Method for reprogramming islet cells into induced pluripotent stem cells

Abstract

The invention provides an innovative method for reprogramming islet cells into induced pluripotent stem cells (iPSCs). The method comprises the following steps: firstly, separating and purifying pancreatic islet cells from pancreatic tissues of mammals to obtain the pancreatic islet cells as starting cells; then, a specific reprogramming factor combination is utilized, and islet cells are introduced through a vector; the reprogramming factors induce islet cells to undergo a series of morphological and gene expression changes, and finally the islet cells are converted into iPSCs with embryonic stem cell (ESCs) characteristics and differentiation potential. In the reprogramming process, optimized cell culture conditions are adopted, so that the reprogramming efficiency is improved; meanwhile, the culture medium also contains specific growth factors to support cell proliferation and pluripotency maintenance. Compared with a traditional iPSCs reprogramming method, the method for reprogramming the islet cells has the advantages that the reprogramming efficiency is higher, the safety is better, the clinical relevance is stronger, and a new hope is brought to research and treatment of metabolic diseases such as diabetes mellitus.

Description

Technical Field

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

[0002] Islet cells are mainly responsible for regulating blood sugar levels in the human body, and their dysfunction is closely related to metabolic diseases such as diabetes1. In order to study the pathogenesis of diabetes and develop new treatments, scientists have been committed to finding ways to obtain large numbers of islet cells in vitro. In recent years, the technology of induced pluripotent stem cells (iPSCs) has brought new hope for regenerative medicine and cell therapy2. iPSCs are cells obtained by reprogramming somatic cells with characteristics similar to those of embryonic stem cells, which can differentiate into a variety of somatic cell types, including islet cells. Therefore, by reprogramming islet cells into iPSCs, it is not only helpful to study the biological characteristics of islet cells, but also provides an important cell source for cell replacement therapy for diabetes.

[0003] Traditional iPSCs preparation methods mostly rely on somatic cells such as skin fibroblasts3, while using pancreatic islet cells as starting cells for reprogramming can more directly obtain cell models related to pancreatic function. In addition, the technology of reprogramming pancreatic islet cells into iPSCs can overcome the problem of immune rejection, especially in autologous transplantation and personalized treatment, which has great application potential. However, there are still challenges such as how to improve reprogramming efficiency, reduce genomic instability, and control differentiation direction. Therefore, studying an efficient and stable method to reprogram pancreatic islet cells into iPSCs has important scientific significance and clinical application value. Summary of the invention

[0004] The present invention relates to a method for reprogramming pancreatic islet cells into induced pluripotent stem cells (iPSCs), which specifically includes using specific reprogramming factors to introduce these factors into pancreatic islet cells through non-viral vectors, so that they are reprogrammed into induced pluripotent stem cells with embryonic stem cell (ESCs) characteristics and differentiation potential under in vitro conditions. This method provides a new cell source that can be used to study the pathogenesis of diabetes and other metabolic diseases, and can also provide a feasible solution for regenerative medicine, cell replacement therapy and personalized treatment.

[0005] The core technology of the present invention includes isolating and purifying pancreatic islet cells from pancreatic tissue of mammals (preferably humans or other higher animals), and then, under culture conditions, introducing reprogramming factors including Oct4, Sox2, Klf4 and c-Myc, so that these pancreatic islet cells regain pluripotency. These reprogramming factors can be introduced into cells in the form of various vectors such as lentiviral vectors, retroviral vectors, plasmids, and mRNA. After the reprogramming factors are introduced, the pancreatic islet cells begin to undergo a series of changes in morphology and gene expression, and eventually transform into induced pluripotent stem cells that exhibit properties similar to embryonic stem cells.

[0006] Compared with the traditional iPSCs reprogramming method, the present invention uses pancreatic islet cells for reprogramming, which has important advantages. First, pancreatic islet cells themselves have endocrine functions, especially β cells can secrete insulin, which has unique value for the study of metabolic diseases such as diabetes4. By reprogramming pancreatic islet cells into iPSCs and further inducing differentiation into functional pancreatic islet cells, a cell source with no rejection risk can be provided for cell replacement therapy of diabetes. Secondly, using pancreatic islet cells as starting cells can effectively overcome the limitations of xenogeneic cell reprogramming, reduce challenges in specific differentiation directions, and help improve the efficiency of reprogramming and differentiation.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] 1. Isolation and purification of pancreatic islet cells

[0009] The first step of the method of the present invention is to separate pancreatic islet cells from mammalian pancreatic tissue. The islet cell separation step comprises:

[0010] 1. Obtaining Pancreatic Tissue

[0011] Tissue source: Pancreatic tissue is obtained surgically from mammals (such as humans, mice or pigs), and healthy and undamaged pancreases are selected. For human tissue, it is usually obtained from organ donations.

[0012] Tissue preservation: Place the harvested pancreatic tissue in cold saline or culture medium containing antibiotics to avoid cell damage and contamination.

[0013] 2. Tissue Processing and Enzymatic Hydrolysis

[0014] Collagenase preparation: Use collagenase (such as collagenase IV) to prepare the enzymatic solution. The concentration of collagenase is usually 1-2 mg / mL.

[0015] Other enzymes: Other digestive enzymes, such as DNase (0.1 mg / mL) and heparin (10 U / mL), can be used in combination to improve the efficiency of cell separation.

[0016] Tissue cutting: Cut the pancreatic tissue into small pieces of approximately 1-2 mm to increase the surface area for enzymatic hydrolysis.

[0017] Digestion reaction: Place the cut pancreatic tissue into a centrifuge tube containing enzymatic solution, shake gently and incubate in a 37°C water bath. The digestion time is 1 hour, and the tube should be gently shaken every 10-15 minutes to ensure that the enzyme is fully in contact with the tissue.

[0018] 3. Cell separation and washing

[0019] Stop the enzymatic reaction: After the digestion is complete, add an equal volume of culture medium (such as DMEM / F12 or RPMI 1640) to stop the enzymatic reaction.

[0020] Filtration: Filter the digested cell suspension through a 70 μm cell strainer to remove undigested tissue fragments.

[0021] Centrifugation: Transfer the filtered cell suspension to a centrifuge tube and centrifuge at 400 x g for 10 minutes to collect the cell pellet.

[0022] Wash: Remove the supernatant, add pre-cooled culture medium (or PBS), gently resuspend the cells, and centrifuge again. This step can be repeated 1-2 times to remove residual enzymes and impurities.

[0023] 4. Enrichment of Pancreatic Islet Cells

[0024] Layered centrifugation: Resuspend the washed cells in an appropriate amount of density gradient medium and centrifuge to separate the islet cells. Centrifuge at 400xg for 30 minutes.

[0025] Collecting islet cells: After centrifugation, islet cells will aggregate in a specific layer (usually in the middle layer of the density medium). Carefully collect these cells and transfer them to a new centrifuge tube.

[0026] 5. Culture and maintenance of pancreatic islet cells

[0027] Cell resuspension: Resuspend the isolated islet cells in a suitable culture medium (such as RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics) to adjust the cell concentration.

[0028] Culture conditions: Transfer cells to a culture dish and place at 37°C and 5% CO 2 Cells were cultured and maintained in an incubator. The culture medium was changed regularly according to experimental requirements, and the cell growth status was observed.

[0029] 2. Selection and introduction of reprogramming factors

[0030] In order to reprogram pancreatic islet cells into iPSCs, the present invention uses a combination of classical reprogramming factors. The reprogramming factors include:

[0031] Oct4 (POU family transcription factor), Sox2 (SRY-box transcription factor), Klf4 (Krüppel-like transcription factor), c-Myc (proto-oncogene), these reprogramming factors work together to induce pancreatic islet cells to restore pluripotency. In addition, the present invention also selects other auxiliary factors according to specific experimental requirements, such as: Nanog: used to further enhance pluripotency maintenance and Lin28: promote the maintenance of stem cell state.

[0032] Introduction of reprogramming factors:

[0033] The present invention adopts plasmid DNA transfection method, and the specific implementation method is as follows:

[0034] Material preparation: pancreatic islet cells, reprogramming factor plasmids (such as pCAGGS-Oct4, pCAGGS-Sox2, etc.), transfection reagents (such as Lipofectamine 2000 or JetPEI), culture medium (DMEM, high glucose, fetal bovine serum, etc.).

[0035] Experimental steps:

[0036] Plasmid construction: Clone the reprogramming factor gene into an appropriate plasmid vector and confirm the correctness of the inserted fragment.

[0037] Cell preparation: Pancreatic islet cells were seeded in 6-well plates and cultured to 70-80% confluence.

[0038] Transfect cells: Prepare transfection complexes according to the instructions of the transfection reagent. Add the transfection complexes to the cells, mix gently, and continue culturing.

[0039] Culture and screening: After 24 hours, replace the culture medium and continue to culture the cells. Regularly observe the changes in cell morphology during the culture process.

[0040] Cell expansion and characterization: Analyze the expression of reprogramming factors using RT-qPCR or Western blot. Perform immunofluorescence staining to detect the expression of pluripotency markers.

[0041] III. Reprogramming Culture Conditions

[0042] During the reprogramming process, the present invention provides optimized cell culture conditions to improve the reprogramming efficiency and ensure the quality of iPSCs. The culture medium includes the following components:

[0043] Basal medium: Use a medium suitable for the growth of pancreatic islet cells and stem cells, preferably containing fetal bovine serum (FBS), L-glutamine and penicillin-streptomycin.

[0044] Small molecule compounds: Add small molecule compounds that can promote reprogramming, including: Valproic Acid (VPA): an HDAC inhibitor that can enhance reprogramming efficiency. CHIR99021: a GSK3 inhibitor that promotes pluripotency maintenance by activating the Wnt signaling pathway. 3-Deazaneplanocin A (DZNep): a histone methylation inhibitor that can improve the efficacy of reprogramming factors.

[0045] Growth factors: Specific growth factors are added to the culture medium to maintain cell proliferation and pluripotency, including: bFGF (basic fibroblast growth factor): maintains the proliferation of pluripotent cells, EGF (epidermal growth factor): promotes cell proliferation and survival, LIF (leukemia inhibitory factor): specifically used to maintain the pluripotency of mouse iPSCs.

[0046] Culture environment: Reprogrammed cells were placed in 37°C, 5% CO 2 The reprogramming process usually takes 7 to 30 days, preferably 10 to 20 days. During this period, the cell morphology gradually changes from a typical pancreatic islet cell to a morphology similar to that of an embryonic stem cell.

[0047] 4. Monitoring of the Reprogramming Process

[0048] During the reprogramming process, the morphology, gene expression and function of cells are monitored in real time to ensure the success of reprogramming.

[0049] Morphological observation: Observe the changes in cell morphology under a microscope. During the reprogramming process, the cells will gradually take on a morphology similar to that of pluripotent stem cells.

[0050] Immunofluorescence staining: Use antibodies against pluripotent stem cell-specific markers (such as Oct4, SSEA-4, and Nanog) to detect whether the cells successfully express these pluripotency markers.

[0051] RT-qPCR detection: Use real-time fluorescence quantitative PCR technology to detect the gene expression levels of reprogramming factors, such as Oct4, Sox2, etc., to confirm the reprogramming status of cells.

[0052] 5. Differentiation of iPS cells after reprogramming

[0053] The obtained islet cell-derived iPSCs can be further differentiated into different types of functional cells under specific differentiation conditions, including:

[0054] Pancreatic β cells: iPSCs can be induced to differentiate into functional pancreatic β cells through specific differentiation culture conditions, which are used for diabetes model research and treatment. Neurons: iPSCs can be induced to differentiate into neuronal cells by adding neural differentiation factors, which are used for the study of neurodegenerative diseases. Cardiomyocytes: iPSCs can be induced to differentiate into cardiomyocytes through specific cardiac differentiation culture medium, which are used for the study of heart disease and regenerative medicine applications.

[0055] 6. Application fields

[0056] The iPSCs obtained by the method of the present invention have a wide range of application potentials:

[0057] Disease model construction: Use reprogrammed iPSCs to construct a pancreatic β cell model for pathological research of metabolic diseases such as diabetes.

[0058] Drug screening: iPSCs-based in vitro models can be used for high-throughput screening of new drugs, especially in the field of diabetes treatment.

[0059] Cell replacement therapy: Reprogrammed iPSCs can be differentiated into functional pancreatic islet cells for cell replacement therapy of diabetes.

[0060] Personalized medicine: Reprogramming autologous pancreatic islet cells into iPSCs can avoid immune rejection and be used in personalized cell therapy.

[0061] 7. Technical advantages

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] (1) Higher reprogramming efficiency: The reprogramming efficiency of pancreatic islet cells is improved through the synergistic effect of small molecule compounds and growth factors.

[0064] (2) Safety: The use of non-viral vectors avoids safety risks caused by genome integration.

[0065] (3) Clinical relevance: Direct use of pancreatic islet cells as starting cells provides a more clinically relevant cell model for the study and treatment of metabolic diseases such as diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 Brightfield image of a representative pancreatic islet isolated from a mouse

[0067] Figure 2 Fluorescence images of representative pancreatic islets isolated from mice

[0068] Figure 3 For isolated pancreatic islet cells

[0069] Figure 4 Reprogrammed induced pluripotent stem cells DETAILED DESCRIPTION

[0070] 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.

[0071] Example

[0072] Example 1: Construction of a recombinant plasmid for islet cell reprogramming

[0073] Table 1 Materials and reagents

[0074]

[0075] Specific steps

[0076] 1. PCR amplification of gene fragments

[0077] Specific PCR primers for Pdx1, Ngn3 and Mafa were designed. Restriction endonuclease sites compatible with the plasmid multiple cloning site were designed at both ends of the primers. PCR amplification of these three gene fragments was performed using cDNA templates.

[0078] Table 2 PCR reaction system

[0079]

[0080] Table 3 PCR reaction conditions

[0081]

[0082] 2. Enzyme digestion of vector

[0083] The vector pCDH was double-digested with restriction endonucleases EcoRI and XhoI at 37°C for 2 hours to provide a cloning site for gene insertion.

[0084] Table 4

[0085]

[0086] 3. Enzyme digestion of gene fragments

[0087] The Pdx1, Ngn3 and Mafa gene fragments obtained by PCR amplification were double-digested with EcoRI and XhoI respectively, under the same conditions as above. The digestion products were separated by agarose gel electrophoresis, and the target bands were recovered using a gel recovery kit.

[0088] 4. Ligation of recombinant plasmid

[0089] The digested vector and gene fragment were ligated using T4 DNA ligase and reacted at 16°C for 16 hours.

[0090] Table 5

[0091]

[0092] 5. Transformation into Competent Bacteria

[0093] The ligation product was transformed into competent DH5α bacteria.

[0094] Competent bacteria: 50 μL, ligation product: 5 μL, heat shock at 42°C for 45 seconds, place on ice for 2 minutes, add 500 μL SOC medium, shake culture at 37°C for 1 hour, spread on LB medium plates containing ampicillin, and incubate at 37°C overnight.

[0095] 6. Colony PCR screening of positive clones

[0096] Select multiple single colonies, extract plasmids using a plasmid extraction kit, and perform colony PCR to screen positive clones. The PCR amplification conditions are the same as in the first step, and the purpose is to verify the correctness of the inserted fragment.

[0097] 7. Verification of recombinant plasmid

[0098] The plasmid extracted from the positive clone was double-digested and verified, and the plasmid was cut again with EcoRI and XhoI to observe whether the expected size of the inserted fragment was obtained. Finally, the recombinant plasmid was sent to a sequencing company for sequencing to ensure the correctness of the inserted sequence.

[0099] Example 2: Flow cytometry detection of reprogrammed induced pluripotent stem cells

[0100] 1. Cell Collection and Preparation

[0101] Collect cells from reprogramming-induced iPSCs culture: Gently treat cells with trypsin or EDTA to avoid excessive treatment that may cause cell damage. Treat cells into a single cell suspension of about 1×10 6 cells.

[0102] 2. Cell fixation and permeabilization (only for intracellular marker detection)

[0103] For the detection of intracellular markers (such as OCT4, NANOG), cells need to be fixed and permeabilized: add cells to 4% paraformaldehyde solution and fix for 10 minutes. Wash twice with PBS to remove the fixative. Add 0.1% Triton X-100 to permeabilize the cell membrane for 5 minutes to allow the antibody to enter the cell.

[0104] 3. Blocking and Antibody Staining

[0105] For both cell surface and intracellular markers, cells need to be blocked to prevent nonspecific antibody binding: Add the cell suspension to a PBS solution containing 1% BSA and block for 30 minutes. Add specific primary antibody (OCT4) and incubate for 30 minutes to 1 hour (4°C) according to the antibody dilution ratio recommended by the manufacturer. Wash the cells three times with PBS to remove unbound antibodies.

[0106] 4. Fluorescent secondary antibody incubation

[0107] Add fluorescently labeled secondary antibodies (such as antibodies labeled with Alexa Fluor 488) to the cells and incubate for 30 minutes to 1 hour according to the dilution ratio. Wash again three times with PBS to ensure that unbound secondary antibodies are removed.

[0108] 5. Flow Cytometry

[0109] Add the stained cell suspension to a FACS tube and use a flow cytometer to detect fluorescently labeled cells, setting the appropriate lasers and filters to detect the fluorescent dyes used. Set up appropriate negative controls (unstained cells or cells stained with secondary antibodies only) and positive controls (iPSCs known to express specific markers) to distinguish nonspecific signals.

Claims

1. A method for reprogramming pancreatic islet cells into induced pluripotent stem cells, characterized in that: The following steps are involved: a) isolating and purifying pancreatic islet cells from mammalian pancreatic tissue; b) exposing the pancreatic islet cells to a vector system containing reprogramming factors, wherein the reprogramming factors include Oct4, Sox2, Klf4 and c-Myc; c) The reprogrammed pancreatic islet cells are cultured in a specific cell culture medium to reprogram them into induced pluripotent stem cells (iPSCs).

2. The method for reprogramming pancreatic islet cells into induced pluripotent stem cells according to claim 1, characterized in that: The reprogramming factors are introduced into pancreatic islet cells via a viral vector, preferably a lentiviral vector or a retroviral vector, and the viral vector can achieve stable integration of the target gene.

3. The method for reprogramming pancreatic islet cells into induced pluripotent stem cells according to claim 1, characterized in that: The introduction of reprogramming factors is achieved through non-viral vectors, and the plasmid or mRNA can temporarily express the reprogramming factors in cells to avoid genome integration.

4. The method for reprogramming pancreatic islet cells into induced pluripotent stem cells according to claim 1, characterized in that: The medium used to culture the reprogrammed cells is a defined medium containing small molecule compounds to improve the reprogramming efficiency, including but not limited to: Valproic Acid, an HDAC inhibitor, is used to promote cell reprogramming; CHIR99021, a GSK3 inhibitor, activates the Wnt signaling pathway to enhance the reprogramming process; 3-Deazaneplanocin A, a histone methylation inhibitor.

5. The method for reprogramming pancreatic islet cells into induced pluripotent stem cells according to claim 1, characterized in that: The culture medium also contains specific growth factors to support cell proliferation and reprogramming, including but not limited to: bFGF basic fibroblast growth factor, used to maintain cell pluripotency; EGF epidermal growth factor, used to promote cell proliferation; LIF leukemia inhibitory factor is used to maintain the pluripotent state of mouse iPSCs.

6. The method for reprogramming pancreatic islet cells into induced pluripotent stem cells according to any one of claims 1 to 5, characterized in that: During the reprogramming process, the reprogramming progress of the pancreatic islet cells is monitored, and the monitoring step comprises: a) Morphological changes: monitoring the transition of cell morphology from primitive islet cells to pluripotent stem cell-like phenotypes under a microscope; b) Immunofluorescence labeling detection, using antibodies against embryonic stem cell markers, including Oct4, SSEA-4, Nanog, etc.; c) RT-qPCR analysis to detect the expression levels of reprogramming genes, such as Oct4 and Sox2, and evaluate the effects of reprogramming factors.

7. The method for reprogramming pancreatic islet cells into induced pluripotent stem cells according to claim 1, characterized in that: The pancreatic islet cells are derived from humans or other mammals, preferably human, mouse or pig pancreatic islet cells, and a purified pancreatic islet cell population is obtained by islet separation technology.

8. The method for reprogramming pancreatic islet cells into induced pluripotent stem cells according to claim 1, characterized in that: The pancreatic islet cells are pre-treated before reprogramming, and the pre-treatment steps include: a) Purification of pancreatic islet cells, enrichment of pancreatic β cells by flow cytometry or antibody labeling; b) Transfection optimization, including using electro-transfection or lipofectamine transfection techniques to improve the transfection efficiency of reprogramming factors; c) Culture medium pretreatment, by adding specific growth factors or compounds to improve cell viability and reprogramming potential.

9. The method for reprogramming pancreatic islet cells into induced pluripotent stem cells according to claim 1, characterized in that: The culture time of the reprogramming process is 7 to 30 days, preferably 10 to 20 days, and the reprogramming efficiency is improved by regulating the temporal expression of reprogramming factors; wherein, Klf4 and Sox2 are highly expressed in the early stage, and c-Myc expression is reduced in the late stage to promote the establishment of cell homeostasis.

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