Preparation method for inducing and differentiating pancreas islet beta cells by using double-gene modified stem cells

Abstract

The invention relates to a preparation method for inducing and differentiating pancreatic beta cells by using double-gene modified stem cells, relates to the technical field of biology, and provides a double-gene expression pAdxsi-CMV-PDXI / CMV-NeuroD1 adenovirus vector. A three-dimensional culture system for efficiently inducing the hUSCs to be differentiated into the pancreas islet beta cells is constructed by recombining pAdxsi-GFP-PDXI / NeuroD1 adenovirus modified urine-derived stem cell combined cell factors and combining NunclonSphera 3D culture consumables, so that the problems that the pancreas islet beta cell induced differentiation rate is low due to the fact that an optimal three-dimensional cell microenvironment cannot be provided for pancreas islet cell growth and clustering by a traditional two-dimensional culture method; meanwhile, the problems of poor insulin synthesis and secretion functions and the like of a traditional cytokine induction method are solved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing pancreatic beta cells induced and differentiated by double-gene modified stem cells. Background Art

[0002] Pdx1 (pancreatic duodenal homeobox gene 1) is one of the key transcription factors related to pancreatic development. It can promote early pancreatic development and late cell differentiation during embryonic development, and help maintain the morphology and function of B cells in adults. At the same time, it can also activate its own genes and regulate the expression of downstream genes, and participate in regulating a variety of genes specifically expressed in islet cells, including glucose kinase (GK), neurogenin 3 (Neurogenin3, NGN3), insulin (insulin), glucagon (glucagon), glucose transporter-2 (glucose transporter-2, Glut2), etc., and these genes are involved in the differentiation and maturation of pancreatic cells. A large number of studies have confirmed that mice with PDX1 expression deficiency have undeveloped pancreatic tissue and severe diabetes, suggesting that the PDX1 gene plays an important role in the development of the pancreas and in maintaining the normal physiological function of cells.

[0003] The NeuroD gene is located on the long arm of human chromosome 2, region 3, band 2 (2q32). Its gene location is adjacent to the type 1 diabetes gene IDDM7 (D2S152) site. People have conducted a series of studies on the relationship between its gene mutation and diabetes. The results showed that the G(A mutation at the 211th nucleotide of codon 45 of the NeuroD gene resulted in a change in amino acids (Ala45(Thr), but its relationship with type 2 diabetes is still unclear. Experiments have confirmed that NeuroD has an important influence on the expression and regulation of the insulin gene, and animal experiments have also confirmed that NeuroD plays a vital role in the development of the pancreas. The pancreas of mice begins to develop at 9.5 days of embryonic period, and NeuroD protein can be detected in β cells until the entire pancreas is fully developed. After birth, protein expression is observed in all mature β cells and a small number of α cells.

[0004] Diabetes has become a major public health problem that seriously endangers national health and brings a heavy economic burden to society. Restoring the number and function of pancreatic β cells in patients is the fundamental way to treat diabetes, among which β cell regeneration is a research direction with great potential application value. Due to the two reasons of donor source and immune rejection, human islet transplantation is difficult to promote on a large scale, and finding new potential sources of β cells has become a goal of people's efforts. However, due to the unique three-dimensional structure of pancreatic cell spheres, the current method (two-dimensional cytokine induction culture method) still cannot provide the best three-dimensional cell microenvironment for pancreatic cell growth and clustering, resulting in a low induced differentiation rate of pancreatic β cells, and their insulin synthesis and secretion functions are also relatively poor. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for preparing double-gene modified stem cells that induce differentiation of pancreatic β cells.

[0006] In a first aspect, the present invention provides a dual-gene expression pAdxsi-CMV-PDXI / CMV-NeuroD1 adenovirus vector, and the preparation method of the dual-gene expression pAdxsi-CMV-PDXI / CMV-NeuroD1 adenovirus vector comprises the following steps:

[0007] pUC57-PDX1 and pUC57-NeuroD1 were digested with BglII and XhoI restriction endonucleases, respectively, and PDX1 and NeuroD1 were ligated to the pShuttle-GFP-CMV recombinant shuttle vector to obtain pShuttle-GFP-CMV-PDX1 / pShuttle-GFP-CMV-NeuroD1 plasmids;

[0008] Transferring the pShuttle-GFP-CMV-PDX1 / pShuttle-GFP-CMV-NeuroD1 plasmid to the pAdxsi vector, and harvesting the pAdxsi-CMV-PDX1 / NeuroD1 recombinant viral plasmid;

[0009] The pAdxsi-CMV-PDX1 / NeuroD1 recombinant virus plasmid was transfected into 293 cells to package a complete adenovirus, thereby obtaining a dual-gene expression pAdxsi-CMV-PDXI / CMV-NeuroD1 adenovirus vector.

[0010] In a second aspect, the present invention provides a method for preparing double-gene modified stem cells that induce differentiation of pancreatic β cells, the method comprising the following steps:

[0011] Obtain hUSCs;

[0012] Transfecting the hUSCs with the dual-gene expression pAdxsi-CMV-PDXI / CMV-NeuroD1 adenoviral vector according to claim 1;

[0013] hUSCs were infected with adenovirus carrying pAdxsi-CMV-PDXI / CMV-NeuroD1 and differentiated into pancreatic β cells by cytokine induction.

[0014] Furthermore, the step of obtaining hUSCs includes the following process:

[0015] Primary stem cells hUSCs were extracted from fresh human urine using the adherence method;

[0016] The primary stem cells hUSCs are expanded and subcultured, and hUSCs can be obtained after three subcultures.

[0017] Furthermore, the steps of infecting hUSCs with adenovirus carrying pAdxsi-CMV-PDXI / CMV-NeuroD1 and inducing differentiation into pancreatic β cells by cytokines include pre-induction differentiation, one-stage induction differentiation, two-stage induction differentiation and three-stage induction differentiation.

[0018] Furthermore, the working condition parameters of the pre-induction differentiation include: 30-80ug / ml of rat insulinoma cell lysis extract, 0.5-2% insulin transferrin-selenium ITS, and culture medium DMEMF / 12 induction for 1-3 days.

[0019] Furthermore, the working condition parameters for inducing differentiation in the first stage include: 10-100 ng / ml EGF, 0.5-5% (v / v) B27, 20-60 ng / ml glucagon-like peptide-1 (GLP-1) + DMEMF / 12 induction for 2-4 days.

[0020] Furthermore, the working condition parameters of the second stage induction differentiation include: 20-60ng / mlGLP-1, 40-60ng / ml human cytokine, 0.5-5% (v / v) B27, 10-100ng / mlEGF, 5-30ng / mlHGF+DMEMF / 12 containing 1-10%FBS for induction for 6-8 days.

[0021] Furthermore, the working condition parameters of the three-stage differentiation induction include: 5-20 mM nicotinamide (NIC), 0.1-1.0 mmol / L β-mercaptoethanol, 5-30 ng / ml HGF+1-10% FBS DMEMF / 12 induction for 6-8 days.

[0022] In a third aspect, the present invention provides a pancreatic β cell, wherein the pancreatic β cell is prepared by the method for preparing pancreatic β cells induced to differentiate by using double-gene modified stem cells as described in any one of the second aspects.

[0023] The above technical solution provided by the embodiment of the present invention has at least the following advantages compared with the prior art:

[0024] The embodiment of the present invention provides a preparation method for inducing differentiation of pancreatic β cells by dual-gene modified stem cells, by constructing a dual-gene expression pAdxsi-CMV-PDXI / CMV-NeuroD1 adenovirus vector combined with multiple cytokines to induce hUSCs to differentiate into pancreatic β cells in the NunclonSphera3D culture system. NunclonSphera3D culture consumables can control cells to spontaneously form a three-dimensional multicellular spheroid structure by regulating multiple mechanical properties such as cell adhesion ability, elastic modulus and stress relaxation, which can better provide the nutrients required by pancreatic cells, exchange substances, and be closer to the optimal growth conditions of pancreatic β cells; at the same time, the Pdx1, NeuroD1 and other genes related to insulin synthesis and secretion of pancreatic β cells (IPCs) induced by the dual-gene expression pAdxsi-CMV-PDXI / CMV-NeuroD1 adenovirus vector are significantly higher than the current conventional two-dimensional culture method of multiple cytokines in terms of transcription level and protein expression level, as well as in terms of insulin and C-peptide secretion. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0027] Figure 1 This is a comparison of the insulin secretion amounts of the experimental group and the control group at different stages in the present invention.

[0028] Figure 2 This is a comparison of the C-peptide secretion levels of the experimental group and the control group at different stages in the present invention.

[0029] Figure 3 This is an image of hUSCsP0 inverted microscope 10x objective lens in an embodiment of the present invention.

[0030] Figure 4 This is the hUSCsP0 inverted microscope 10x objective lens imaging in the embodiment of the present invention Figure 1 .

[0031] Figure 5 This is the hUSCsP0 inverted microscope 10x objective lens imaging in the embodiment of the present invention Figure 2 .

[0032] Figure 6 This is an image of hUSCsP1 imaged using an inverted microscope with a 10x objective lens in an embodiment of the present invention.

[0033] Figure 7 This is an image of hUSCsP2 imaged using an inverted microscope with a 10x objective lens in an embodiment of the present invention.

[0034] Figure 8 This is an image of hUSCsP3 imaged using an inverted microscope with a 10x objective lens in an embodiment of the present invention.

[0035] Fig. 9 This is a diagram of the morphological changes during hUSCs 3D differentiation in an embodiment of the present invention.

[0036] Fig.10 This is a diagram of the DTZ staining (10X) results in an embodiment of the present invention.

[0037] Fig.11 This is a diagram of the immunofluorescence staining results in an embodiment of the present invention. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. 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.

[0039] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0040] The present invention provides a method for preparing pancreatic β cells induced by differentiation of dual-gene modified stem cells, which specifically includes the following contents:

[0041] 1. The primary stem cells hUSCs were extracted from fresh human urine by the adherence method and then expanded, cultured and identified.

[0042] Use a sterile urine bottle to collect about 200-250mL of fresh midstream urine from healthy male volunteers aged 24-28 years, and transfer it to 4 50mL centrifuge tubes aseptically in a biosafety cabinet. After 5-10 minutes, centrifuge at 1000-2000 rpm, remove the supernatant with a pipette, leaving only about 2-5mL of the bottom solution, and add PBS to dilute to 15-25mL according to the turbidity of the urine. Use a pipette to blow the bottom of the centrifuge tube and the surrounding tube wall thoroughly, merge the 4 tubes into 2 tubes, centrifuge again for 5-10 minutes, 1000-2000 rpm, and remove the supernatant. This time, keep about 2-5mL of the bottom solution, add PBS again to dilute to 15-25mL, and use a pipette to absorb the bottom solution and blow the bottom and wall of the tube. After blowing evenly, merge the 2 tubes into 1 tube. Centrifuge again for 5-10 minutes at 1000-2000 rpm. Discard the supernatant and keep 2-5ml of the bottom solution. Add 2-5mL hUSCs complete medium (Tianjin Haoyang Huake Biotechnology Co., Ltd. Catalog No.: SC2013-G) and blow evenly. Then add to T25 or T75 culture flask containing 2-5mL hUSCs medium. Culture in a cell culture incubator. Observe the growth of hUSCs under a microscope regularly. After observing the formation of hUSCs colonies, replace the hUSCs medium every 2-3 days, culture for 7-15 days until the hUSCs P0 generation cell fusion reaches 80-90%, then collect and passage, add 5-10ml PBS (Tianjin Haoyang Huake Biotechnology Co., Ltd. PB2004Y) to rinse 1-2 times, add 0.5-1mL trypsin (Tianjin Haoyang Huake Biotechnology Co., Ltd. TE2004Y), and place in a 37°C incubator for digestion reaction for about 1-2 minutes. After digestion is completed, add 3-5mL stop solution (Tianjin Haoyang Huake Biotechnology Co., Ltd. SC2013-GE) to terminate digestion. Transfer to a centrifuge tube, centrifuge at 1000-1500 rpm for 5-10 minutes, remove the supernatant, and resuspend the precipitate in 2-5 mL of culture medium. Pipette 10-15 mL of hUSCs complete culture medium into a T75cm2 cell culture flask, and transfer the resulting cell suspension to the same culture flask for continued culture. At this time, the hUSCs are P1 generation. Culture for 3-4 days, and when the cell fusion reaches 80%-90%, repeat the above steps and pass it again at a ratio of 1:3-1:5. After three passages, P3 generation hUSCs can be obtained.

[0043] 2. Construct a dual-gene expression adenoviral vector pAdxsi-CMV-PDXI / CMV-NeuroD1 with high infection efficiency and synergistic effect on hUSCs.

[0044] pUC57-PDX1 bacterial solution and pUC57-NeuroD1 containing the target gene were provided by the Pancreatic Disease Research Institute of Union Hospital, Tongji Hospital, Huazhong University of Science and Technology; pAdxsi recombinant adenovirus backbone vector and pShuttle-GFP-CMV recombinant shuttle vector were provided by Nosai Genomic Research Center Co., Ltd. PDX1 and NeuroD1 were cut with BglI / XhoI enzymes from pUC57-PDX1 and pUC57-NeuroD1, and connected to the pShuttle-GFP-CMV recombinant shuttle vector to harvest pShuttle-GFP-CMV-PDX1 / pShuttle-GFP-CMV-NeuroD1 plasmids, and pShuttle-GFP-CMV.PDX1 / NeuroD1 was transferred to the pAdxsi vector, and the pAdxsi-CMV-PDX1 / NeuroD1 recombinant virus plasmid was harvested. The recombinant virus infected 293 cells to package the complete adenovirus.

[0045] 3. hUSCs were infected with adenovirus carrying pAdxsi-CMV-PDXI / CMV-NeuroD1 and induced to differentiate into pancreatic β cells by cytokine culture.

[0046] 3.1 The experimental plan is as follows:

[0047] The experiment was divided into two groups: experimental group: hUSCs transfected with adenovirus carrying PDX1 / NeuroD1 gene combined with cytokine 3D culture induction group; control group: hUSCs not genetically modified combined with cytokine 2D culture induction group.

[0048] Induction scheme of experimental group:

[0049] 1) Adenovirus transfection of hUSCs with PDX1 / NeuroD1 genes: P3 hUSCs were cultured using Nunclon Sphera 3D ultra-low adsorption 6-well culture plates as culture carriers, and P3 human urine-derived hUSCs were infected with recombinant adenovirus (Adxsi-CMV-PDX1 / CMV-NeuroD1) carrying PDX1 and NeuroD1 genes (5.0-6.0x10^6 / ml of cells were inoculated in a culture flask and cultured in DMEM medium containing 5-10% FBS. After the cells reached 80-90% confluence, pAdxsi-CMV-PDXI / CMV-NeuroD1 virus was directly added to the culture flask at 100-150 PFU / cell, and repeated 2-3 times) and infected for 6-8 days.

[0050] 2) Pre-induction of differentiation: 30-80ug / ml of rat insulinoma cell lysate extract, 0.5-2% insulin transferrin-selenium ITS, and induction in culture medium DMEMF / 12 for 1-3 days.

[0051] 3) Three-stage differentiation induction:

[0052] Phase 1: 10-100 ng / ml EGF, 0.5-5% (v / v) B27, 20-60 ng / ml glucagon-like peptide-1 (GLP-1) + DMEMF / 12 induction for 2-4 days;

[0053] Phase II: 20-60 ng / ml GLP-1, 40-60 ng / ml human cytokinin, 0.5-5% (v / v) B27, 10-100 ng / ml EGF, 5-30 ng / ml HGF + DMEMF / 12 containing 1-10% FBS for 6-8 days;

[0054] Three stages: 6-8 days of induction with DMEMF / 12 containing 5-20 mM nicotinamide (NIC), 0.1-1.0 mmol / L β-mercaptoethanol, 5-30 ng / ml HGF+1-10% FBS.

[0055] The control group was hUSCs without genetic modification combined with cytokine 2D culture induction group. The cytokine induction scheme was the same as that of the experimental group but Corning 6-well culture plates (2D culture) were used.

[0056] 3.2 hUSCs induced differentiation into pancreatic β cells (IPCs)

[0057] 3.2.1 Adenovirus transfection of PDX1 / NeuroD1 genes in hUSCs: P3 generation hUSCs were cultured using Nunclon Sphera 3D ultra-low adsorption 6-well culture plates as culture carriers, and P3 generation human urine-derived hUSCs were infected with recombinant adenovirus (Adxsi-CMV-PDX1 / CMV-NeuroD1) carrying PDX1 and NeuroD1 genes (5.0-6.0x10^6 / ml cells were inoculated in the culture plate and cultured in 10ml of 10% FBS DMEM culture medium. After the cells reached 85% confluence, pAdxsi-CMV-PDXI / CMV-NeuroD1 virus was directly added to the culture flask at 100 PFU / cell, and repeated 2-3 times) and infected for 6-8 days (the control group was not treated with this treatment).

[0058] 3.2.2 Inducing hUSCs to differentiate into IPCs through three stages:

[0059] (1) After observing that hUSCs form compact spheres in the 6-well plate, aspirate and discard the DMEMF / 12 complete medium; add the first stage differentiation medium and cytokines according to the experimental protocol, 100-200 μL per well.

[0060] (2) After 2-3 days, discard the first stage differentiation medium and replace it with the second stage differentiation medium and cytokines, 100-200uL per well. Replace the second stage differentiation medium and cytokines every 2-3 days (the second stage is cultured for 6-8 days).

[0061] (3) After 6-8 days, discard the second stage differentiation medium again and replace it with the third stage differentiation medium and cytokines, 100-200 μL per well. Replace the third stage differentiation medium and cytokines every 2-3 days (the second stage is cultured for 6-8 days).

[0062] 4. Identification of pancreatic β cells induced by hUSCs

[0063] The PDX1 gene was correctly inserted into the shuttle plasmid and recombined with the viral backbone plasmid by sequencing and restriction digestion, and the recombinant adenovirus could efficiently infect hUSCs. The induced cells were stained with dithizone (DTZ) and the cytoplasm was bright red: RT-PCR, immunocytochemistry, immunofluorescence and Western blot could detect the expression of PDX1, insulin and glut-2 islet-related genes and proteins in the induced cells. In the experimental group, the secretion of insulin and IC peptide in the supernatant was 273.42 + 18.57 mU / L and 0.36 ± 0.11 ng / mL on the 10th day of induction, and the secretion of insulin and C peptide on the 17th day was 497.27 ± 55.96 mU / L and 2.85 ± 0.37 ng / mL, respectively. After 1 hour of synergistic action of 25 mmol / L high glucose, the contents of insulin and C peptide reached 985.23 ± 66.30 mU / L and 4.31 ± 0.68 ng / mL, respectively. The insulin secretion (132.75±8.16mU / L, 213.36±13.42mU / L, 397.34±46.21mU / L) and C-peptide (0.13±0.06ng / mL, 0.89±0.12ng / mL, 1.07±0.16ng / mL) of the control group on the 10th day, 17th day and 17th day + high glucose were significantly lower than those of the experimental group. The differences between the experimental group and the control group and in different time periods of the experimental group were statistically significant (P<0.05). The experimental group detected the percentage of insulin{+ cells after induction by flow cytometry was 25.34±5.13%, and the control group was 8.12±3.87% respectively) which was significantly lower than that of the experimental group (P<0.05).

[0064] like Figure 1 and Figure 2The results shown are: After the constructed pAdxsi-CMV-PDXI / CMV-NeuroD1 adenovirus infects hUSCs to modify human urine-derived stem cells hUSCs, combined with NunclonSphera3D culture flasks and multiple cytokine induction, they can be induced to differentiate into insulin-secreting cells in vitro, with high levels of insulin and C-peptide secretion, and can respond to high glucose stimulation.

[0065] It should be noted that the components and raw materials involved in the preparation method of the dual-gene expression pAdxsi-CMV-PDXI / CMV-NeuroD1 adenovirus vector and the dual-gene modified stem cell-induced differentiation of pancreatic β cells provided in the embodiments of the present invention, unless otherwise specified or specified, can be directly commercially available products or homemade using existing public preparation methods; at the same time, the steps and parameters involved, unless otherwise specified or specified, can be carried out according to the existing adenovirus vector preparation process and the existing dual-gene modified stem cell-induced differentiation of pancreatic β cells preparation method or directly using existing equipment, and the present document will not repeat them one by one.

[0066] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

[0067] Example

[0068] This example provides a method for preparing a dual-gene expression pAdxsi-CMV-PDXI / CMV-NeuroD1 adenovirus vector and a dual-gene modified stem cell-induced differentiation pancreatic β cells, which specifically includes the following processes:

[0069] 1. Extract primary stem cells hUSCs from fresh human urine and expand, culture and identify them.

[0070] 1.1 Acquisition, extraction and culture of primary hUSCsP0 cells: Primary hUSCs were obtained by adherence screening. Use a sterile urine bottle to collect about 240 mL of fresh midstream urine from healthy male volunteers aged 24-28 years. Avoid contamination of the specimen during the operation. Use a pipette to transfer the collected urine specimens to 4 50 mL centrifuge tubes in a biosafety cabinet. After 5 minutes of centrifugation at 2000 rpm, the supernatant is aspirated with a pipette, leaving only about 3 mL of the bottom liquid. PBS is added to dilute to 20 mL according to the turbidity of the urine. Use a pipette to blow the bottom of the centrifuge tube and the surrounding tube wall thoroughly, merge the 4 tubes into 2 tubes, and centrifuge again for 5 minutes at 2000 rpm to aspirate the supernatant. This time, about 3 mL of the bottom liquid is retained. PBS is added again to dilute to 15 mL, and the bottom liquid is aspirated with a pipette to blow the bottom and wall of the tube. After blowing evenly, the 2 tubes are merged into 1 tube. Centrifuge again for 5 minutes at 2000 rpm. Discard the supernatant and keep 3 ml of base liquid. Add 3 mL of hUSCs complete culture medium (Tianjin Haoyang Huake Biotechnology Co., Ltd. Catalog No.: SC2013-G) and pipette evenly. Then add to a T25 culture flask containing 3 mL of hUSCs culture medium. Culture in a cell culture incubator. Observe the growth of hUSCs under a microscope regularly. After observing the formation of hUSCs colonies, replace the hUSCs culture medium every 3 days and culture for 15 days. The obtained cells are recorded as primary hUSCs (P0) and photographed. The hUSCs P0 inverted microscope 10x objective lens image (D0 day inoculation image) is as follows Figure 3 As shown, hUSCsP0 inverted microscope 10x objective lens imaging (fusion degree 50-60%) Figure 4 As shown, hUSCsP0 inverted microscope 10x objective lens imaging (fusion degree 80-85%) Figure 5 shown.

[0071] 1.2 Subculture of hUSCsP0 cells (P1-P3): hUSCs at the P0 generation can be subcultured after they grow adherently to the bottom of the flask and become 80%-90% confluent.

[0072] Add 5 ml PBS (PB2004Y, Tianjin Haoyang Huake Biotechnology Co., Ltd.) to rinse twice, add 1 mL of trypsin (TE2004Y, Tianjin Haoyang Huake Biotechnology Co., Ltd.), and place in a 37°C incubator for digestion reaction for about 2 minutes. After digestion is complete, add 5 mL of stop solution (SC2013-GE, Tianjin Haoyang Huake Biotechnology Co., Ltd.) to stop digestion. Transfer to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, aspirate the supernatant, and resuspend the precipitate in 5 mL of culture medium. Use a pipette to draw 10 mL of hUSCs complete culture medium into a T75cm2 cell culture flask, and transfer the resulting cell suspension to the same culture flask and continue culturing. The hUSCs at this time are the P1 generation. After 3 days of culture, when the cells are fused to 80%-90%, repeat the above steps and pass them again at a ratio of 1:3. After three passagings, P3 generation hUSCs can be obtained. The hUSCs P1 inverted microscope 10x objective lens image (fusion degree 80-85%) is as shown Figure 6 As shown, hUSCsP2 inverted microscope 10x objective lens imaging (fusion degree 80-85%) Figure 7 As shown, hUSCsP3 inverted microscope 10x objective lens imaging (fusion degree 80-85%) Figure 8 shown.

[0073] 1.3P3 generation urine-derived stem cells hUSCs identification

[0074] 1) Morphological structure: Use an inverted biological microscope to carefully observe the morphological structure of the cells. They grow adherently to the wall, most of them grow like fibroblasts, and a few are spindle-shaped or irregular triangles.

[0075] 2) Cell growth curve: 1. Cell preparation: After digestion and washing, P3 mesenchymal stem cells were prepared into different cell densities (8×10 4 / ml, 4×10 4 / ml, 2×10 4 / ml, 1×10 4 / ml, 5×10 3 / ml) 2. Use RTCAS16 system for detection: add 50μl culture medium to the well of E-Plate16, detect the baseline, and then add 100ul of cell suspension of different densities. The number of cells inoculated in each well is 8000, 4000, 2000, 1000, and 500, and there are 2 replicates for each inoculation density. 3. Detect a value every 15min, and detect continuously for 5d to obtain the proliferation curve of MSCs with different inoculation densities. Conclusion: RCTA (real-time label-free cell analysis technology) was used to analyze the proliferation curve of hUSCs. When the inoculation density was 4000-5000 / cm2, the cells reached the plateau between 72-80h, that is, 3-4 days for passage.

[0076] 3) Cell viability and purity analysis (flow cytometry analysis): The positive rates of CD105, CD73 and CD90 were ≥90%; while CD45, CD34, CD14, CD19 and HLA-DR were negative, with positive rates ≤5%. The statistical results of hUSCs cell viability and purity are shown in Table 1.

[0077] Table 1

[0078] Live rate CD90+ CD105+ CD73+ CD45- CD34- HLA-DR- CD19- CD14- 99.02％ 99.53％ 99.73％ 99.72％ 100％ 99.93％ 98.9％ 98.9％ 98.39％

[0079] 4) Stem cell specific markers SSEA-4 and Oct-4: RT-PCR detected the expression of stem cell markers SSEA-4 and Oct-4 in human hUSCsP3.

[0080] 5) Adipogenic / osteogenic differentiation ability: After induction with adipogenic / osteogenic differentiation medium, adipogenic induction was stained red with Oil Red O and osteogenic induction was stained red with Alizarin Red.

[0081] 2. Construction of a dual-gene expression pAdxsi-CMV-PDXI / CMV-NeuroD1 adenovirus vector with high infection efficiency and synergistic effect on hUSCs. (pUC57-PDX1 bacterial solution and pUC57-NeuroD1 containing the target gene were provided by the Pancreatic Disease Research Institute of Union Hospital, Tongji Hospital, Huazhong University of Science and Technology; pAdxsi recombinant adenovirus backbone vector and pShuttle-GFP-CMV recombinant shuttle vector were provided by Nosai Genomics Research Center Co., Ltd.)

[0082] 2.1 Construction of pAdxsi-GFP-PDX1 and pAdxsi-GFP-NeuroD1

[0083] 2.1.1 First, construct the recombinant shuttle vector, cut Pdx1 and NeuroD1 from pUC57-PDX1 and pUC57-NeuroD1, and connect them to the pShuttle-GFP-CMV recombinant shuttle vector to obtain the pShuttle-GFP-CMV-PDX1 recombinant shuttle plasmid and the pShuttle-GFP-CMV-NeuroD1 recombinant shuttle plasmid. The process is as follows: ① pUC57-PDX1 / NeuroD1 was digested with BglI and Xhol, the target fragment was cut out after electrophoresis, the target fragment was recovered by DNA gel, and the 5.2kb vector fragment was recovered by purification kit. ② Recover the approximately 0.9Kb insert fragment from the gel run, digest the pShuttle-GFP-CMV recombinant shuttle vector with BglI and XhoI, and then dephosphorylate; ③ Connect the vector fragment and the insert fragment with T4 DNA ligase; ④ Take 3pL of the ligation product to transform the chemically competent cell DH5a strain, pick out several monoclonal colonies, inoculate them into kanamycin-resistant liquid culture medium, and culture them in a shaker at 37℃ and 300rpm overnight; ⑤ Extract the plasmid pShuttle-GFP-CMV-PDXI / NeuroD1.

[0084] 2.1.2 Construction of recombinant adenovirus vector plasmid, transfer of verified pShuttle-GFP-CMV-PDX1 / NeuroD1 plasmid to pAdxsi vector, harvesting pAdxsi-GFP-PDX1 / NeuroD1 virus plasmid. The process is as follows: ① I-CeuI and I-SceI double digestion of pAdxsi vector, dephosphorylation; ② Inactivation with 10% SDS75C for about 10 minutes and then recovery of the vector by ethanol precipitation; ③ After I-CeuI and I-Scel digestion, insert the fragment into pShuttle-GFP-CMV-PDX1 / NeuroD1, use DNA gel to recover the target fragment, and purify the kit to recover the target fragment: ④ Connect the treated vector fragment and the insert fragment with T4DNA ligase; ⑤ Take 3plDH5a strain and put it on ampicillin LB plate: ⑥ Incubate overnight, pick an appropriate amount of monoclonal colonies, and inoculate them into ampicillin anti-penicillin medium. ⑦ Add 2 ml of bacterial solution in the logarithmic growth phase to 100 ml of LB medium containing 100 ug / ml Amp, then shake at 37'℃ overnight to extract the plasmid.

[0085] 2.2 Recombinant adenovirus packaging, amplification, and titer determination

[0086] 293 cells were inoculated in a six-well plate, the culture medium was DMEM plus 5% FBS, and placed in a 37°C incubator containing 5% cO2 for overnight culture. The virus was inoculated in the culture plate, and the cell growth was observed after 18-24 hours. After harvesting the lesion cell suspension, centrifuge at 2000rpm for 5 minutes, add 5-10ml STbuffer to mix, freeze and thaw three times, and centrifuge at 3000rpm for 5min. When the cell confluence is greater than 80%, pAdxsi-GFP-PDX1 / NeuroD1 is linearized with PacI restriction endonuclease, and 293 cells are infected with Lipofectamine2000 liposomes. After 45 days, the cells become larger, round or oval, and obvious plaques begin to appear. When most of the cells are lesions and fall off from the bottom, they are collected, and the supernatant is collected by centrifugation. The supernatant is used to continue to infect 293 cells to amplify the virus in large quantities. When all cells fall off the bottom, they can be collected, placed in a centrifuge tube, and frozen in a -86°C refrigerator for aliquoting. Strictly follow the operating procedures of the AdEasy adenoviral Vector System (Adenovirus Vector Operating Manual English Version) to determine the TCID50 value of the virus product and the virus titer.

[0087] Among them, the enzyme digestion system and reaction conditions are: shuttle plasmid DNA 2ug, 10xBuffer 5uL, I-CeuI (NEB, 5U / μL) 2uL, 10xBSA 5uL, I-SceI (NEB, 5U / μL) 2μL, ddH2O to 50μL, 37℃ for 3 hours, total volume 50uL.

[0088] 3. hUSCs were infected with adenovirus carrying pAdxsi-CMV-PDXI / CMV-NeuroD1 and induced to differentiate into pancreatic β cells by cytokine culture.

[0089] 3.1 Experimental plan

[0090] The experiment was divided into two groups: experimental group: hUSCs transfected with adenovirus carrying PDX1 / NeuroD1 gene combined with cytokine 3D culture induction group; control group: hUSCs not genetically modified combined with cytokine 2D culture induction group.

[0091] Induction scheme of experimental group:

[0092] 1) Adenovirus transfection of PDX1 / NeuroD1 genes into hUSCs: P3 generation hUSCs were cultured using Nunclon Sphera3D ultra-low adsorption 6-well culture plates as culture carriers, and P3 generation human urine-derived hUSCs were infected with recombinant adenovirus (Adxsi-CMV-PDX1 / CMV-NeuroD1) carrying PDX1 and NeuroD1 genes (5.0x10^6 / ml cells were inoculated in the culture plate and cultured in 10 ml of 10% FBS DMEM medium. After the cells reached 85% confluence, pAdxsi-CMV-PDXI / CMV-NeuroD1 virus was directly added to the culture flask at 100 PFU / cell, and repeated 3 times) and infected for 7 days.

[0093] 2) Pre-induction of differentiation: 50ug / ml of rat insulinoma cell lysate extract, 0.5% insulin transferrin-selenium ITS, and induction in culture medium DMEMF / 12 for 1 day.

[0094] 3) Three-stage differentiation induction: Stage 1: 100 ng / ml EGF, 3% (v / v) B27, 10 ng / ml glucagon-like peptide-1 (GLP-1) + DMEMF / 12 induction for 2 days;

[0095] Phase II: 10 ng / ml GLP-1, 10 ng / ml human β-cell regulatory factor, 3% (v / v) B27, 100 ng / ml EGF, 10 ng / ml HGF + 5% FBS DMEMF / 12 induction for 7 days;

[0096] Three stages: 10 mM nicotinamide (NIC), 0.1 mmol / L β-mercaptoethanol, 10 ng / ml HGF+5% FBS in DMEMF / 12 for 7 days of induction.

[0097] The control group was hUSCs without genetic modification combined with cytokine 2D culture induction group. The cytokine induction scheme was the same as that of the experimental group but Corning 6-well culture plates (2D culture) were used.

[0098] 3.2 hUSCs induced differentiation into pancreatic β cells (IPCs)

[0099] 3.2.1 Adenovirus transfection of PDX1 / NeuroD1 genes in hUSCs: P3 generation hUSCs were cultured using Nunclon Sphera3D ultra-low adsorption 6-well culture plates as culture carriers, and P3 generation human urine-derived hUSCs were infected with recombinant adenovirus (Adxsi-CMV-PDX1 / CMV-NeuroD1) carrying PDX1 and NeuroD1 genes (5.0x10^6 / ml cells were inoculated in the culture plate and cultured in 10ml of 10% FBS DMEM culture medium. After the cells reached 85% confluence, pAdxsi-CMV-PDXI / CMV-NeuroD1 virus was directly added to the culture flask at 100 PFU / cell, and repeated 3 times) and infected for 7 days (the control group was not treated with this treatment).

[0100] 3.2.2 Inducing hUSCs to differentiate into IPCs through three stages:

[0101] (1) After observing that hUSCs form compact spheres in the 6-well plate, aspirate and discard the DMEMF / 12 complete medium; add the first stage differentiation medium and cytokines according to the experimental protocol, 200 μL per well.

[0102] (2) After 2 days, discard the first stage differentiation medium and replace it with the second stage differentiation medium and cytokines, 200uL per well. Replace the second stage differentiation medium and cytokines every 3 days (the second stage is cultured for 7 days).

[0103] (3) After 7 days, the second stage differentiation medium was discarded again and replaced with the third stage differentiation medium and cytokines, 200 μL per well. The third stage differentiation medium and cytokines were replaced every 3 days.

[0104] 4. Identification of pancreatic β cells (IPCs) and their secretions

[0105] 4.1 Morphological observation of IPCs

[0106] The adherent hUMSCs were placed in an ultra-low adsorption 96-well plate for 3D culture, and the single cells gradually gathered into cell clusters. After 24 hours, it was observed that the HUCMSCs single cells formed a cell cluster with a diameter of 280-300mm, with a small number of single cells on the edge; after 48 hours, the HUCMSCs cell cluster became more compact, with a diameter reduced to 250mm, and the cell morphology was relatively uniform, close to the size of the real islet cluster. Induction was carried out according to the three-step differentiation scheme, and the differentiation medium was replaced regularly. As the number of days of differentiation increased, the morphology of the cell cluster became slightly loose (such as Fig. 9 As shown, Fig. 9Middle: A is the result of the three-stage differentiation experiment group of the present invention; B is the result of hUSCs (control), most of the differentiated cell clusters began to change from spherical to ellipsoidal. There were some diffuse cells around the undifferentiated HUCMSCs cell clusters in the control group, but the whole was relatively compact and the morphology changed little.

[0107] 4.2 Dithizone (DTZ) staining

[0108] 4.2.1 Experimental methods

[0109] (1) After the three-stage differentiation, IPCs were aspirated from the ultra-low adsorption 6-well plate using a cut pipette tip. About 10-20 cell clusters were collected in the 6-well plate; the control group was an undifferentiated hUSCs cell cluster.

[0110] (2) Rinse with PBS 2-3 times and discard the PBS.

[0111] (3) Dilute the prepared DTZ stock solution with PBS at a ratio of 1:10 and add it to a 6-well plate.

[0112] (4) Incubate at 37°C for 30 min, rinse 2-3 times with PBS, and observe and photograph under a microscope.

[0113] 4.2.2 Staining results

[0114] Dithizone (DTZ) staining is one of the methods for identifying pancreatic islets isolated in vitro from mice, pigs, and humans. It can specifically bind to the Zn2* of insulin molecules in pancreatic β cells to form a red complex, which stains the pancreatic islet clusters scarlet. This is also a basic and important identification indicator for the successful differentiation of hUSCs into IPCS. We stained the differentiated IPCS and undifferentiated hUSCs with DTZ, and we could clearly observe the coloration of IPCs (e.g. Fig.10 As shown, Fig.10 Middle: A is the undifferentiated cell cluster before staining; B and C are the undifferentiated cell clusters after staining; D is the IPCs cell cluster before staining; E and F are the IPCs after staining). The cell clusters were stained scarlet, indicating that insulin was produced in the cells. The control group did not show DTZ staining, indicating that the undifferentiated cell clusters did not produce insulin in the cells. The preliminary identification of the DTZ staining results showed that our induced differentiated IPCs were able to produce insulin.

[0115] 4.3 Immunofluorescence staining

[0116] 4.3.1 Method of immunofluorescence staining of IPCs cell clusters

[0117] (1) Make a mouth pipette for picking up cell clusters.

[0118] (2) After the three-stage differentiation, IPCs were aspirated from the ultra-low attachment 6-well plate using a cut pipette tip. About 10-20 cell clusters were collected in the 6-well plate. The control group was an undifferentiated UhSCs cell cluster.

[0119] (3) Add PBS and soak the cell pellet 2-3 times.

[0120] (4) Pick out the cell clusters with a pipette, place them in 4% PFA, and fix them at room temperature for 1 hour; pick out the cell clusters and soak them in PBS 2-3 times.

[0121] (5) Pick out the cell clusters, add 0.3% TritonX-100 solution, incubate at room temperature for 1-2 hours, and soak in PBS 2-3 times. (6) Pick out the cell clusters, add 3% BSA and 10% FBS mixture, and store in a refrigerator at 4°C overnight. Rinse in PBS 2-3 times.

[0122] (6) Add primary antibody (10 ug / mL Insulin Antibody) and incubate overnight at 4°C.

[0123] (7) Rinse with PBS 2-3 times, add 10ug / mL PDX-1 Antibody, and incubate at 4°C overnight.

[0124] (8) Rinse with PBS 2-3 times, add 10ug / mL NKX6.1 Antibody, and incubate at 4°C overnight.

[0125] (9) Rinse with PBS 2-3 times, add secondary antibodies, Anti-mouse IgG-NL493, Anti-rabbit AF 647,

[0126] Goat anti-guinea pig IgG H&L, 5ug / mL each, and DAPI were added at the same time, and incubated at room temperature for 1h in the dark.

[0127] (10) Rinse with PBS 2-3 times, wrap the 12-well plate with tin foil, and take pictures with a laser confocal microscope.

[0128] 4.3.2 Immunofluorescence staining of IPCs cell cluster embedded sections

[0129] (1) After the three-stage differentiation, IPCs were aspirated from the ultra-low attachment 6-well plate using a cut pipette tip. About 30-40 cell clusters were collected in a 1.5 m EP tube. The control group was an undifferentiated hUSCs cell cluster.

[0130] (2) Pick out the cell clusters and rinse them with PBS 2-3 times.

[0131] (3) Pick out the cell clusters, add 4% PFA solution, fix at room temperature for 30 min, and rinse with PBS 2-3 times.

[0132] (4) Aspirate and discard PBS as much as possible, select an embedding box of appropriate size (1 cm), and move the cell clusters into the embedding box with as little liquid as possible. (5) Add OCT to fill the embedding box, use the needle of a syringe to collect the cell clusters together, and mark the bottom of the embedding box with a pen.

[0133] (6) Place in a -20℃ freezer overnight.

[0134] (7) Use a microtome to cut the embedded cell mass into 8 μm / slice and prepare frozen sections of the cell mass. (8) Leave the frozen sections at room temperature for a while, add PBS to gently rinse to remove the OCT embedding agent, and add 0.2%

[0135] The membrane was permeabilized with TritonX-100 solution and allowed to stand at room temperature for 1 h.

[0136] (9) Add PBS to rinse the membrane, add 3% BSA, and seal the membrane at 4°C for 1 h.

[0137] (10) Rinse with PBS 2-3 times, and add primary antibodies PDX-1Antibody, InsulinAntibody, and NKX6.1

[0138] Antibody, place the sections in a humidified chamber and incubate at 4°C overnight.

[0139] (11) Rinse with PBS 2-3 times, add secondary antibody and DAPI, and incubate in a wet box at room temperature for 1 h.

[0140] (12) Rinse with PBS 2-3 times, slowly add anti-fluorescence quenching reagent to the frozen sections, and aspirate the excess liquid.

[0141] (13) Avoid creating bubbles, press the coverslip firmly, and apply sealing fluid to seal the slide.

[0142] (14) Observe and photograph the slices under a laser confocal microscope.

[0143] 4.3.3 Immunofluorescence staining results

[0144] PDX-1 (Pancreas / duodenumhomeobox protein1), also known as insulin initiation factor 1 (IPF1), is a homeodomain transcription factor required for pancreatic development and maturation of pancreatic cell function, and is involved in glucose-dependent regulation of insulin gene transcription. NKX6-1 (NK6homeobox1) is specific to adult cells that produce insulin, and is involved in the regulation of islet growth and endocrine activity. It is also involved in the lineage specification of early pancreatic progenitor cell tissue and regulates the expression of multiple pancreas-related genes. The INS (Insulin) gene encodes preproinsulin, which is converted into insulin by enzymes. Insulin is produced by pancreatic B cells, and the production of insulin is strictly regulated by specific DNA elements present within the -400bp proximal region of the INS promoter, and is also involved in the regulation of glucose metabolism. After the three-stage differentiation, IPCS and hUSCs were identified by immunofluorescence staining: three specific markers of pancreatic B cells, PDX1, Insulin, and NKX6-1, were selected for immunofluorescence staining (such as Fig.11 As shown, Fig.11 Middle: A is the IPCS differentiation group; B is the undifferentiated group; C is the IPCs section staining). We observed under an immunofluorescence microscope that in IPCs, the important pancreatic B cell transcription factors PDX1 (red fluorescence), NKX6-1 (silver fluorescence) and Insulin (green fluorescence) were co-expressed. In comparison, NKX6-1 was strongly expressed, with a large staining area and strong color. In the control group, the undifferentiated hUSCs cell clusters only expressed specific markers of pancreatic B cells under non-specific staining with DAPI. Combined with the morphological changes that occurred during the induction of differentiation, we observed that IPCS may form a cell cavity, so we performed 3D layer scanning and section staining of the cell clusters ( Fig.11 ) found that IPCs do undergo some morphological changes, differentiating into ellipsoids, with a diameter of 200-250um but a thickness of 80-100um. Immunofluorescence staining of frozen sections of cell clusters showed that IPCs cell clusters were not hollow, and Imnsulin (green fluorescence) was expressed in large quantities inside the cell clusters.

[0145] 4.4 Chemiluminescence detection of insulin and C-peptide levels

[0146] The cell culture supernatant of each experimental group was stored at -20°C and tested for insulin and C. After induction, the islet-like cell clusters were removed from the culture medium and serum-free H-DMEM and 25 mmol glucose were added. After incubation for 1 hour, the supernatant was taken and tested for C-peptide and insulin.

[0147] On the 10th day of induction, the secretions of insulin and IC peptide in the supernatant of the experimental group were 273.42+18.57m U / L and 0.36±0.11ng / mL, respectively. On the 17th day, the secretions of insulin and C peptide were 497.27±55.96mU / L and 2.85±0.37ng / mL, respectively. After the synergistic effect of 25mmol / L high glucose for 1 hour, the contents of insulin and C peptide reached 985.23±66.30mU / L and 4.31±0.68ng / mL, respectively. The control group had significantly lower insulin secretion (132.75±8.16m U / L, 213.36±13.42m U / L, 397.34±46.21m U / L) and C-peptide (0.13±0.06ng / mL, 0.89±0.12ng / mL, 1.07±0.16ng / mL) than the experimental group on the 10th day, 17th day and 17th day+high glucose. The differences between the experimental group and the control group and in different time periods of the experimental group were statistically significant (P<0.05).

[0148] 4.5 Detection of insulin (+) cell rate by flow cytometry

[0149] The cells induced by combined cytokines for 17 days were digested with 0.25% trypsin (mass fraction), washed with PBS and centrifuged, and the supernatant was discarded. The dye insulin-CY5 was added, and 10 cells were counted and detected by flow cytometry. The instrument was equipped with software for analysis. The experimental group detected insulin after induction by flow cytometry {the percentage of insulin+ cells was 25.34±5.13%, and the control group was 8.12±3.87% respectively) was significantly lower than that of the experimental group (P<0.05).

[0150] Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0151] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A dual gene expression adenoviral vector pAdxsi-CMV-PDXI / CMV-NeuroD1, It is characterized in that The preparation method of the dual-gene expression pAdxsi-CMV-PDXI / CMV-NeuroD1 adenovirus vector comprises the following steps: pUC57-PDX1 and pUC57-NeuroD1 were digested with BglII and XhoI restriction endonucleases, respectively, and PDX1 and NeuroD1 were ligated to the pShuttle-GFP-CMV recombinant shuttle vector to obtain pShuttle-GFP-CMV-PDX1 / pShuttle-GFP-CMV-NeuroD1 plasmids; Transferring the pShuttle-GFP-CMV-PDX1 / pShuttle-GFP-CMV-NeuroD1 plasmid to the pAdxsi vector, and harvesting the pAdxsi-CMV-PDX1 / NeuroD1 recombinant viral plasmid; The pAdxsi-CMV-PDX1 / NeuroD1 recombinant virus plasmid was transfected into 293 cells to package a complete adenovirus, thereby obtaining a dual-gene expression pAdxsi-CMV-PDXI / CMV-NeuroD1 adenovirus vector.

2. A method for preparing double-gene modified stem cells for inducing differentiation of pancreatic β cells, It is characterized in that The method comprises the following steps: Obtain hUSCs; Transfecting the hUSCs with the dual-gene expression pAdxsi-CMV-PDXI / CMV-NeuroD1 adenoviral vector according to claim 1; hUSCs were infected with adenovirus carrying pAdxsi-CMV-PDXI / CMV-NeuroD1 and differentiated into pancreatic β cells by cytokine induction.

3. The method for preparing the double-gene modified stem cells for inducing differentiation of pancreatic β cells according to claim 2, It is characterized in that The step of obtaining hUSCs includes the following process: Primary stem cells hUSCs were extracted from fresh human urine using the adherence method; The primary stem cells hUSCs are expanded and subcultured, and hUSCs can be obtained after three subcultures.

4. The method for preparing the double-gene modified stem cells for inducing differentiation of pancreatic β cells according to claim 2, It is characterized in that The steps of infecting hUSCs with adenovirus carrying pAdxsi-CMV-PDXI / CMV-NeuroD1 and inducing differentiation into pancreatic β cells by cytokine include pre-induction differentiation, one-stage induction differentiation, two-stage induction differentiation and three-stage induction differentiation.

5. The method for preparing the double-gene modified stem cells for inducing differentiation of pancreatic β cells according to claim 4, It is characterized in that The working condition parameters of the pre-induction differentiation include: 30-80ug / ml of rat insulinoma cell lysis extract, 0.5-2% insulin transferrin-selenium ITS, and culture medium DMEMF / 12 induction for 1-3 days.

6. The method for preparing the double-gene modified stem cells for inducing differentiation of pancreatic β cells according to claim 4, It is characterized in that The working condition parameters for the first stage of differentiation induction include: 10-100 ng / ml EGF, 0.5-5% (v / v) B27, 20-60 ng / ml glucagon-like peptide-1 (GLP-1)+DMEMF / 12 induction for 2-4 days.

7. The method for preparing the double-gene modified stem cells for inducing differentiation of pancreatic β cells according to claim 4, It is characterized in that The working condition parameters of the second stage induction differentiation include: 20-60ng / ml GLP-1, 40-60ng / ml human cytokine, 0.5-5% (v / v) B27, 10-100ng / ml EGF, 5-30ng / ml HGF + DMEMF / 12 containing 1-10% FBS for induction for 6-8 days.

8. The method for preparing the double-gene modified stem cells for inducing differentiation of pancreatic β cells according to claim 4, It is characterized in that The working condition parameters of the three-stage differentiation induction include: 5-20 mM nicotinamide (NIC), 0.1-1.0 mmol / L β-mercaptoethanol, 5-30 ng / ml HGF+1-10% FBS DMEMF / 12 induction for 6-8 days.

9. A pancreatic beta cell, It is characterized in that The pancreatic β cells are prepared by the preparation method of inducing differentiation of pancreatic β cells by double-gene modified stem cells as described in any one of claims 2 to 8.

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