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

Abstract

The present invention provides a method and culture medium for inducing the generation of insulin-secreting cells from stem cells using a specific culture medium and method. This method precisely regulates each stage of differentiation, improving differentiation efficiency and yielding more functional insulin-secreting cells. These cells can be used in diabetes research and treatment, providing a valuable reference and application for subsequent diabetes treatments.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, specifically 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. The resulting insulin-secreting cells have excellent functional properties and can effectively address the treatment needs of diabetes. Background Art

[0002] Diabetes is a metabolic disease characterized by chronic hyperglycemia, with insufficient insulin secretion or defective insulin action as 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 inducing the generation of insulin-secreting cells from stem cells offers new hope for a fundamental treatment of diabetes. However, current induction methods have problems such as low efficiency, long induction time, and poor function of the 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, in one aspect, the present invention discloses a monoclonal antibody, which 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 further 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 expanding and maintaining stem cells in step (1) of the present invention are: after resuscitation of human iPSCs, 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 passage 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%, replacing 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; continuing the culture 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 Activin A and 50 ng / ml FGF10; continuing to culture for 4 days, and 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, 0.1 mM β-mercaptoethanol, 10 ng / ml GLP-1, 10 mM nicotine amide and 20 ng / ml HGF; continuing to culture for 7 days, changing the culture medium every day, to obtain 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, wherein the culture medium includes endoderm induction medium, insulin-secreting cell precursor induction medium, and insulin-secreting cell maturation medium, wherein the endoderm induction medium includes 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 Activin A 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 the generation of insulin-secreting cells from stem cells using a specific culture medium and method. By precisely controlling each stage of differentiation, this method improves differentiation efficiency and produces more functional insulin-secreting cells. These cells can induce higher titers of insulin and rapidly lower blood sugar in mice. Therefore, these cells can be used for diabetes research and treatment, providing new directions and applications for diabetes treatment. 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 commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated 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 steps

[0028] (1) Stem cell expansion and maintenance: 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 80% confluence, 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 70% confluence, 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 cell differentiation 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: The cells were further transferred 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. The culture was continued for 7 days, with the culture medium changed daily. During this period, the cell morphology was observed daily. After the culture was completed, the cells were expanded, tested, frozen, or used directly.

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

[0033] During the cell preparation process in Example 1, it was found that the addition of 3 μM CHIR99021 had an effect on cell growth. Therefore, we screened a monoclonal antibody against GSK3, which was generated using conventional mouse hybridoma technology (e.g. Figure 1 ) was prepared. Through sequencing and analysis, the amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody were 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, step (2) of "1.2 Experimental Procedures" in Example 1 was adjusted as follows: When the stem cells grew to 70% confluence, the culture medium was replaced with DMEM / F12, 1% fetal bovine serum, 1% glutamine, 0.1 mM β-mercaptoethanol, 0.5 μM CHIR99021, and 50 ng / ml monoclonal antibody to activate the Wnt signaling pathway and induce cell differentiation into endoderm; the culture was continued for 3 days, with the culture medium replaced daily. The other steps were identical to those in Example 1 until the insulin-secreting cells matured. After the culture was completed, the cells were expanded, tested, frozen, or used directly.

[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 impact of CHIR99021 on cell culture.

[0036] Example 3: Functional detection of cells

[0037] 3.1 Collect the cultured insulin-secreting cells and test their functionality through an insulin release assay. Measure insulin release under glucose stimulation and quantify it using an enzyme-linked immunosorbent assay (ELISA). Simultaneously, test C-peptide secretion to confirm cell maturity. 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 Examples 1 and 2) were collected from the culture flask and rinsed three times with KRB buffer to remove 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: Aliquot the cells into a 96-well plate and add an equal amount of cell suspension to each well. Add low-glucose KRB buffer to maintain a consistent volume per well. 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] ③ High Glucose Stimulation: After collecting the supernatant from the low glucose stimulation, add high glucose KRB buffer to each well. Return the 96-well plate to the incubator and incubate for 1 hour. After incubation, collect the supernatant from each well, label it, and store it at -20°C.

[0045] ④ Insulin and C-peptide Detection: Use insulin ELISA kits and C-peptide ELISA kits, prepare standard curves and samples according to the kit instructions. Perform ELISA on the collected supernatant according to the kit instructions to determine the insulin concentration in the supernatant. Read the OD value using a microplate reader and calculate the insulin concentration in the sample based on the standard curve.

[0046] 3.2 Test results

[0047] (1) Significant changes in insulin release: Under high glucose conditions, insulin release increased significantly. This indicates that both groups of cells have a strong response 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, approximately four times that under low glucose conditions. C-peptide secretion reflects the production of endogenous insulin in cells, which is consistent with the results of insulin testing. (See Table 1.)

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

[0050]

[0051] Example 4: Application of cells

[0052] (1) Animal Preparation: Thirty healthy NOD / SCID mice or immunodeficient mice were randomly divided into three groups, each containing 10 mice: a control group (injected with PBS), an experimental group 1 (injected with the cells prepared in Example 1), and an 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: Collect differentiated insulin-secreting cells from the culture flasks (prepared in Examples 1 and 2) and rinse the cells with PBS or a suitable culture medium. Resuspend the cells in an appropriate amount of PBS or culture medium to ensure that the number of cells injected into each mouse is consistent (1 × 10^6 cells per mouse).

[0054] (3) Cell transplantation: Anesthetize mice with isoflurane or an appropriate anesthetic. Under sterile conditions, inject the cell suspension into the subrenal capsule or subcutaneous region of the mouse. Observe the mouse to ensure it recovers from anesthesia.

[0055] (4) Blood glucose monitoring: The fasting blood glucose levels of mice were measured before transplantation and on day 1, 3, 7, 14, 21, and 28 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 concentrations of insulin and C-peptide in serum were detected using ELISA kits.

[0057] (6) Test results

[0058] ① Blood glucose test results: Blood glucose levels in mice in experimental groups 1 and 2 gradually decreased after transplantation, with the rate of decrease being faster in experimental group 2. However, blood glucose levels in mice in the control group remained unchanged. This indicates that the transplanted insulin-secreting cells are able to effectively regulate blood glucose. (See 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: Serum insulin and C-peptide levels in mice in Experimental Groups 1 and 2 increased significantly after transplantation, indicating that the transplanted cells not only survived but also maintained the ability to continuously secrete insulin and C-peptide. Furthermore, Experimental Group 2 showed a significantly better effect than 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 considered as equivalent replacement methods and are included in the scope of protection 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) Stem cell expansion and maintenance: After human iPSCs are revived, they are placed in serum-free DMEM / F12 medium for expansion, and the medium is changed every 2 to 3 days. When the cells grow to 80% confluence, TrypLE enzymatic solution is used for cell passage to ensure that the cells remain in an undifferentiated state. (2) Induction of endoderm: When the stem cells grow to 70% confluence, replace the culture medium with DMEM / F12, 1% fetal bovine serum, 1% glutamine, 0.1 mM β-mercaptoethanol, 0.5 μM CHIR99021, and 50 ng / ml of the monoclonal antibody of claim 1 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; (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 daily; (4) Maturation of insulin-secreting cells: Further transition to a new culture medium containing DMEM / F12, 1% fetal bovine serum, 1% glutamine, 0.1mM β-mercaptoethanol, 10ng / ml GLP-1, 10mM nicotineamide, and 20ng / ml HGF; continue culturing for 7 days, changing the culture medium every day, and insulin-secreting cells are obtained.

3. 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; 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; 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; 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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