Method for inducing directional differentiation of pluripotent stem cells (iPSCs) into pancreatic beta cells

Abstract

The invention relates to a method for directionally differentiating induced pluripotent stem cells (iPSCs) into functional pancreas islet beta cells. According to the method, the iPSCs are induced into pancreatic endoderm precursor cells through activation of Activin A and Wnt signal channels; the endoderm cells are processed by growth factors (such as FGF10 and RA) and are further differentiated into pancreatic precursor cells for expressing key pancreatic markers (such as Pdx1); subsequently, the pancreatic precursor cells are gradually matured to functional pancreatic beta cells by regulating specific transcription factors (such as Ngn3, MafA and Nkx6.1) and using a combination of compounds (such as Nicotinamide and Exendin-4). The obtained pancreas islet beta cell can secrete insulin in a high-glucose environment, and has a function similar to that of a natural pancreas islet beta cell; in addition, these differentiated cells exhibit a blood glucose regulation ability to diabetic mice in an in vivo transplantation experiment. According to the invention, efficient and stable generation of pancreatic beta cells is realized, and a potential cell replacement therapy is provided for diabetics.

Description

Technical Field

[0001] The present invention belongs to the technical field of somatic cell reprogramming and relates to a method for directing differentiation of induced pluripotent stem cells into pancreatic beta cells. Background Art

[0002] The technical background of induced pluripotent stem cells (iPSCs) directed differentiation into pancreatic β cells involves multiple fields such as stem cell biology, developmental biology and diabetes treatment. iPSCs are a type of stem cell that induces somatic cells into a pluripotent state through gene reprogramming technology, and have the potential to differentiate into almost all cell types in the body. The pancreatic β cell differentiation technology based on iPSCs aims to provide cell replacement therapy for diabetes, especially type 1 diabetes patients, and provide a new model for drug screening and diabetes mechanism research.

[0003] Pancreatic beta cells are cells located in the pancreas. Their main function is to synthesize and secrete insulin to regulate blood sugar levels. Diabetes is a metabolic disease caused by insufficient insulin secretion or cell insensitivity to insulin. Among them, patients with type 1 diabetes lack sufficient insulin in their bodies and their blood sugar levels are out of control because their immune system attacks pancreatic beta cells. Therefore, the generation and transplantation of replacement pancreatic beta cells has become an important research direction for the treatment of diabetes.

[0004] In 2006, Shinya Yamanaka and his team successfully reprogrammed somatic cells into iPSCs for the first time by introducing specific transcription factors (such as Oct4, Sox2, Klf4, and c-Myc) into somatic cells. This breakthrough allowed researchers to obtain stem cells from individuals themselves, solving ethical issues related to embryonic stem cells and providing the potential for customized cell therapy. iPSCs can differentiate into a variety of cell types, including neurons, cardiomyocytes, and pancreatic beta cells, which has promoted the rapid development of regenerative medicine. Summary of the invention

[0005] The present invention aims to provide a method for directing the differentiation of induced pluripotent stem cells (iPSCs) into functional pancreatic β cells. The method simulates the pancreatic development path during embryonic development through a series of optimized differentiation steps to achieve efficient conversion of iPSCs into pancreatic β cells. The present invention provides a way to generate a large number of functional pancreatic β cells in vitro, which can be used for cell replacement therapy, drug screening and the establishment of disease models for diabetes.

[0006] Experimental materials and reagents

[0007] 1. Cells:

[0008] Human induced pluripotent stem cells (hiPSCs)

[0009] 2. Culture medium:

[0010] iPSCs culture medium (mTeSR1 or StemFit)

[0011] Endoderm Differentiation Medium (with Activin A)

[0012] Pancreatic progenitor differentiation medium (with Noggin, Retinoic Acid, etc.)

[0013] Pancreatic β cell maturation medium (containing factors such as IGF-1, GLP-1, etc. that promote pancreatic β cell maturation)

[0014] 3. Reagents:

[0015] Activin A

[0016] FGF7 (Fibroblast Growth Factor 7)

[0017] BMP4 (bone morphogenetic protein 4)

[0018] Noggin

[0019] Retinoic Acid

[0020] IGF-1 (Insulin-like Growth Factor 1)

[0021] GLP-1 (Glucagon-like peptide 1)

[0022] Forskolin (adenylate cyclase activator)

[0023] 4. Equipment:

[0024] CO 2 Incubator

[0025] Cell culture plates (6-well plates, 24-well plates, etc.)

[0026] Inverted microscope

[0027] Flow cytometry

[0028] Insulin ELISA Kit

[0029] IV. Experimental Procedure

[0030] 1. Experimental Background

[0031] This experiment aims to differentiate human induced pluripotent stem cells (hiPSCs) into functional pancreatic β cells through a multi-step differentiation process. The experimental process is divided into four main stages: iPSCs expansion, endoderm differentiation, pancreatic precursor differentiation, and pancreatic β cell maturation. Each stage of the experiment requires the use of specific culture media and differentiation factors, and the differentiation efficiency is verified by detecting corresponding markers.

[0032] 2. Experimental Procedure

[0033] Step 1: Expansion and maintenance of iPSCs

[0034] (1) Culture medium preparation

[0035] Use mTeSR1 or StemFit medium and add appropriate amounts of cytokines, antibiotics and other necessary components before use.

[0036] (2) Cell inoculation

[0037] hiPSCs were seeded in 6-well plates coated with Matrigel at a density of 2-3 × 10 4 cells / well.

[0038] (3) Cell maintenance

[0039] The cells were placed at 37°C and 5% CO 2 Culture in an incubator and observe the cell status regularly. Replace the culture medium every 2 days until the cell confluence reaches 70%-80%.

[0040] (4) Subculture

[0041] When the cells reach about 80% fusion, use enzyme-free digestion solution (such as EDTA) to digest and subculture the cells to ensure uniform cell proliferation. Subculture once every 3-4 days to ensure the health of the cells.

[0042] (5) Cell monitoring

[0043] Using an inverted microscope to observe cell morphology, healthy iPSCs showed a typical morphology of tight clusters and clear edges.

[0044] Step 2: Endoderm differentiation

[0045] (1) Preparation of endoderm induction medium

[0046] The endoderm induction medium was prepared. The basic medium was RPMI 1640, Activin A (100 ng / mL) and 1% B27 supplement were added, and a small amount of L-glutamine and antibiotics were added when necessary.

[0047] (2) Adjustment of culture conditions

[0048] iPSCs at 70-80% confluence were switched to endoderm induction medium. The medium was changed daily and incubated at 37°C, 5% CO 2 Continue to culture for 3-4 days.

[0049] (3) Marker detection

[0050] On day 4, the expression of endoderm cell markers Sox17 and FoxA2 was detected by immunofluorescence, indicating successful induction to the endoderm stage.

[0051] (4) Cell morphology observation

[0052] Endoderm cells should exhibit a more dispersed, flattened morphology and gradually lose the tight clustering morphology typical of iPSCs.

[0053] Step 3: Pancreatic precursor differentiation

[0054] (1) Preparation of pancreatic progenitor differentiation medium

[0055] The basal culture medium was DMEM / F12 supplemented with Noggin (50 ng / mL), Retinoic Acid (1 μM), FGF7 (50 ng / mL), 1% B27 supplement, and antibiotics.

[0056] (2) Cell metastasis

[0057] The cells differentiated into endoderm were replaced with pancreatic precursor differentiation medium and cultured for 4-6 days, with the medium being replaced every two days.

[0058] (3) Marker detection

[0059] On the 4th day of differentiation, the expressions of pancreatic progenitor cell-specific markers Pdx1 and Nkx6.1 were detected by immunofluorescence or flow cytometry.

[0060] (4) Cell morphology observation

[0061] Pancreatic progenitor cells will show polygonal, tightly connected morphology, and some cells will be clustered. At this point, the cells should have the potential to differentiate into pancreatic islet cells.

[0062] Step 4: Pancreatic β-cell maturation

[0063] (1) Preparation of pancreatic β-cell maturation culture medium

[0064] A basic culture medium (such as RPMI 1640) is used, and factors that promote pancreatic β cell maturation are added: IGF-1 (10 ng / mL), GLP-1 (10 nM), Forskolin (10 μM), as well as 1% B27 supplement and antibiotics.

[0065] (2) Induction of pancreatic β-cell maturation

[0066] The pancreatic precursor cells were transferred to pancreatic β-cell maturation medium and cultured for 10-14 days, with the medium changed every two days.

[0067] (3) Marker detection

[0068] On days 10 and 14, the expressions of β-cell-specific markers such as insulin, Pdx1, and MafA were detected by immunofluorescence to confirm the successful generation of pancreatic β cells.

[0069] (4) Insulin secretion function test

[0070] Prepare insulin detection solution: Use an insulin ELISA kit to prepare glucose solutions of different concentrations (2.8 mM and 16.7 mM) for detecting the insulin secretion function of cells.

[0071] Detection steps: The mature pancreatic β cells were incubated in low (2.8 mM) and high (16.7 mM) concentrations of glucose for 1 hour, the supernatant was collected and the insulin secretion was detected using an ELISA kit.

[0072] Result analysis: Normally functioning pancreatic β cells should significantly increase insulin secretion in a high glucose concentration environment.

[0073] Step 5: Data analysis and result recording

[0074] (1) Analysis of marker expression

[0075] Cells at each stage were analyzed by immunofluorescence, qPCR or flow cytometry to record the expression levels of markers for endoderm (Sox17, FoxA2), pancreatic precursors (Pdx1, Nkx6.1) and pancreatic β cells (Insulin, MafA, Pdx1).

[0076] (2) Analysis of insulin secretion function

[0077] The ELISA test results were plotted to compare the insulin secretion levels under different glucose concentrations. A significant increase in insulin secretion under high glucose conditions indicated that the cells were functional.

[0078] Note:

[0079] Quality of culture medium and aseptic operation: The culture medium must be freshly prepared for each experiment to avoid contamination and ensure the success rate of the differentiation process.

[0080] Monitor cell status regularly: Replace culture medium in time to avoid excessive fusion or detachment of cells, which may affect differentiation efficiency.

[0081] Determination of experimental endpoint: The endpoint of each differentiation stage should be confirmed by marker detection to ensure differentiation efficiency and cell quality.

[0082] Through these steps, functional pancreatic β cells can be obtained, which are suitable for further diabetes research or therapeutic applications.

[0083] The following is a detailed table of the culture media and their components required for each stage of the process of directed differentiation of induced pluripotent stem cells (iPSCs) into pancreatic β cells:

[0084]

[0085] illustrate:

[0086] L-Glutamine: Provides nitrogen source for cells and supports protein and nucleic acid synthesis.

[0087] B27 supplements: Contains multiple vitamins, antioxidants and hormones to support cell growth and differentiation.

[0088] Activin A, Noggin, Retinoic Acid and FGF7: These growth factors and differentiation factors are used to promote the different stages of differentiation.

[0089] IGF-1, GLP-1 and Forskolin: used to enhance the maturation and functionality of pancreatic β cells.

[0090] By formulating the culture medium with the components provided in the above table, the directed differentiation of cells can be promoted according to the requirements of each stage, and functional pancreatic β cells can be finally obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 It is the early stage of differentiation of pluripotent stem cells in the S1 stage;

[0092] Figure 2 The S2 stage is the definitive endoderm stage;

[0093] Figure 3 It is the primitive intestinal stage of S3;

[0094] Figure 4 It is the S4 stage pancreatic progenitor cell stage 2;

[0095] Figure 5 It is the S5 endocrine progenitor cell stage;

[0096] Figure 6 These are pancreatic β cells differentiated at the S6 stage. DETAILED DESCRIPTION

[0097] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0098] The present application is described in detail below in conjunction with the accompanying drawings and specific embodiments. The following embodiments are only used to illustrate the present application and are not used to limit the scope of the present application.

[0099] Example 1

[0100] Research purpose: To develop a method to efficiently differentiate human iPSCs into functional pancreatic β cells for potential cell replacement therapy for diabetes.

[0101] Experimental process:

[0102] Phase I: Endoderm Induction

[0103] Human iPSCs were induced to differentiate into pancreatic endoderm cells using signaling pathway regulators such as Wnt and Activin A. This is the initial source of pancreatic progenitor cells and is crucial.

[0104] Phase II: Formation of pancreatic precursor cells

[0105] Next, by regulating pancreas-related signaling pathways (such as FGF and RA signaling pathways), endoderm cells are further induced into pancreatic progenitor cells, which express key pancreatic developmental genes, such as Pdx1.

[0106] Phase III: β-cell maturation

[0107] Using a specific combination of growth factors, such as Nicotinamide, Exendin-4, etc., these precursor cells are further promoted to differentiate into functional β cells. These cells not only express β cell markers (such as Insulin and C-peptide), but also can secrete insulin under high glucose conditions.

[0108] Reagents

[0109] stage Reagents Differentiation of iPSCs into endoderm Activin A,Wnt3a Endoderm differentiation into pancreatic precursors FGF10,RA Pancreatic precursor maturation Nicotinamide,Exendin-4,DAPT,Pdx1,Ngn3 Functional β-cell maturation Insulin, MafA, Glucose stimulation (high sugar stimulation)

[0110] Experimental results:

[0111] In vitro, these beta cells were able to regulate insulin secretion according to glucose concentration, demonstrating similar functions to natural beta cells. In addition, after being transplanted into mice, these cells could improve the blood sugar levels of mice, indicating that they have potential therapeutic effects.

[0112] Contribution: This study demonstrated for the first time that iPSCs were successfully converted into functional β cells through a step-by-step approach, marking an important progress in the application of iPSCs in diabetic cell therapy.

[0113] Example 2

[0114] Research purpose: To explore the possibility of using small molecule compounds to efficiently induce the directed differentiation of iPSCs into functional pancreatic β cells.

[0115] Experimental process:

[0116] Phase I: Endoderm Induction

[0117] By using a combination of Wnt signaling activators and Activin A, iPSCs were first directed to become pancreatic endoderm cells.

[0118] Phase II: Differentiation of pancreatic endoderm into pancreatic precursors

[0119] The researchers used signaling molecules such as RA (retinoic acid) and FGF10 to induce the formation of pancreatic precursors and ensured the effectiveness of differentiation by detecting key markers of pancreatic development, such as Pdx1 and Sox9.

[0120] Phase III: Differentiation and maturation of β cells

[0121] In the final step, the researchers used small molecule compounds (such as GSK3 inhibitors and TGF-β inhibitors) and some key β cell development factors (such as Ngn3, MafA and Pdx1) to further induce the precursor cells into pancreatic β cells. Mature β cells express insulin genes and can secrete insulin in a high glucose environment.

[0122] Reagents:

[0123]

[0124]

[0125] Experimental results:

[0126] The obtained pancreatic β cells were able to secrete insulin in vitro, and after being transplanted into a diabetic mouse model, they successfully restored some blood sugar regulation function.

[0127] Contribution: Through precise regulation of small molecule compounds, the research team simplified the steps of β-cell differentiation and improved the efficiency, making this process promising for large-scale production of functional β-cells in the future.

[0128] Example 3

[0129] Research purpose: To establish an efficient and stable differentiation system to induce human iPSCs into functional β cells for use in diabetes treatment research.

[0130] Experimental process:

[0131] Stage 1: Pancreatic endoderm induction

[0132] Using growth factors such as Activin A and Wnt signaling regulators, iPSCs were differentiated into cells expressing pancreatic endoderm markers such as FoxA2 and Sox17.

[0133] Phase II: Formation of pancreatic precursor cells

[0134] Through the synergistic action of FGF and RA signaling pathways, the formation of pancreatic progenitor cells is induced, and these cells express key pancreatic developmental genes, including Pdx1 and Nkx6.1.

[0135] Phase III: β-cell maturation

[0136] In the final differentiation step, nicotineamide and a variety of small molecule drugs were used to promote the differentiation of these precursor cells into mature beta cells. Beta cells can effectively secrete insulin under high glucose conditions and show the ability to regulate blood sugar in in vivo experiments.

[0137] Reagents:

[0138] stage Reagents Differentiation of iPSCs into endoderm Activin A,Wnt3a,CHIR99021 Endoderm differentiation into pancreatic precursors FGF7,KGF,RA Pancreatic precursor maturation EGF, DAPT, Pdx1, Ngn3, NKX6.1 Functional β-cell maturation Insulin, MafA, Glucose stimulation (high sugar stimulation)

[0139] Experimental results:

[0140] The study showed that the beta cells generated by this step not only had good insulin secretion function in vitro, but also could effectively improve blood sugar levels in a diabetic mouse model, which proved that the beta cells differentiated from iPSCs have the potential to replace natural pancreatic islet cells in vivo.

[0141] Contribution: Methods Through an optimized differentiation process and a specific small molecule drug combination, the goal of large-scale generation of functional β cells was successfully achieved, laying the foundation for the development of cell therapy for diabetes.

Claims

1. A method for directing differentiation of induced pluripotent stem cells (iPSCs) into pancreatic β cells, characterized in that: The following steps are involved: a) preliminarily inducing differentiation of iPSCs under culture conditions containing an endoderm differentiation medium to obtain endoderm cells; b) further inducing differentiation of the endoderm cells under culture conditions containing a pancreatic precursor differentiation medium to obtain pancreatic precursor cells; c) finally inducing the pancreatic precursor cells under culture conditions containing a pancreatic β cell maturation medium to obtain mature pancreatic β cells.

2. A method for directed differentiation of induced pluripotent stem cells (iPSCs) into pancreatic β cells according to claim 1, characterized in that: The germ layer endoderm differentiation medium contains Activin A at a concentration of about 50 to 100 ng / mL.

3. A method for directed differentiation of induced pluripotent stem cells (iPSCs) into pancreatic β cells according to claim 1, characterized in that: The pancreatic precursor differentiation culture medium contains Noggin, Retinoic Acid and FGF7, and is used to induce the generation of pancreatic precursor cells.

4. A method for directed differentiation of induced pluripotent stem cells (iPSCs) into pancreatic β cells according to claim 1, characterized in that: The pancreatic β cell maturation culture medium contains IGF-1, GLP-1 and Forskolin, and is used to promote the maturation of pancreatic β cells.

5. The method for directed differentiation of induced pluripotent stem cells (iPSCs) into pancreatic β cells according to claim 1, characterized in that: It also includes monitoring the expression of specific markers of cells at each differentiation stage by flow cytometry or immunofluorescence detection.

6. A method for directed differentiation of induced pluripotent stem cells (iPSCs) into pancreatic β cells according to claim 1, characterized in that: The endoderm cells generated in step a) express Sox17 and FoxA2.

7. A method for directed differentiation of induced pluripotent stem cells (iPSCs) into pancreatic β cells according to claim 1, characterized in that: The pancreatic precursor cells generated in step b) express Pdx1 and Nkx6.

1.

8. The method for directed differentiation of induced pluripotent stem cells (iPSCs) into pancreatic β cells according to claim 1, characterized in that: The pancreatic β cells generated in step c) express insulin, MafA and Pdx1, and can secrete insulin under glucose stimulation.

9. The method for directed differentiation of induced pluripotent stem cells (iPSCs) into pancreatic β cells according to claim 1, characterized in that: iPSCs are human induced pluripotent stem cells hiPSCs.

10. The method for directed differentiation of induced pluripotent stem cells (iPSCs) into pancreatic β cells according to claim 1, characterized in that: The method further comprises performing an insulin secretion function test on the pancreatic β cells in step c) to evaluate their functional response ability under different glucose concentrations.

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