Method for inducing and differentiating islet cells by iPSCs (induced pluripotent stem cells)

Abstract

The invention provides a method for inducing and differentiating islet cells through iPSCs, and belongs to the technical field of genetic engineering. The method comprises the following steps: inducing iPSCs (induced pluripotent stem cells) into definitive endoderm cells, inducing the definitive endoderm cells into pancreatic progenitor cells, and inducing and differentiating the pancreatic progenitor cells into islet cells; the method for inducing and differentiating the pancreatic progenitor cells into the islet cells comprises the following steps: mixing the pancreatic progenitor cells, HUVEC cells and hADSC cells, culturing in a differentiation culture medium containing factors, and inducing to obtain the islet cells. The time for inducing the islet cells is 24 days, the INS positive rate in the induced islet cells is 89.7%, the insulin secretion amount is 246.1 ng / 105 cells under the stimulation of 22mmol / L glucose, and the insulin secretion amount is 81.5 ng / 105 cells under the stimulation of 2mmol / L glucose.

Description

Technical Field

[0001] The invention relates to a method for inducing differentiation of pancreatic islet cells from iPSCs, and belongs to the technical field of genetic engineering. Background Art

[0002] Diabetes is a common metabolic disease, which is mainly manifested by long-term elevated blood sugar levels and seriously endangers human health. Type 1 diabetes is an autoimmune disease. In patients with type 1 diabetes, their immune system mistakenly attacks and destroys the insulin-producing B cells in the pancreas, causing abnormal insulin secretion. As a method of treating diabetes, islet cell transplantation has shown unique advantages over pancreas transplantation and islet transplantation in clinical applications guided by basic research. However, its long-term effects are not ideal. Islet cell transplantation faces the challenges of poor long-term effects and severe shortage of donor organs.

[0003] The use of induced pluripotent stem cells (iPSCs) to induce differentiation into pancreatic islet cells not only eliminates ethical issues, but also significantly reduces the risk of immune rejection as an autologous therapy. Patent CN104911141A provides a method for inducing the differentiation of iPSCs into pancreatic β-like cells by selecting suitable small molecule compounds or culture media, but the conversion efficiency of pancreatic islet cells induced by this method is 25%, and the conversion efficiency needs to be improved. Patent CN117511847A discloses a method for preparing pancreatic islet cell clusters in vitro from autologous induced pluripotent stem cells, but it takes up to 64 days. There are existing literatures that use 2D and 3D mixed culture methods, which use low-adsorption culture plates for shaking culture when inducing pancreatic progenitor cells to differentiate into pancreatic islet cells to construct a 3D environment (Chen Xinya et al. Establishment of an in vitro directed differentiation method for functional pancreatic β cells derived from human induced pluripotent stem cells (J). Chinese Journal of Biochemistry and Molecular Biology, 2024, 40 (5): 664-673), and the insulin and C-peptide secretion of the induced pancreatic islet cells is low. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a method for inducing the differentiation of pancreatic islet cells from iPSCs to achieve the following invention objectives: the induced pancreatic islet cells have a high INS positive rate and enhanced secretion capacity for insulin and C-peptide.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions: A method for inducing iPSCs to differentiate into islet cells, the method comprising inducing iPSCs into definitive endoderm cells, inducing definitive endoderm cells into pancreatic progenitor cells, and inducing pancreatic progenitor cells to differentiate into islet cells; the method for inducing pancreatic progenitor cells to differentiate into islet cells comprises mixing pancreatic progenitor cells, HUVEC cells, and hADSC cells, culturing in a differentiation medium containing LDN193189 at a final concentration of 98-102nM, XXI at a final concentration of 98-102nM, 8-12µM SB431542, and 0.8-1.2µM T3 factor, and after culturing for 7 days, replacing the culture medium with a differentiation medium containing a vitamin E analog at a final concentration of 9-11µM, 1.8-2.2µM R428, 0.9-1.1mM N-acetylcysteine, 8-12µM SB431542, and 0.9-1.1µM T3, and continuing culturing for 5 days to induce islet cells.

[0006] The initial number ratio of the pancreatic progenitor cells, HUVEC cells, and hADSC cells is 20:10:1.

[0007] The seeding density of pancreatic progenitor cells is (0.8-1.2)×10 7 / mL, the seeding density of the HUVEC cells is (4.8-5.2)×10 6 / mL, the seeding density of the hADSC cells is (4.8-5.2)×10 5 Pieces / mL.

[0008] In the process of inducing the differentiation of pancreatic islet cells from iPSCs, a differentiation medium containing factors is used. The differentiation medium is DMEM / F12 supplemented with BSA with a final concentration of 18-22 mmol / L, 0.8-1.2% Vol glutamine, 0.55-0.57 g / L NaHCO3, 0.32-0.34 g / L thiamine, 0.08-0.12 g / L reduced glutathione, 3.2-3.4 g / L vitamin C, (6.8-7.2)×10 -4 Vol% β-ME, 0.18-0.22Vol% LiCl, 0.0018-0.0022g / L insulin, 2.8-3.2g / L methylcellulose, 0.14-0.16g / L low acyl gellan gum.

[0009] The method for inducing iPSCs into definitive endoderm cells is to expand and culture iPSCs cells into cell clusters, culture them in a differentiation medium containing Activin-A with a final concentration of 98-102 ng / mL and 2.8-3.2 µM CHIR99021 for 1 day, replace the culture medium with a differentiation medium containing Activin-A with a final concentration of 98-102 ng / mL on the second day, and continue to culture for 2 days to obtain definitive endoderm cells.

[0010] The diameter of the cell cluster is 50nm-100nm.

[0011] The method for inducing definitive endoderm cells into pancreatic progenitor cells is to induce the definitive endoderm cells in a medium containing a final concentration of 48-52 ng / mL The cells were cultured in a differentiation medium containing FGF7 for 2 days to induce the differentiation of endoderm cells into primitive gut tube cells. The primitive gut tube cells were then cultured in a differentiation medium containing a final concentration of 48-52 ng / mL FGF7, 0.24-0.26 µM SANT-1, 0.8-1.2 µM retinoic acid RA, 98-102 nM LDN193189, and 98-102 nM TPPB for 3 days to induce the differentiation of primitive gut tube cells into pancreatic progenitor cells. The pancreatic progenitor cells were then cultured in a differentiation medium containing a final concentration of 0.24-0.26 µM SANT-1, 48-52 nM retinoic acid, 8-12 µM SB431542, and 0.8-1.2 µM T3 for 4 days to induce the differentiation of pancreatic progenitor cells into pancreatic progenitor cells.

[0012] Compared with the prior art, the present invention achieves the following beneficial effects: The present invention adds methylcellulose and low-acyl gellan gum to the differentiation medium, and the differentiation medium is a milky medium, which is more conducive to the suspension culture of cells; when pancreatic progenitor cells are induced into islet cells, HUVEC cells and hADSC cells are added for mixed culture, and islet cells are induced under 3D conditions, thereby increasing the INS positive rate in the islet cells and increasing the secretion of insulin and C-peptide.

[0013] The time for inducing pancreatic islet cells in the present invention is 24 days, the INS positive rate in the induced pancreatic islet cells is 89.7%, and the insulin secretion amount is 246.1ng / 10 5 cells, under 2mmol / L glucose stimulation, insulin secretion is 81.5ng / 10 5 cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a microscope image of iPSCs cells revived in Example 1; Figure 2This is a microscopic picture of pancreatic islet cells induced by the present invention; Figure 3 It is a line graph of the relative expression levels of the endoderm cell markers SOX17 and FOXA2 genes in the experimental group and the control group; A is a line graph of the relative expression of the endoderm cell marker SOX17 gene in the experimental group and the control group; B is a line graph of the relative expression of the endoderm cell marker FOXA2 gene in the experimental group and the control group; Figure 4 It is a line graph of the relative expression levels of pancreatic progenitor cell markers PDX1 and NKX6.1 genes in the experimental group and the control group; A is a line graph of the relative expression of the pancreatic progenitor cell marker NKX6.1 gene in the experimental group and the control group; B is a line graph of the relative expression of the pancreatic progenitor cell marker PDX1 gene in the experimental group and the control group; Figure 5 It is a line graph of the relative expression of islet cell markers INS, GCG and SST genes in the experimental group and the control group; A is a line graph of the relative expression of the pancreatic islet cell marker INS gene in the experimental group and the control group; B is a line graph of the relative expression of the pancreatic islet cell marker GCG gene in the experimental group and the control group; C is a line graph of the relative expression of the pancreatic islet cell marker SST gene in the experimental group and the control group; Figure 6 It is a bar graph of the positive rate of INS in pancreatic islet cells of the experimental group and the control group; Figure 7 It is a bar graph of the C-peptide secretion of pancreatic islet cells in the experimental group and the control group under low-glucose and high-glucose stimulation. DETAILED DESCRIPTION

[0015] Example 1 Recovery and Passaging of iPSCs The preserved iPSCs were revived and passaged according to conventional methods. A tube of iPSCs cells was taken out of liquid nitrogen, thawed in a 40°C water bath, and transferred to a 50mL centrifuge tube. Then 10mL of mTeSR medium was added dropwise. The cells were gently shaken to mix during the process. Centrifuged at 850rpm for 5min. The supernatant was discarded, and 10mL of mTeSR medium (purchased from STEMCELL Technology) containing Y-27632 (purchased from MCE) at a final concentration of 10μM was added. The cells were mixed and blown as little as possible to obtain cell culture medium. The mixed cell culture medium was inoculated into a T75 bottle coated with matrix gel at a rate of 10mL / bottle, and cultured in an incubator at 37°C and 5% CO2. After 24 hours, new mTeSR medium was replaced (10mL was added to each bottle), and then mTeSR complete medium was replaced every day. When the confluence of iPSCs cells reaches about 85% (such as Figure 1) for subculture at a ratio of 1:5. When the diameter of the iPSCs cell clusters at the second generation was between 50nm and 100nm, they were used for induction of differentiation.

[0016] Example 2 Inducing differentiation of iPSCs into pancreatic islet cells in a 3D environment The specific steps are as follows: 1. Induction of iPSCs into definitive endoderm cells On the first day, iPSCs cell pellets were collected and centrifuged at 400 g for 5 min. The pellets were resuspended in differentiation medium containing 100 ng / mL Activin-A (purchased from MCE, Catalog No.: HY-P70311) and 3 µM CHIR99021 (purchased from MCE, Catalog No.: HY-10182) at a final concentration of 2 × 10 6 Cells / mL were inoculated into cell culture flasks and cultured for 1 day. On the second day, the culture medium was replaced with a differentiation medium containing Activin-A at a final concentration of 100 ng / mL, and culture was continued for 2 days to obtain definitive endoderm cells. The induction time at this stage was 3 days.

[0017] 2. Induction of definitive endoderm cells into pancreatic progenitor cells This stage is divided into three steps. The first step is to induce differentiation into primitive intestinal tube cells for 2 days, the second step is to induce differentiation into pancreatic precursor cells for 3 days, and the third step is to induce differentiation into pancreatic progenitor cells for 4 days.

[0018] On day 4, definitive endoderm cells were collected, centrifuged at 400 g for 5 min, and resuspended in differentiation medium containing FGF7 (purchased from MCE, catalog number: HY-P70597) at a final concentration of 50 ng / mL. The cell density was 2 × 10 6 The cells were cultured at 37°C in a 5% CO2 incubator for 2 days to induce the endoderm cells to differentiate into primitive gut tube cells.

[0019] On the 6th day, primitive gut cells were collected, centrifuged at 400 g for 5 min, and resuspended in differentiation medium containing 50 ng / mL FGF7, 0.25 μM SANT-1 (purchased from MCE, Catalog No.: HY-100224), 1 μM retinoic acid RA (purchased from MCE, Catalog No.: HY-14649), 100 nM LDN193189 (purchased from MCE, Catalog No.: HY-12071), and 100 nM TPPB (purchased from MCE, Catalog No.: HY-12359). 6 The cells were inoculated into culture flasks and cultured for 3 days. Fresh differentiation medium containing the above factors was replaced every other day to induce the primitive gut tube cells to differentiate into pancreatic progenitor cells.

[0020] On day 9, pancreatic progenitor cells were collected, centrifuged at 400 g for 5 min, and resuspended in differentiation medium containing 0.25 μM SANT-1, 50 nM retinoic acid, 10 μM SB431542 (purchased from MCE, catalog number: HY-10431), and 1 μM T3 (purchased from MCE, catalog number: HY-A0070A) at a final concentration of 2 × 10 6 The cells were seeded into culture flasks at a concentration of 10 cells / mL and cultured for 4 days. Fresh differentiation medium containing the above factors was replaced every other day to induce pancreatic precursor cells to differentiate into pancreatic progenitor cells.

[0021] 3. Induction of pancreatic progenitor cells to differentiate into pancreatic islet cells On day 13, pancreatic progenitor cells were collected, digested into single cells with Accutase digestion solution, and transferred into 50 mL centrifuge tubes for cell counting. HUVEC cells and hADSC cells were revived and cultured, digested into single cells with TrypLE™ Select enzyme, and transferred into 50 mL centrifuge tubes for cell counting. 1×10 8 Pancreatic progenitor cells, 5×10 7 HUVEC cells and 5×10 6 hADSC cells were mixed and added to 10 mL of differentiation medium containing 100 nM LDN193189, 100 nM XXI (purchased from MCE), 10 µM SB431542, and 1 µM T3 factor at a final concentration. The culture medium was replaced every other day for 7 days. After 7 days, the medium was replaced with a differentiation medium containing 10 µM vitamin E analog (Trolox, purchased from MCE, catalog number: HY-101445), 2 µM R428 (purchased from MCE, catalog number: HY-15150), 1 mM N-acetylcysteine ​​(purchased from MCE, catalog number: HY-B0125), 10 µM SB431542, and 1 µM T3 at a final concentration of 10 µM. The culture medium was continued for 5 days to isolate and obtain pancreatic islet cells (such as Figure 2 as shown).

[0022] The differentiation medium is DMEM / F12 supplemented with 20 mmol / L BSA, 1% Vol glutamine, 0.56 g / L NaHCO3, 0.33 g / L thiamine, 0.1 g / L reduced glutathione, 3.3 g / L vitamin C, 7×10 -4 Vol% β-ME, 0.2Vol% LiCl, 0.002g / L insulin, 3g / L methylcellulose, 0.15g / L low acyl gellan gum.

[0023] Example 3 Comparative Example The steps of the control example are basically the same as those of Example 2, except that: ① The differentiation medium is different. The differentiation medium in the control example does not contain methylcellulose and low acyl gellan gum. During the entire induction differentiation process, it is placed on a shaker for shaking culture (100rpm / min, 37°C, 5% CO2) to achieve suspension culture. ② The process of inducing pancreatic progenitor cells into islet cells is different. HUVEC cells and hADSC cells are not added at this stage in the control example. The rest of the process is the same as in Example 2.

[0024] Example 4 RT-qPCR method to detect the expression of cell-specific markers at each stage The definitive endoderm cells, pancreatic progenitor cells and pancreatic islet cells prepared in Example 2 (as the experimental group) and Example 3 (as the control group) were collected, and total RNA was extracted, cDNA was synthesized, and target genes were amplified by q-PCR using conventional methods. The specific markers of cells at each stage detected included SOX17, FOXA2, PDX1, NKX6.1, INS, GCG, and SST.

[0025] Depend on Figure 3-5 It can be seen that the specific markers of endoderm cells, SOX17 and FOXA2, are highly expressed at this stage, and the relative expression levels of the experimental group are higher than those of the control group; the cell markers of pancreatic progenitor cells, PDX1 and NKX6.1, are significantly expressed at this stage, and the relative expression levels of the experimental group in the present invention are higher than those of the control group; the cell markers of islet cells, INS, GCG, and SST are significantly expressed, and the relative expression levels of the experimental group in the present invention are higher than those of the control group. This indicates that the induction rate of islet cells induced by the present invention is higher than that of the control group at each stage.

[0026] Example 5 Flow cytometry to detect the positive rate of INS in pancreatic islet cells The pancreatic islet cells induced in Example 2 (as the experimental group) and Example 3 (as the control group) were collected, digested into single cells, fixed with 4% paraformaldehyde, and the INS positive rate in the pancreatic islet cells was detected using INS flow cytometry antibody.

[0027] The results are as follows Figure 6 As shown, the INS positive rate in the pancreatic islet cells of the control group was 76.9%, and the INS positive rate in the pancreatic islet cells of the experimental group of the present invention was 89.7%, indicating that the β cell content in the pancreatic islet cells induced by the present invention was higher than that in the pancreatic islet cells induced by the control group.

[0028] Example 6 Experiment on Glucose Stimulation of Insulin Content and C-Peptide Secretion 1. Insulin secretion experiment (1) Experimental groups: the pancreatic islet cell group prepared in Example 2 (as the experimental group) and the pancreatic islet cell group prepared in Example 3 (as the control group).

[0029] (2) Cell preparation: 5×10 pancreatic islet cells were collected from the control group and the experimental group. 5 Each well was mixed with 2 mL of sugar-free KRB buffer (Merck), added to a six-well plate, and cultured in a 37°C, 5% CO2 incubator for 1 h.

[0030] (3) Centrifuge and remove the supernatant. Add 2 mL of KRB buffer containing 2 mmol / L (low glucose) or 22 mmol / L (high glucose) glucose. Continue incubation for 1 h and collect the supernatant into an EP tube.

[0031] (4) Wash the cells three times with 1× PBS, add 50 µL / well (24-well plate) of RIPA lysis buffer to fully lyse the cells, collect the lysate in a low-temperature centrifuge, centrifuge at 4°C, 12,000 rpm for 20 min, and collect the supernatant into an EP tube.

[0032] (5) The insulin content in the supernatant was detected using a human insulin ELISA kit purchased from Beijing Solebow Biotechnology Co., Ltd. The insulin content in the cell supernatant was determined using an ELISA kit. The insulin content secreted by the two groups of cells under low sugar and high sugar stimulation is shown in the following table:

[0033] 2. C-peptide secretion detection experiment According to the Mercodia ultrasensitive c-peptide ELISA kit, the amount of C-peptide secretion in the supernatant was detected: 25 μL of the standard and the cell supernatant samples of the experimental group and the control group collected above were added to the 96-well plate coated with c-peptide antibody, and three replicate wells were set for each group. The OD value was measured at a wavelength of 450 nm on the microplate reader and the results were calculated.

[0034] The C-peptide secretion in the cell supernatant of the control group and the experimental group was measured by ELISA kit. Figure 7 As shown, under high glucose stimulation, the C-peptide secretion of the control group cells was 320pmol / mg, and the C-peptide secretion of the experimental group cells was 510pmol / mg. Under low glucose stimulation, the C-peptide secretion of the control group cells was 140pmol / mg, and the C-peptide secretion of the experimental group cells was 230pmol / mg.

[0035] The results of insulin secretion and C-peptide secretion tests showed that the islet cells induced in the experimental group of the present invention secreted higher amounts of insulin and C-peptide than those in the control group.

Claims

1. A method for inducing differentiation of pancreatic islet cells from iPSCs, characterized in that: The method includes inducing iPSCs into definitive endoderm cells, inducing definitive endoderm cells into pancreatic progenitor cells, and inducing pancreatic progenitor cells to differentiate into islet cells; the method for inducing pancreatic progenitor cells to differentiate into islet cells is to mix pancreatic progenitor cells, HUVEC cells, and hADSC cells, and culture them in a differentiation medium containing LDN193189 with a final concentration of 98-102nM, XXI with a final concentration of 98-102nM, 8-12µM SB431542, and 0.8-1.2µM T3 factor, and after 7 days of culture, replace it with a differentiation medium containing a final concentration of 9-11µM vitamin E analog, 1.8-2.2µM R428, 0.9-1.1mM N-acetylcysteine, 8-12µM SB431542, and 0.9-1.1µM T3, and continue to culture for 5 days to induce islet cells.

2. The method for inducing differentiation of pancreatic islet cells from iPSCs according to claim 1, characterized in that: The initial number ratio of the pancreatic progenitor cells, HUVEC cells, and hADSC cells is 20:10:

1.

3. The method for inducing differentiation of pancreatic islet cells from iPSCs according to claim 1, characterized in that: The seeding density of pancreatic progenitor cells is (0.8-1.2)×10 7 / mL, the seeding density of the HUVEC cells is (4.8-5.2)×10 6 / mL, the seeding density of the hADSC cells is (4.8-5.2)×10 5 Pieces / mL.

4. The method for inducing differentiation of pancreatic islet cells from iPSCs according to claim 1, characterized in that: In the process of inducing the differentiation of pancreatic islet cells from iPSCs, a differentiation medium containing factors is used. The differentiation medium is DMEM / F12 supplemented with BSA with a final concentration of 18-22 mmol / L, 0.8-1.2% Vol glutamine, 0.55-0.57 g / L NaHCO3, 0.32-0.34 g / L thiamine, 0.08-0.12 g / L reduced glutathione, 3.2-3.4 g / L vitamin C, (6.8-7.2)×10 -4 Vol% β-ME, 0.18-0.22Vol% LiCl, 0.0018-0.0022g / L insulin, 2.8-3.2g / L methylcellulose, 0.14-0.16g / L low acyl gellan gum.

5. The method for inducing differentiation of pancreatic islet cells from iPSCs according to claim 1, characterized in that: The method for inducing iPSCs into definitive endoderm cells is to expand and culture iPSCs cells into cell clusters, culture them in a differentiation medium containing Activin-A with a final concentration of 98-102 ng / mL and 2.8-3.2 µM CHIR99021 for 1 day, replace the culture medium with a differentiation medium containing Activin-A with a final concentration of 98-102 ng / mL on the second day, and continue to culture for 2 days to obtain definitive endoderm cells.

6. The method for inducing differentiation of pancreatic islet cells from iPSCs according to claim 5, characterized in that: The diameter of the cell cluster is 50nm-100nm.

7. The method for inducing differentiation of pancreatic islet cells from iPSCs according to claim 1, characterized in that: The method for inducing definitive endoderm cells into pancreatic progenitor cells is to culture the definitive endoderm cells in a differentiation medium containing FGF7 with a final concentration of 48-52 ng / mL for 2 days to induce the endoderm cells to differentiate into primitive gut tube cells; then, the primitive gut tube cells are cultured in a medium containing FGF7 with a final concentration of 48-52 ng / mL, 0.24-0.26 µM SANT-1, 0.8-1.2 µM retinoic acid RA, 98-102 nM L The primitive gut tube cells were cultured in a differentiation medium containing DN193189 and 98-102 nM TPPB for 3 days to induce differentiation into pancreatic progenitor cells, and then the pancreatic progenitor cells were cultured in a differentiation medium containing a final concentration of 0.24-0.26 µM SANT-1, 48-52 nM retinoic acid, 8-12 µM SB431542, and 0.8-1.2 µM T3 for 4 days to induce differentiation of pancreatic progenitor cells into pancreatic progenitor cells.

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