphology did not change significantly compared with the uninduced differentiation hAESC (S0), showing a typical long spindle morphology and some cell death. In the second stage, the cells were further induced to differentiate by adding a basic induction solution containing dorsomorphin, retinoic acid and SB431542. The cell morphology gradually shortened and thickened from a long spindle. In the third stage of induction, the cells were further shortened and thickened from the initial long spindle morphology by adding a basic induction solution containing forskolin, dexamethasone, Alk5 inhibitor II and nicotinamide, and even aggregated into sheets. Finally, the cell morphology changed from sheets to islet-like clusters.

[0079] 2. qRT-PCR detection of mRNA expression levels related to differentiation induction

[0080] The cells in the induction process of each stage (S0, S1, S2, S3) of Example 1 were collected for RNA extraction, reverse transcription and real-time fluorescence quantitative PCR reaction. Figure 3The results showed that during the induction process, the cell stemness index OCT4 was gradually downregulated, and significantly decreased in S2 and S3 compared with the S0 stage, with a statistically significant difference (p<0.05). The marker genes Foxa2 and Sox17 of the endoderm in the third stage of induction (S3) were significantly increased compared with the S0 stage, with a statistically significant difference (p<0.05). During the second stage of induction (S2), the pancreatic progenitor cell marker genes pdx1 and Nkx6.1 were significantly increased compared with the S0 stage, with a statistically significant difference (p<0.05). Finally, in the third stage of induction, the pancreatic β-like cell-specific marker genes insulin, Glut2, and MAFA were significantly increased compared with the S0 stage, with a statistically significant difference (p<0.05), indicating the successful induction of pancreatic β-like cells.

[0081] 3. Cell immunofluorescence detection of the expression of related proteins at each stage of the induced differentiation process

[0082] The cells at stages S1, S2, and S3 of Example 1 were collected for immunofluorescence experiments. Figure 4 The figure shows the expression changes of different marker proteins in the S1, S2 and S3 stages. It can be seen from the figure that the expression of Sox17 is high in the S1 stage, indicating that the pancreatic progenitor cells are successfully induced. The expression of PDX1 protein increases in the S2 stage, and the expression of insulin protein is obvious in the S3 stage, which is consistent with the gene expression and is consistent with the function of mature β cells.

[0083] 4. Western blot detection of the expression of related proteins at each stage of the induced differentiation process

[0084] The cells at each stage S0, S1, S2, and S3 of Example 1 were collected for Western blot experiments. Figure 5 The graph shows the changes in the expression of β-cell functional proteins in stages S0, S1, S2, and S3. As can be seen from the graph, with the process of cell induction, the expression of functional indicators Glut2, Pdx1, MAFA, and Insulin proteins gradually increased, which is similar to gene expression and consistent with the function of mature β-cells. In addition, the expression of METTL3 also showed an upward trend.

[0085] 5. In vitro glucose stimulation test to detect the function of IPCs

[0086] The supernatants of the hAESC (S0) and the 21st day IPCs (S3) cells in Examples 1-4 and Comparative Examples 1-5 were incubated in low-sugar and high-sugar solutions for 2 hours and the insulin release experiment was performed using a human insulin ELISA kit. The insulin release amounts are shown in Table 1.

[0087] Table 1

[0088]

[0089] The results showed that the amount of insulin released by the cells at the S3 stage in Example 1 in a high-sugar 25 mM glucose solution was greater than that of the other groups, and the difference was statistically significant (P<0.05). In summary, it was shown that the cells were successfully induced to differentiate into pancreatic β-like cells that can secrete insulin, indicating that Example 1 is the best.

[0090] 6. Function of IPCs in vitro C-peptide content detection

[0091] The supernatants of the cells at each stage S0, S1, S2, and S3 of Examples 1-4 and Comparative Examples 1-5 were collected respectively, and the C-peptide content was detected by using a human C-peptide ELISA kit. The C-peptide content is shown in Table 2.

[0092] Table 2

[0093] Induction method S0(pM) S1(pM) S2(pM) S3(pM) Example 1 4.1±0.014 9.7±0.023 99.9±0.041 133.5±0.026 Example 2 3.4±0.025 5.7±0.039 73.4±0.048 98.4±0.086 Example 3 1.8±0.071 9.5±0.053 87.6±0.081 116.8±0.078 Example 4 2.4±0.066 8.2±0.055 74.5±0.023 105.1±0.054 Comparative Example 1 1.3±0.069 6.4±0.057 42.3±0.034 65.1±0.038 Comparative Example 2 1.1±0.022 5.5±0.026 82.3±0.015 106.6±0.024 Comparative Example 3 2.4±0.018 7.4±0.039 64.2±0.022 83.4±0.053 Comparative Example 4 2.5±0.044 8.2±0.062 83.9±0.044 97.3±0.057 Comparative Example 5 2.7±0.013 7.6±0.027 77.3±0.026 105.4±0.039

[0094] The results showed that with the process of cell induction, the C-peptide content in Example 1 gradually increased, the amount of C-peptide in the S3 stage was more than that in the S0 stage, and was also higher than that in other groups, and the difference was statistically significant (P<0.05). In summary, Example 1 is the best.

[0095] Experimental Example 2: Overexpression of METTL3 gene to achieve maturation of β-like cells

[0096] hAESCs were transfected according to the instructions of TransIntro EL transfection reagent (Transgen, FT201). When the cells reached 80-90% confluence during transfection, 0.8 μg of plasmid DNA (overexpressed METTL3 plasmid from Genechem) was added to 50 μl of Opti-MEM medium and gently mixed; 16 μl of TransIntroEL was added to the diluted plasmid DNA, gently mixed, and allowed to stand at room temperature for 15-20 minutes. The plasmid DNA-TransIntro EL complex was added to the cells and cultured in an incubator. The medium was replaced 4-6 hours after transfection and cultured for 24 hours. After transfection overnight, induction culture was performed according to the method of Example 1.

[0097] 1. Verification after overexpression of METTL3 gene

[0098] Figure 6A is the protein expression of METTL3 in normal stem cell induction (WT) and induction after overexpression of METTL3 gene (METTL3-OE). The results showed that the expression level of METTL3 protein increased after overexpression of METTL3, and the difference was statistically significant (P<0.05); Figure 6 B: Two groups of cells were collected separately, and the results of real-time fluorescence quantitative PCR reaction showed that the mRNA level of METTL3 in the METTL3 overexpression group also increased, and the difference was statistically significant (P<0.05); in summary, it was shown that the expression level of METTL3 in hAESCs was increased by transfection of METTL3 overexpression plasmid.

[0099] 2. IPCs function induced by overexpression of METTL3 gene

[0100] Figure 7 A shows the islet spheroidization results and insulin immunofluorescence expression of normal induction (WT) and induction after overexpression of METTL3 gene (METTL3-OE). The results show that islets overexpressing METTL3 gene have more spheroids and higher insulin expression levels. Figure 7 B is the experiment of collecting the supernatants of cells from the two groups at each stage S0, S1, S2, and S3, and detecting the C-peptide content using a human C-peptide ELISA kit. The results showed that the C-peptide content of the genome overexpressing METTL3 increased, and the difference was statistically significant (P<0.05); Figure 7 C The results showed that the amount of insulin released by cells in the S3 stage overexpressing the METTL3 gene in a high-glucose 25mM glucose solution was greater than that released by cells in the normally induced S3 stage in a high-glucose solution. Compared with the WT group, the content increased by about 40%, and the difference was statistically significant (P<0.05). In summary, it was shown that overexpression of the METTL3 gene can increase the ability of hAESCs to differentiate into pancreatic β-like cells that can secrete insulin.

[0101] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A method for inducing human amniotic epithelial stem cells, characterized in that: The method comprises: hAESCs cells were plated in a six-well plate, and when the cells filled 70%-80% of the six-well plate, the first stage induction solution, the second stage induction solution and the third stage induction solution were used for culture in sequence; wherein, the first stage induction solution included basic induction solution, 80-120ng / mL activinA and 1-5μM CHIR99021, the second stage induction solution included basic induction solution, 0.5-1.5μM dorsomorphin, 1-4μM retinoic acid and 5-15μM SB431542, and the third stage induction solution included basic induction solution, 8-12μM forskolin, 6-10μM dexamethasone, 3-6μM Alk5 inhibitorII and 10-12mM nicotinamide.

2. The method for inducing human amniotic epithelial stem cells according to claim 1, characterized in that: The composition of the basic induction solution is: Knockout-DMEM, containing 1-3% knockout-serum, 0.5-1.5mM β-mercaptoethanol, 0.5-1% non-essential amino acids, 1-2% B27, 0.5-2% GlutaMax, 1.5-2.5% N-2, 10-30ng / ml basic fibroblast growth factor, 15-35ng / ml epidermal growth factor, and 1% penicillin-streptomycin.

3. The method for inducing human amniotic epithelial stem cells according to claim 1, characterized in that: The preparation method of the hAESCs cells comprises: Human amniotic tissue was cultured using LG-DMEM medium containing 10 v / v% fetal bovine serum, 1 v / v% L-GlutaMAX, 1 v / v% non-essential amino acids and 10 ng / mL EGF. When the cell fusion rate reached 70%-80%, the cells were passaged, and primary cultured hAESCs cells were obtained at the second generation.

4. The method for inducing human amniotic epithelial stem cells according to claim 1, characterized in that: When the first-stage induction solution is used for culturing, the culture conditions are: culturing at 37° C. in a 5% CO 2 cell culture incubator for 3 days.

5. The method for inducing human amniotic epithelial stem cells according to claim 1, characterized in that: When the second-stage induction medium is used for culturing, the culture conditions are: culturing at 37° C. in a 5% CO 2 cell culture incubator for 7 days, and replacing half of the induction medium every 3 days.

6. The method for inducing human amniotic epithelial stem cells according to claim 1, characterized in that: When the third stage induction medium is used for culture, the culture conditions are: culture at 37° C. in a 5% CO 2 cell culture incubator for 11 days, and replace half of the induction medium every 3 days.

7. The method for inducing human amniotic epithelial stem cells according to any one of claims 1 to 6, characterized in that: The hAESCs cells are hAESCs cells overexpressing the METTL3 gene.

8. Use of the method for inducing human amniotic epithelial stem cells according to any one of claims 1 to 7 in the preparation of a product for treating diabetes.

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