Method for inducing stem cells to generate insulin-secreting cells and stem cell culture medium

Abstract

The invention provides a method and a culture medium for inducing stem cells to generate insulin-secreting cells through a specific culture medium and method. According to the method, each stage of differentiation is accurately regulated and controlled, so that the differentiation efficiency is improved, and the insulin-secreting cells with stronger functionality are obtained. The cells can be used for research and treatment of diabetes mellitus and provide good reference and application for subsequent treatment of diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for inducing stem cells to generate insulin-secreting cells and a corresponding stem cell culture medium. The culture medium provided by the present invention can effectively promote the differentiation of stem cells into insulin-secreting cells, and the generated insulin-secreting cells have good functional characteristics and can effectively meet the treatment needs of diabetes. Background Art

[0002] Diabetes is a metabolic disease characterized by chronic hyperglycemia, and insufficient insulin secretion or defective insulin action is its main cause. Existing treatments for diabetes include insulin injections and oral hypoglycemic drugs, but these methods can only relieve symptoms and cannot cure the disease. Research on the induction of insulin-secreting cells from stem cells provides new hope for the fundamental treatment of diabetes. However, current induction methods have problems such as low efficiency, long induction time, and poor function of generated cells. There is an urgent need to develop new and more effective induction methods and culture media. Summary of the invention

[0003] The purpose of the present invention is to provide a method for effectively inducing stem cells to generate insulin-secreting cells and a corresponding stem cell culture medium, thereby providing a new solution for the treatment of diabetes.

[0004] Therefore, the present invention discloses a monoclonal antibody on one hand. The monoclonal antibody is an anti-GSK3 monoclonal antibody, and the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0005] In another aspect, the present invention also discloses a method for inducing stem cells to generate insulin-secreting cells, the method comprising the following steps:

[0006] (1) Expansion and maintenance of stem cells;

[0007] (2) induction of endoderm, wherein the culture medium used in this step includes the monoclonal antibody;

[0008] (3) induction of insulin-secreting cell precursors;

[0009] (4) Maturation of insulin-secreting cells.

[0010] Preferably, the specific steps of the expansion and maintenance of stem cells in step (1) of the present invention are: after the human iPSCs are revived, they are placed in serum-free DMEM / F12 culture medium for expansion, and the medium is changed every 2 to 3 days; when the cells grow to a confluence of 80%, TrypLE enzymatic solution is used for cell passaging to ensure that the cells remain in an undifferentiated state.

[0011] Preferably, the specific steps of inducing the endoderm in step (2) of the present invention are: when the stem cells grow to a confluence of 70%, replace the culture medium containing DMEM / F12, 1% fetal bovine serum, 1% glutamine, 0.1mMβ-mercaptoethanol, 0.5μMCHIR99021, and 50ng / ml monoclonal antibody to activate the Wnt signaling pathway and induce the cells to differentiate into the endoderm; continue culturing for 3 days, replacing the culture medium every day.

[0012] Preferably, the specific steps of inducing insulin-secreting cell precursors in step (3) of the present invention are: transitioning to a new differentiation medium containing DMEM / F12, 2% fetal bovine serum, 1% glutamine, 0.1 mM β-mercaptoethanol, 100 ng / ml ActivinA and 50 ng / ml FGF10; continuing to culture for 4 days, changing the medium every day.

[0013] Preferably, the specific steps of inducing the maturation of insulin-secreting cells in step (4) of the present invention are: further transitioning to a new culture medium containing DMEM / F12, 1% fetal bovine serum, 1% glutamine, 0.1mM β-mercaptoethanol, 10ng / ml GLP-1, 10mM nicotine amide and 20ng / ml HGF; continuing to culture for 7 days, changing the culture medium every day, and obtaining insulin-secreting cells.

[0014] On the other hand, the present invention also discloses a culture medium for inducing stem cells to generate insulin-secreting cells, the culture medium comprising endoderm induction medium, insulin-secreting cell precursor induction medium, and insulin-secreting cell maturation medium, wherein the endoderm induction medium comprises the monoclonal antibody.

[0015] Preferably, the endoderm induction medium of the present invention is a medium containing DMEM / F12, 1% fetal bovine serum, 1% glutamine, 0.1 mM β-mercaptoethanol, 0.5 μM CHIR99021, and 50 ng / ml monoclonal antibody.

[0016] Preferably, the insulin-secreting cell precursor induction medium of the present invention is a medium comprising DMEM / F12, 2% fetal bovine serum, 1% glutamine, 0.1 mM β-mercaptoethanol, 100 ng / ml ActivinA and 50 ng / ml FGF10.

[0017] Preferably, the insulin-secreting cell maturation medium of the present invention is a medium comprising DMEM / F12, 1% fetal bovine serum, 1% glutamine, 0.1 mM β-mercaptoethanol, 10 ng / ml GLP-1, 10 mM nicotine amide and 20 ng / ml HGF.

[0018] The present invention provides a method for inducing stem cells to generate insulin-secreting cells by using a specific culture medium and method. The method improves differentiation efficiency and obtains more functional insulin-secreting cells by precisely regulating each stage of differentiation. The cells can induce higher titers of insulin and can quickly lower blood sugar in mice. Therefore, these cells can be used for the research and treatment of diabetes, providing a new direction and application for the treatment of diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of mouse hybridoma technology. DETAILED DESCRIPTION

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0021] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0022] Example 1: Method for inducing stem cells to generate insulin-secreting cells

[0023] 1.1 Materials

[0024] (1) Stem cells: human iPSCs (derived from umbilical cord blood) (Jinzhun Biopharmaceuticals, catalog number: 907005-CBP35)

[0025] (2) Culture medium: Basic culture medium: DMEM / F12, fetal bovine serum (FBS) (GIBCO), glutamine, glucose, sodium pyruvate (Sinopharm, etc.).

[0026] (3) Growth factors and small molecule compounds: basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor (IGF-1), CHIR99021 (GSK3 inhibitor), Retinoic Acid (vitamin A acid), Noggin (BMP inhibitor), Activin A, FGF10, GLP-1, nicotine amide, hepatocyte growth factor (HGF), etc. are all commercial reagents (MCE, etc.).

[0027] 1.2 Experimental procedures

[0028] (1) Expansion and maintenance of stem cells: After human iPSCs were revived, they were placed in serum-free DMEM / F12 medium for expansion, and the medium was changed every 2 to 3 days. When the cells grew to a confluence of 80%, TrypLE enzymatic solution was used for cell passage to ensure that the cells remained in an undifferentiated state.

[0029] (2) Induction of endoderm: When the stem cells grow to a confluence of 70%, replace the culture medium containing DMEM / F12, 1% fetal bovine serum, 1% glutamine, 0.1 mM β-mercaptoethanol, and 3 μM CHIR99021 to activate the Wnt signaling pathway and induce the cells to differentiate into endoderm; continue culturing for 3 days, changing the culture medium every day.

[0030] (3) Induction of insulin-secreting cell precursors: Transition to a new differentiation medium containing DMEM / F12, 2% fetal bovine serum, 1% glutamine, 0.1 mM β-mercaptoethanol, 100 ng / ml Activin A and 50 ng / ml FGF10; continue culturing for 4 days, changing the medium every day.

[0031] (4) Maturation of insulin-secreting cells: Further transition to a new culture medium containing DMEM / F12, 1% fetal bovine serum, 1% glutamine, 0.1 mM β-mercaptoethanol, 10 ng / ml GLP-1, 10 mM nicotine amide, and 20 ng / ml HGF; continue culturing for 7 days, changing the culture medium every day; during this period, observe cell morphological changes every day. After the culture is completed, the cells are expanded or tested or frozen or used directly.

[0032] Example 2: Method for inducing stem cells to generate insulin-secreting cells

[0033] In the process of preparing cells in Example 1, it was found that the addition of 3 μM CHIR99021 had an effect on the growth of cells. Therefore, we screened a monoclonal antibody against GSK3, which was produced by conventional mouse hybridoma technology (e.g. Figure 1 ). Through sequencing and analysis, the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively. The activity of the monoclonal antibody was detected by ELISA, and its dilution could reach 10 7 , indicating that the monoclonal antibody has good activity.

[0034] Next, the step (2) of "1.2 Experimental Steps" in Example 1 is adjusted as follows: when the stem cells grow to a confluence of 70%, replace the culture medium containing DMEM / F12, 1% fetal bovine serum, 1% glutamine, 0.1mM β-mercaptoethanol, 0.5μM CHIR99021, and 50ng / ml monoclonal antibody to activate the Wnt signaling pathway and induce the cells to differentiate into the endoderm; continue to culture for 3 days, and replace the culture medium every day. The other steps are completely consistent with Example 1 until the insulin-secreting cells mature. After the culture is completed, the cells are expanded or tested or frozen or directly used.

[0035] By increasing the amount of monoclonal antibody and reducing the amount of CHIR99021 used, the cell culture resumed normal growth. Therefore, the monoclonal antibody can reduce the effect of CHIR99021 on cell culture.

[0036] Example 3: Functional detection of cells

[0037] 3.1 Collect the cultured insulin-secreting cells and test the functionality of the cells by insulin release experiment. Detect the amount of insulin released under glucose stimulation and quantify it by enzyme-linked immunosorbent assay (ELISA). At the same time, detect the secretion of C-peptide to confirm the maturity of the cells. The specific method is as follows:

[0038] (1) Materials and reagents

[0039] Krebs-Ringer buffer (KRB): 115 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.16 mM MgSO4, 25 mM NaHCO3, 2.5 mM CaCl2, 10 mM HEPES (pH 7.4).

[0040] Low glucose KRB buffer: contains 2.8 mM glucose. High glucose KRB buffer: contains 16.7 mM glucose. Insulin ELISA kit (Wuhan Saipei Biotechnology Co., Ltd.). C-peptide ELISA kit (Wuhan Aidikang Biotechnology Co., Ltd.).

[0041] (2) Experimental steps:

[0042] ① Cell preparation: The differentiated insulin-secreting cells (prepared in Example 1 and Example 2) were collected from the culture flask, and the cells were rinsed three times with KRB buffer to remove the residual culture medium components; the cells were resuspended in low glucose KRB buffer and incubated at 37° C. for 1 hour to stabilize the cells.

[0043] ② Low glucose stimulation: Dispense the cells into a 96-well plate and add an equal amount of cell suspension to each well. Add low glucose KRB buffer to keep the volume of each well consistent. Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 1 hour. Collect the supernatant from each well, label and store at -20°C for subsequent insulin determination.

[0044] ③High glucose stimulation: After collecting the supernatant after low glucose stimulation, add high glucose KRB buffer to each well. Place the 96-well plate back in the incubator and incubate for 1 hour. After incubation, collect the supernatant from each well, label and store at -20°C.

[0045] ④ Insulin and C-peptide detection: Use insulin ELISA kit and C-peptide ELISA kit to prepare standard curve and samples according to the instructions of the kit. Perform ELISA test on the collected supernatant according to the steps in the kit instructions to determine the insulin concentration in the supernatant. Use an ELISA reader to read the OD value and calculate the insulin concentration in the sample according to the standard curve.

[0046] 3.2 Test results

[0047] (1) Significant changes in insulin release: Under high glucose conditions, the amount of insulin released increased significantly. This indicates that both groups of cells have a strong ability to respond to high glucose stimulation, and the cells prepared in Example 2 have a better effect. As shown in Table 1.

[0048] (2) Significant changes in C-peptide release: C-peptide release also increased significantly under high glucose conditions, about 4 times that under low glucose conditions. The secretion of C-peptide reflects the production of endogenous insulin in cells, which is consistent with the results of insulin detection. As shown in Table 1.

[0049] Table 1 Insulin and C-peptide test results

[0050]

[0051] Example 4: Application of cells

[0052] (1) Animal preparation: 30 healthy NOD / SCID mice or immunodeficient mice were selected and randomly divided into 3 groups, each with 10 mice, one group as the control group (injected with PBS), one group as the experimental group 1 (injected with the cells prepared in Example 1), and one group as the experimental group 2 (injected with the cells prepared in Example 2). Adaptive feeding was performed for one week. The mice were fasted for 6 hours, but provided with drinking water.

[0053] (2) Cell preparation: The differentiated insulin-secreting cells were collected from the culture flask (prepared in Example 1 and Example 2), and the cells were rinsed with PBS or a suitable culture medium. The cells were suspended in an appropriate amount of PBS or culture medium to ensure that the number of cells injected into each mouse was consistent (1×10^6 cells were injected into each mouse).

[0054] (3) Cell transplantation: Anesthetize mice with isoflurane or an appropriate anesthetic. Inject the cell suspension under the renal capsule or subcutaneously of the mice under sterile conditions. Observe the mice to ensure that they recover from anesthesia.

[0055] (4) Blood glucose monitoring: The fasting blood glucose levels of mice were measured before transplantation and 1 day, 3 days, 7 days, 14 days, 21 days and 28 days after transplantation.

[0056] (5) Insulin and C-peptide detection: Blood samples were collected from mice at 1 week and 4 weeks after transplantation, and the insulin and C-peptide concentrations in the serum were detected using ELISA kits.

[0057] (6) Test results

[0058] ① Blood sugar test results: The blood sugar levels of mice in experimental group 1 and experimental group 2 gradually decreased after transplantation, and the rate of decrease in experimental group 2 was faster. However, the blood sugar levels of mice in the control group did not change significantly. This indicates that the transplanted insulin-secreting cells can effectively regulate blood sugar. As shown in Table 2.

[0059] Table 2 Blood glucose test results (mmol / L)

[0060] Collection time Control group Experimental Group 1 Experimental Group 2 Before transplantation 22.5±1.2 22.3±1.3 22.1±1.1 1 day after transplantation 21.8±1.1 20.5±1.2 18.6±0.9 3 days after transplantation 21.5±1.2 18.5±1.1 15.8±1.0 7 days after transplantation 21.0±1.0 15.2±1.0 11.1±1.0 14 days after transplantation 20.8±1.3 10.5±1.2 8.3±0.8 21 days after transplantation 20.5±1.2 7.8±0.9 5.6±0.8 28 days after transplantation 20.2±1.4 5.5±0.8 4.3±0.5

[0061] ② Serum insulin and C-peptide test results: The serum insulin and C-peptide levels of mice in experimental group 1 and experimental group 2 increased significantly after transplantation, indicating that the transplanted cells not only survived, but also had the ability to continuously secrete insulin and C-peptide. And the effect of experimental group 2 was better than that of experimental group 1! As shown in Table 3.

[0062] Table 3 Serum insulin and C-peptide test results

[0063]

[0064] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A monoclonal antibody, characterized in that The monoclonal antibody is an anti-GSK3 monoclonal antibody, and the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

2. A method for inducing stem cells to generate insulin-secreting cells, characterized in that: The method comprises the following steps: (1) Expansion and maintenance of stem cells; (2) induction of endoderm, wherein the culture medium used in this step comprises the monoclonal antibody according to claim 1; (3) induction of insulin-secreting cell precursors; (4) Maturation of insulin-secreting cells.

3. The method according to claim 2, characterized in that The specific steps of the expansion and maintenance of stem cells in step (1) are as follows: after the human iPSCs are revived, they are placed in serum-free DMEM / F12 culture medium for expansion, and the medium is changed every 2 to 3 days; when the cells grow to a confluence of 80%, TrypLE enzymatic solution is used for cell passaging to ensure that the cells remain in an undifferentiated state.

4. The method according to claim 2, characterized in that: The specific steps of inducing the endoderm in step (2) are as follows: when the stem cells grow to a confluence of 70%, replace the culture medium containing DMEM / F12, 1% fetal bovine serum, 1% glutamine, 0.1mM β-mercaptoethanol, 0.5μM CHIR99021, and 50ng / ml monoclonal antibody to activate the Wnt signaling pathway and induce the cells to differentiate into the endoderm; continue culturing for 3 days, replacing the culture medium every day.

5. The method according to claim 2, characterized in that: The specific steps of inducing insulin-secreting cell precursors in step (3) are: transitioning to a new differentiation medium containing DMEM / F12, 2% fetal bovine serum, 1% glutamine, 0.1 mM β-mercaptoethanol, 100 ng / ml ActivinA and 50 ng / ml FGF10; continuing to culture for 4 days, changing the medium every day.

6. The method according to claim 2, characterized in that The specific steps of inducing the maturation of insulin-secreting cells in step (4) are: further transitioning to a new culture medium containing DMEM / F12, 1% fetal bovine serum, 1% glutamine, 0.1mM β-mercaptoethanol, 10ng / ml GLP-1, 10mM nicotine amide and 20ng / ml HGF; continuing to culture for 7 days, changing the culture medium every day, and obtaining insulin-secreting cells.

7. A culture medium for inducing stem cells to generate insulin-secreting cells, characterized in that: The culture medium comprises an endoderm induction medium, an insulin-secreting cell precursor induction medium, and an insulin-secreting cell maturation medium, wherein the endoderm induction medium comprises the monoclonal antibody according to claim 1.

8. The culture medium according to claim 7, characterized in that The endoderm induction medium is a medium containing DMEM / F12, 1% fetal bovine serum, 1% glutamine, 0.1 mM β-mercaptoethanol, 0.5 μM CHIR99021, and 50 ng / ml monoclonal antibody.

9. The culture medium according to claim 7, characterized in that The insulin secreting cell precursor induction medium is a medium containing DMEM / F12, 2% fetal bovine serum, 1% glutamine, 0.1 mM β-mercaptoethanol, 100 ng / ml Activin A and 50 ng / ml FGF10.

10. The culture medium according to claim 7, characterized in that The insulin secreting cell maturation medium is a medium containing DMEM / F12, 1% fetal bovine serum, 1% glutamine, 0.1 mM β-mercaptoethanol, 10 ng / ml GLP-1, 10 mM nicotine amide and 20 ng / ml HGF.

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