A method and application of inducing differentiation of human umbilical cord mesenchymal stem cells into pancreatic islet microsphere organoids
By adopting a three-dimensional induction scheme on hUC-MSCs, a specific culture medium is prepared to gradually induce the formation of pancreatic islet organoids, which solves the problem of low efficiency under the existing two-dimensional culture method, and achieves efficient and stable acquisition of pancreatic islet cell microspheres with normal insulin secretion function, with broad clinical application prospects.
Patent Information
- Application Number
- CN202410824951.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-25
- Publication Date
- 2025-04-04
AI Technical Summary
Under the existing two-dimensional culture method, the induction of hUC-MSCs into insulin-secreting cells has low efficiency, low induction success rate, poor stability, and is difficult to achieve large-scale mass production and wide clinical application.
A three-dimensional induction scheme based on hUC-MSCs was adopted, and by preparing culture medium with five different components, the formation of endoderm cells, pancreatic group cells, pancreatic endocrine progenitor cells and mature pancreatic islet organoids were gradually induced to form spherical pancreatic islet organoids.
The induction efficiency was significantly improved. The C-peptide+ cell rate of three-dimensional islet organoid C-peptide+ was 71.17%±0.40%, and a large number of mature islet cell microspheres with normal insulin secretion were obtained. The operation was simple, the process was repetitive, and there was no rejection after transplantation, which was highly safe.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedical technology, and more specifically relates to the construction of a technical solution for inducing the differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) into pancreatic islet organoids. Background Art
[0002] Type 1 Diabetes Mellitus (T1DM) is a metabolic disease in which the insulin-producing pancreatic β cells are destroyed by autoimmune reactions, resulting in insufficient endogenous insulin secretion. Although intensive insulin therapy can regulate blood sugar to a certain extent, it cannot effectively prevent the occurrence and development of diabetes, causing many patients to need islet transplantation. However, islet transplantation in clinical practice currently faces many problems such as donor shortage and post-transplant immune rejection.
[0003] Stem cell technology provides an effective way to solve the above problems. Some researchers have induced embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) to differentiate into insulin-producing cells (IPCs) for islet replacement therapy. However, since these two types of cells have the risk of tumorigenesis, there are great risks in clinical application. Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a relatively primitive adult stem cell with the potential for cross-germ layer differentiation and no tumorigenic risk.
[0004] At present, the induction methods for inducing stem cells to differentiate into insulin-secreting cells are all two-dimensional flat culture, and the establishment of the induction culture medium system for the directional induction and differentiation of hUC-MSCs into IPCs in various laboratories is not standardized. The induction operation is complicated, the induction success rate is low, the stability is poor, the number of IPCs obtained is small, and the insulin secretion function is only 1% of that of normal pancreatic islets. Therefore, the efficiency of inducing hUC-MSCs to generate IPCs in the existing two-dimensional culture method is very low and cannot be used for clinical research. In order to improve the induction efficiency of hUC-MSCs to differentiate into IPCs, a three-dimensional growth method closer to normal pancreatic islets should be used for induction culture to better simulate the growth environment of normal pancreatic islets in vivo. Most methods for constructing three-dimensional pancreatic islet organoids require the use of foreign companies' technology and the use of matrix gel. Due to its complex operation and high cost, it is difficult to achieve large-scale mass production and widespread clinical application of hUC-MSCs directional induction and differentiation into pancreatic islet organoids using matrix gel, which will make it difficult to ensure the clinical application of the generated three-dimensional pancreatic islet organoids based on hUC-MSCs. How to break these limitations has become a key issue in cell therapy. Summary of the invention
[0005] In view of the above-mentioned problems existing in the existing induction technology when inducing hUC-MSCs to differentiate into pancreatic islet organoids, the present invention proposes a three-dimensional induction scheme for pancreatic islet organoids based on hUC-MSCs. Each induction component is safe and non-toxic, with high induction efficiency and strong stability, and a large number of mature pancreatic islet cell microspheres with normal insulin secretion function can be obtained.
[0006] The technical solution adopted by the present invention is as follows:
[0007] Prepare the following 5 culture media:
[0008] Solution A: DMEM basal medium supplemented with 5-10 ng / ml EGF, 0.2% BSA, 0.3-0.5 mM vitamin C, and 2% FBS;
[0009] Solution B: DMEM basal medium supplemented with 5-10 ng / ml EGF, 20-50 ng / ml nitazoxanide, 20-50 ng / ml Wnt-C59, 1-4 μM dorsomorphin, 0.3-0.5 mM vitamin C, 1% non-essential amino acids, 4-6 mmol / L glutamine, and 2% FBS;
[0010] Solution C: DMEM basal medium supplemented with 5-10 ng / ml bFGF, 20-50 ng / ml nitazoxanide, 20-50 ng / ml Wnt-C59, 2-6 ng / ml ISX9, 2-4 μmol / L Purmorphamine, and 5-7% FBS;
[0011] Solution D: DMEM basal medium supplemented with 5-10 ng / ml bFGF, 10-20 μg / ml ascorbic acid, 1-4 μM dorsomorphin, and 10% FBS:
[0012] Solution E: DMEM basal medium supplemented with 5-10 ng / ml bFGF, 1-4 μM dorsomorphin, and 10% FBS;
[0013] S1. Induction of endoderm cells The human umbilical cord mesenchymal stem cells were placed in an adhesion six-well plate and cultured with solution A, wherein activin A (100 ng / ml), nicotinamide (5-10 mM), and double antibody (1%) were added to solution A;
[0014] S2. Induction of pancreatic progenitor cells The endoderm cells obtained above are placed in a microwell plate to form a three-dimensional cell microsphere, which is then placed in a low-adsorption six-well plate and cultured with B solution, wherein nicotinamide (5-10 mM) and double antibody (1%) are added to the B solution;
[0015] S3, induction of pancreatic endocrine progenitor cells The pancreatic progenitor cell microspheres obtained above were placed in a low-adsorption six-well plate and cultured with C solution, wherein nicotinamide (5-10 mM), ITS-X (1%) and dual antibody (1%) were added to the C solution;
[0016] S4. Induction of pancreatic endocrine cells The pancreatic progenitor cell microspheres obtained above were placed in a low-adsorption six-well plate and cultured with D solution, wherein ascorbic acid (40 μg / ml), ITS-X (1%) and double antibody (1%) were added to the D solution;
[0017] S5. Induction of mature pancreatic islet organoids The pancreatic endocrine cell microspheres obtained above were placed in a low-adsorption six-well plate and cultured with E solution, wherein nicotinamide (5-10 mM), Exendin-4 (40 ng / ml), ITS-X (1%) and dual antibody (1%) were added to the E solution;
[0018] Further, the cells were cultured in induction differentiation solution A for 3 days, in induction differentiation solution B for 4 days, in induction differentiation solution C for 6 days, in induction differentiation solution D for 4 days, and induction differentiation solution D for 7 days, with the culture medium being changed every day.
[0019] Compared with the prior art, the present invention provides an induction scheme for inducing hUC-MSCs to differentiate into spherical pancreatic islet organoids in a three-dimensional culture mode, which has the following advantages:
[0020] 1. The present invention induces hUC-MSCs to differentiate into spherical pancreatic islet organoids in a three-dimensional culture method, which significantly improves the induction efficiency compared with the traditional two-dimensional culture method. + The cell rate was 71.17%±0.40%, which was more conducive to obtaining a large number of mature islet cell microspheres with normal insulin secretion function, and provided a culture method that was more suitable for the development and differentiation of islet cell microspheres.
[0021] 2. The present invention changes the sugar content of the hUC-MSCs system from high sugar to low sugar during the differentiation induction process, which significantly improves the insulin secretion function of the three-dimensional pancreatic islet organoids compared to the induction scheme in which the sugar content remains unchanged during the induction process. The amount of insulin released under the stimulation of high sugar (25mM) solution is 7.48 times that of the insulin released under the stimulation of low sugar (5.5mM) solution, and the amount of C-peptide released under the stimulation of high sugar (25mM) solution is 14.43 times that of the C-peptide released under the stimulation of low sugar (5.5mM) solution.
[0022] 3. The spherical pancreatic islet organoids induced by the present invention are closer to the cell sphere size, β cell ratio and insulin secretion function of natural pancreatic islets.
[0023] 4. The present invention is simple to operate and has a high process repetition rate; the present invention uses three-dimensional culture of human umbilical cord mesenchymal stem cells in the stage of inducing pancreatic progenitor cells, and the pancreatic islet organoids have good uniformity and high repeatability.
[0024] 5. The spherical pancreatic islet organoids induced by the present invention have no rejection after transplantation, are highly safe, and have broad prospects for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 Schematic diagram of cell morphology changes at different stages of hUC-MSCs directed differentiation into pancreatic islet organoids in an example of the present invention;
[0026] Figure 2 Schematic diagram of dithizone staining of pancreatic islet organoids induced and differentiated from hUC-MSCs in an example of the present invention;
[0027] Figure 3 This is a schematic diagram of AO / EB staining of pancreatic islet organoids induced and differentiated from hUC-MSCs in an example of the present invention;
[0028] Figure 4This is a schematic diagram of flow cytometry staining of pancreatic islet organoids induced and differentiated from hUC-MSCs in an example of the present invention;
[0029] Figure 5 It is a schematic diagram of the in vitro glucose-stimulated insulin and C-peptide release of pancreatic islet organoids differentiated into hUC-MSCs in an example of the present invention;
[0030] Figure 6 The figure shows the decrease in fasting blood glucose of mice after the islet organoids differentiated by hUC-MSCs were injected into the renal capsule of T1DM mouse model in the example of the present invention. DETAILED DESCRIPTION
[0031] The following will be combined with the drawings in the embodiments of the present disclosure to clearly and completely describe the technical solutions in the embodiments of the present disclosure. The described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present disclosure.
[0032] Example: A three-dimensional pancreatic islet organoid induction scheme based on hUC-MSCs, the preparation of the induction differentiation medium and the induction differentiation scheme are as follows: Prepare the following 5 culture media: Prepare the following 5 culture media: Solution A: DMEM basal medium supplemented with 5-10 ng / ml EGF, 0.2% BSA, 0.3-0.5 mM vitamin C, and 2% FBS; Solution B: DMEM basal medium supplemented with 5-10 ng / ml EGF, 20-50 ng / ml nitazoxanide, 20-50 ng / ml Wnt-C59, 1-4 μM dorsomorphin, 0.3-0.5 mM vitamin C, 1% non-essential amino acids, 4-6 mmol / L glutamine, and 2% FBS; Solution C: DMEM basal medium supplemented with 5-10 ng / ml bFGF, 20-50 ng / ml nitazoxanide, 20-50 ng / ml Wnt-C59, 2-6 ng / ml ISX9, 2-4 μmol / L Purmorphamine, and 5-7% FBS; Solution D: DMEM basal medium supplemented with 5-10 ng / ml bFGF, 10-20 μg / ml ascorbic acid, 1-4 μM dorsomorphin, and 10% FBS; Solution E: DMEM basal medium supplemented with 5-10 ng / ml bFGF, 1-4 μM dorsomorphin, and 10% FBS; In vitro differentiation induction: S1. Induction of endoderm cells The human umbilical cord mesenchymal stem cells were placed in an adhesion six-well plate and cultured with solution A, wherein activin A (100 ng / ml), nicotinamide (5-10 mM), and double antibody (1%) were added to solution A; S2. Induction of pancreatic progenitor cells The endoderm cells obtained above are placed in a microwell plate to form a three-dimensional cell microsphere, which is then placed in a low-adsorption six-well plate and cultured with B solution, wherein nicotinamide (5-10 mM) and double antibody (1%) are added to the B solution; S3, induction of pancreatic endocrine progenitor cells The pancreatic progenitor cell microspheres obtained above were placed in a low-adsorption six-well plate and cultured with C solution, wherein nicotinamide (5-10 mM), ITS-X (1%) and dual antibody (1%) were added to the C solution; S4. Induction of pancreatic endocrine cells The pancreatic endocrine progenitor cell microspheres obtained above were placed in a low-adsorption six-well plate and cultured with D solution, wherein ascorbic acid (40 μg / ml), ITS-X (1%) and double antibody (1%) were added to the D solution. S5. Induction of mature pancreatic islet organoids The pancreatic endocrine cell microspheres obtained above were placed in a low-adsorption six-well plate and cultured with E solution, wherein niacinamide (5-10mM), Exendin-4 (40ng / ml), ITS-X (1%) and double antibody (1%) were added to the E solution; the cells were cultured in the induction differentiation solution A for 3 days, in the induction differentiation solution B for 4 days, in the induction differentiation solution C for 6 days, in the induction differentiation solution D for 4 days, and in the induction differentiation solution D for 7 days, and the culture medium was changed every day. Characterization of pancreatic islet organoids induced by differentiation: 1. According to the method of the embodiment, hUC-MSCs were induced to differentiate into three-dimensional pancreatic islet organoids for 24 days. The morphology of the induced cells was observed by an inverted microscope. Pictures were taken at the end of each stage to observe whether islet-like cell clusters were formed. The number and diameter of islet-like cell clusters generated in the S4 stage were counted and measured. Figure 1 As shown; 2. According to the method of the embodiment, the induced three-dimensional pancreatic islet organoids were stained with dithizone: about 10-20 induced cell spheres were collected, the S4 stage induction solution in the well plate was discarded, and the cells were washed 3 times with PBS. After adding DTZ staining solution, the cells were blown and mixed evenly, and placed in a cell culture incubator at 37°C and 5% CO2 for 20-30 minutes. After taking out, the DTZ staining solution was discarded, PBS was added to suspend evenly, and the cells were observed using an inverted microscope. Figure 2 As shown; 3. According to the method of the embodiment, the induced three-dimensional pancreatic islet organoids were subjected to flow cytometry to detect C-peptide + Cell rate: Absorb the induced cell ball, discard the S4 stage induction solution in the well plate, wash with PBS three times, add preheated 5mL Accutase digestion solution, gently blow and mix the cell suspension, and place it in a cell culture incubator at 37℃ and 5% CO2 for 15min to digest the cell ball into single cells. Centrifuge at 1000rpm for 5min, discard the supernatant, resuspend the cells with PBS, calculate the number of cells, take 6.0×105 cells and add 500uL, 4℃ precooled fixative, incubate at room temperature in the dark for 10min, centrifuge at 1000rpm for 10min, discard the supernatant, wash with PBS three times, centrifuge at 1000rpm for 10min, discard the supernatant, add 1.5mL membrane permeabilization agent, incubate at room temperature for 10-15min, centrifuge at 1000rpm for 5min, discard the supernatant, add 100uL membrane permeabilization agent to resuspend the cells, and add Alexa 647 Mouse Anti-C-Peptide 3uL, incubate at room temperature for 30min, wash 3 times with PBS, add 500uL PBS to resuspend the cells, filter through nylon mesh and test on the machine. Figure 3 As shown; 4. According to the method of the embodiment, the induced three-dimensional pancreatic islet organoids were subjected to qRT-PCR to detect the expression of marker genes at each stage during the hUC-MSCs-directed IPCs induction process: cells at each stage (S0, S1, S2, S3, S4) of the induction process were taken, the induction solution in the well plate was discarded, and the cells were washed 3 times with PBS. After RNA was extracted, cDNA was reverse transcribed using RNA as a template, and then the expression levels of marker genes (FOXA2, PDX1, NKX6.1, NGN3, GLUT2, MAFA and INS) at each stage during the hUC-MSCs-directed IPCs induction process were detected by three-step qPCR, such as Figure 4 As shown; 5. According to the method of the embodiment, the induced three-dimensional pancreatic islet organoids were subjected to cell immunofluorescence staining to detect the expression of induced products PDX1 and INS: the induced cell spheres were aspirated, the S4 stage induction solution in the well plate was discarded, and 10-20 cell spheres were aspirated, and 4% paraformaldehyde was added to fix for 1 hour, and then the cell spheres were aspirated, and PBS was used to wash 3 times, 5 minutes each time, and 0.3% Triton was added. After X-100 permeabilization for 1 hour, the cells were washed with PBS for 3 times, each time for 5 minutes, the cell balls were aspirated, 1% BSA was added, and the cells were placed in a wet box. After blocking at 37°C for 0.5 hours, 200uL of PDX1 (1:50) and INS (1:50) primary antibodies diluted with 1% BSA were added. After overnight incubation at 4°C, the cell balls were aspirated, washed with PBS for 3 times, each time for 5 minutes, and fluorescent secondary antibodies diluted with 1% BSA were added. The cells were placed in a wet box and incubated at 37°C in the dark for 60 minutes. The cells were washed with PBS for 3 times, each time for 5 minutes, and the cell balls were mixed with anti-fluorescence attenuation mounting medium (containing DAPI). The suspension was then dropped on a glass slide. After adding a coverslip, fluorescence was excited at different wavelengths using a fluorescence microscope and photographed. Figure 5 As shown; 6. According to the method of the embodiment, the induced three-dimensional pancreatic islet organoids were subjected to an in vitro glucose stimulation experiment to detect the function of the induced products to secrete insulin and C-peptide: the induced cell spheres were aspirated, the S4 stage induction solution in the well plate was discarded, and the cell spheres were washed 3 times with PBS. 20 cell spheres were grouped into a group and collected in a 12-well plate. The control group was hUC-MSCs that were not induced to differentiate. After adding KRBH buffer containing 5.5 mM glucose, the cells were placed in a cell culture incubator at 37°C and 5% CO2 for 2 h, and the supernatant was collected. After washing twice with sugar-free KRBH buffer, KRBH buffer containing 25 mM glucose was added, and the cells were placed in a cell culture incubator at 37°C and 5% CO2 for 2 h, and the supernatant was collected. The secretion of insulin in the supernatant under the stimulation of different glucose concentrations was detected by ELISA. Figure 6 As shown; 7. Transplantation of three-dimensional islet organoids: A stable T1DM mouse model was established by a single intraperitoneal injection of a high-dose of 150 mg / kg streptozotocin (STZ), and the mice were randomly divided into a model group (Model), hUC-MSCs group (MSCs), IPCs group 1 (IPCs 1), IPCs group 2 (IPCs 2), and IPCs group 3 (IPCs 3). Another group of normal mice was selected as the normal group (Control). The identified islet organoids were transplanted into the left renal capsule of T1DM mice. The fasting blood glucose of the mice was tested every two days starting from the second day after surgery, and the postoperative hypoglycemic effect of each group was analyzed. A fasting glucose tolerance test (IPGTT) was performed on the 12th day after surgery to detect the glucose response function of IPCs in vivo, such as Figure 6 shown. In summary, the special induction culture medium of the present invention can be used to efficiently induce hUC-MSCs into three-dimensional pancreatic islet organoids with sugar concentration-dependent insulin and C-peptide release functions, large numbers and uniform particle sizes, high induction success rate, and simple and easy operation, which creates conditions for the application of hUC-MSCs in biomedical engineering and the establishment of insulin disease treatment models. The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.
Claims
1. A scheme for inducing three-dimensional pancreatic islet organoids based on human umbilical cord mesenchymal stem cells, characterized in that: The method comprises using the following five differentiation induction fluids in sequence, wherein: Solution A: DMEM basal medium supplemented with 5-10 ng / ml EGF, 0.2% BSA, 0.3-0.5mM vitamin C, and 2% FBS; Solution B: DMEM basal medium supplemented with 5-10 ng / ml EGF, 20-50 ng / ml nitazoxanide, 20-50 ng / ml Wnt-C59, 1-4μM dorsomorphin, 0.3-0.5mM vitamin C, 1% non-essential amino acids, 4-6 mmol / L glutamine, and 2% FBS; Solution C: DMEM basal medium supplemented with 5-10 ng / ml bFGF, 20-50 ng / ml nitazoxanide, 20-50 ng / ml Wnt-C59, 2-6 ng / ml ISX9, 2-4 μmol / L Purmorphamine, and 5-7% FBS; Solution D: DMEM basal medium supplemented with 5-10 ng / ml bFGF, 10-20 μg / ml ascorbic acid, 1-4 μM dorsomorphin, and 10% FBS; Solution E: DMEM basal medium supplemented with 5-10 ng / ml bFGF, 1-4μM dorsomorphin, and 10% FBS.
2. The differentiation induction solution according to claim 1, characterized in that The method for culturing three-dimensional pancreatic islet organoids comprises the following steps: S1. Induction of endoderm cells Human umbilical cord mesenchymal stem cells were placed in an adhesive six-well plate and cultured with solution A, wherein activin A (100 ng / ml), nicotinamide (5-10 mM), and double antibody (1%) were added to solution A; S2. Induction of pancreatic progenitor cells The endoderm cells obtained above were placed in a microwell plate to form a three-dimensional cell microsphere, which was then placed in a low-adsorption six-well plate and cultured with B solution, wherein nicotinamide (5-10 mM) and double antibody (1%) were added to the B solution; S3, induction of pancreatic endocrine progenitor cells The pancreatic progenitor cell microspheres obtained above were placed in a low-adsorption six-well plate and cultured with C solution, wherein nicotinamide (5-10 mM), ITS-X (1%) and double antibody (1%) were added to the C solution; S4. Induction of pancreatic endocrine cells The pancreatic endocrine progenitor cell microspheres obtained above were placed in a low-adsorption six-well plate and cultured with D solution, wherein ascorbic acid (40 μg / ml), ITS-X (1%) and double antibody (1%) were added to the D solution. S5. Induction of mature pancreatic islet organoids The pancreatic endocrine cell microspheres obtained above were placed in a low-adsorption six-well plate and cultured with E solution, wherein niacinamide (5-10 mM), Exendin-4 (40 ng / ml), ITS-X (1%) and dual antibody (1%) were added to the E solution.
3. The in vitro culture method of pancreatic islet organoids according to claim 2, characterized in that: The cells were cultured in differentiation induction medium A for 3 days, in differentiation induction medium B for 4 days, in differentiation induction medium C for 6 days, in differentiation induction medium D for 4 days, and in differentiation induction medium D for 7 days. The culture medium was changed every day.
4. The three-dimensional islet cells, microspheres and organoids obtained according to the method of claim 2 are directly transplanted for islet replacement therapy for diabetes and implanted in scaffold materials as medical devices for clinical transplantation.