Pancreatic beta cell precursor cell transplantation chip for diabetes treatment

Abstract

The invention discloses a pancreatic beta cell precursor cell transplantation chip based on human-derived iPSC (induced pluripotent stem cell) as well as a preparation method and application of the pancreatic beta cell precursor cell transplantation chip. The chip is composed of beta cell precursor cells (expressing Pdx1, Nkx6.1 and NeuroD1) and a porous degradable bracket (PCL / hyaluronic acid composite material), wherein the beta cell precursor cells (expressing Pdx1, Nkx6.1 and NeuroD1) are obtained by performing four-stage induction on autologous iPSC. The preparation method comprises the steps of autologous iPSC acquisition, beta cell precursor cell induction, three-dimensional bracket preparation and chip assembly. After the chip is implanted into the pancreas of a patient through minimally invasive surgery, precursor cells are differentiated into mature beta cells in vivo, insulin is secreted, the stent is gradually degraded, the pancreas islet function can be fundamentally improved, and the chip is used for treating type I and type II diabetes mellitus. The invention avoids immunological rejection, improves cell survival and functional stability, and has significant clinical transformation value.

Description

Technical Field

[0001] This invention relates to the fields of regenerative medicine and diabetes treatment technology, and in particular to the construction of a transplantation chip using autologous iPSC-induced differentiated pancreatic β-cell precursor cells combined with a biomaterial scaffold. This chip achieves β-cell regeneration through autologous transplantation, fundamentally repairing pancreatic islet function and providing a radical treatment option for type I and type II diabetes. Background Technology

[0002] Diabetes mellitus is a prevalent metabolic disease worldwide, and its core pathological mechanism is pancreatic β-cell dysfunction or insulin resistance. Current treatment methods mainly include: (1) exogenous insulin injection: lifelong administration is required, it cannot simulate physiological secretion, and it is prone to hypoglycemia or blood glucose fluctuations; (2) oral hypoglycemic agents: only applicable to some type II diabetes, and the efficacy decreases as the disease progresses; (3) islet transplantation: blood glucose can be stabilized, but the donor depends on cadaveric islets, the source is scarce (global donor shortage >90%), and long-term use of immunosuppressants is required, which poses risks of infection and tumors.

[0003] The emergence of human iPSCs provides a new approach to solving the above problems: iPSCs can be obtained by reprogramming autologous somatic cells, have multi-directional differentiation potential, and avoid immune rejection; their induced differentiated β cells can serve as ideal cell substitutes. However, the existing technology has the following limitations: (1) the survival rate of directly transplanted free cells is low (<30%) due to the lack of extracellular matrix support; (2) the function of differentiated mature β cells is unstable after transplantation and is easily affected by the inflammatory microenvironment; (3) there is a lack of degradable carrier materials that are compatible with the pancreatic microenvironment, resulting in low cell colonization efficiency.

[0004] Therefore, developing a transplantation chip that combines autologous iPSC-induced β-cell precursor cells with a biomimetic scaffold to improve cell survival, differentiation, and functional maintenance is key to achieving a radical cure for diabetes. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing diabetes treatment technologies and provide an autologous, immune-free, and highly efficient β-cell regeneration transplantation chip, along with its preparation method and application, to achieve radical treatment of type I and type II diabetes.

[0006] Technical solution:

[0007] The present invention achieves the above objective through the following steps:

[0008] 1. Acquisition and identification of autologous iPSCs: Collect autologous somatic cells (such as peripheral blood mononuclear cells) from the patient, reprogram them to obtain iPSCs, and ensure their pluripotency and safety;

[0009] 2. Directed induction of β-cell precursor cells: iPSCs are directed to differentiate into high-purity β-cell precursor cells through a four-stage step-by-step induction process. These cells retain their differentiation potential and are more tolerant to the transplantation microenvironment than mature β-cells.

[0010] 3. Fabrication of three-dimensional scaffolds: Biodegradable tissue-compatible materials are selected, and biomimetic scaffolds are fabricated by 3D printing or electrospinning to simulate the extracellular matrix structure of pancreatic cells and support cell survival, proliferation and differentiation;

[0011] 4. Assembly of transplanted microarrays: β-cell precursor cells are seeded onto a scaffold to form a "cell-scaffold" composite microarray, ensuring uniform cell distribution and maintaining cell viability;

[0012] 5. Autologous transplantation and functional restoration: The chip is implanted into the patient's pancreas through minimally invasive surgery. The precursor cells differentiate into mature β cells under the induction of the in vivo microenvironment (such as glucagon and glucose signaling), secrete insulin, and the scaffold gradually degrades, ultimately achieving pancreatic islet function restoration.

[0013] Beneficial effects:

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] (1) Immunocompatibility: Using autologous iPSC-derived cells avoids immune rejection and eliminates the need for long-term use of immunosuppressants;

[0016] (2) High cell survival rate: The three-dimensional scaffold provides physical support and nutrient exchange channels, which increases the cell transplantation survival rate to more than 70%.

[0017] (3) Functional stability: β cell precursor cells gradually differentiate in the in vivo microenvironment, making them more adaptable to pancreatic physiological conditions. After maturation, their insulin secretion response is 2-3 times higher than that of directly transplanted mature β cells.

[0018] (4) Radical cure potential: By regenerating endogenous functional β cells, it fundamentally repairs pancreatic function, rather than just controlling symptoms;

[0019] (5) Safety: The stent material is completely degradable with no residual toxicity, and the iPSCs are strictly screened to avoid the risk of tumor formation. Attached Figure Description

[0020] Appendix Figure 1 Abstract image of pancreatic β-cell precursor cells induced by human iPSCs; Detailed Implementation

[0021] (I) Acquisition and Identification of Autologous iPSCs

[0022] 1. Collect 5 mL of peripheral blood from the patient, isolate mononuclear cells, and culture them in RPMI-1640 medium containing 10% FBS;

[0023] 2. A non-integrating Sendai virus vector carrying four factors (Oct4, Sox2, Klf4, and c-Myc) was used to transfect monocytes, which were then cultured at 37°C and 5% CO2.

[0024] 3. After 14 days, embryonic stem cell colonies were selected and passaged to the 5th generation. Immunofluorescence detection showed that the positive rates of OCT4, SOX2, and NANOG were all ≥98%, and the karyotype analysis was normal, confirming that qualified iPSCs were obtained.

[0025] (II) Induction and Differentiation of β-cell Precursor Cells

[0026] 1. Endoderm induction: iPSCs (generations 5-10) were seeded into Matrigel-coated 6-well plates (1×10⁻⁶). 5 Cells / well were cultured in RPMI / B27 medium containing Activin A (100 ng / mL) and Wnt3a (30 ng / mL) for 2 days, and CXCR4 was detected by flow cytometry. + (Endoderm markers) Positive rate ≥90%;

[0027] 2. Differentiation of primitive intestinal segments: The medium was changed to contain FGF7 (20 ng / mL) and Retinoic Acid (1 μM), and cultured for 3 days. HNF1β was then detected. + (Intestinal markers) Positive rate ≥85%;

[0028] 3. Pancreatic fate determination: KGF (40 ng / mL) and DAPT (10 μM) were added, and the pancreas was cultured for 6 days. Pdx1 was then measured. + (Pancreatic precursor markers) positive rate ≥90%;

[0029] 4. β-cell precursor typing: Cells were cultured for 10 days using a medium containing nicotinamide (10 mM), Exendin-4 (30 nM), and T3 (30 nM). Pdx1 was detected by flow cytometry. + Nkx6.1 + The double positivity rate was 96.2%, NeuroD1 + The positive rate was 92.5%, and the target precursor cells were obtained.

[0030] (III) Fabrication of Three-Dimensional Scaffolds

[0031] 1. Material ratio: Mix PCL and hyaluronic acid at a mass ratio of 2:1, dissolve in hexafluoroisopropanol (concentration 10% w / v), and stir until completely dissolved;

[0032] 2. Electrospinning parameters: voltage 15kV, receiving distance 15cm, feed rate 0.8mL / h, ambient temperature 25℃, humidity 40%;

[0033] 3. Support treatment: After spinning, vacuum dry for 24 hours, then cut into round pieces with a diameter of 5mm and a thickness of 1mm. Co 60 Sterilization, pore size 150±30μm, porosity 82%, degradation cycle 9 weeks.

[0034] (iv) Assembly of the transplanted chip

[0035] 1. The β-cell precursor cells obtained in Example 2 were digested with 0.25% trypsin and the concentration was adjusted to 1×10⁻⁶. 6 pcs / cm 2 ;

[0036] 2. Take the scaffold prepared in Example 3, place it in a 24-well plate, add 100 μL of cell suspension to each well, and let it stand at 37°C for 1 hour to allow the cells to attach;

[0037] 3. Add DMEM / F12 medium containing 10% autologous serum and culture for 48 hours. The cell viability was 91.3% as determined by CCK-8 assay, and microscopic examination showed that the cells were evenly distributed within the scaffold.

[0038] (V) Validation of Diabetic Model Animal Transplantation

[0039] 1. Establish an STZ-induced type 1 diabetic rat model (blood glucose >16.7 mmol / L, lasting for 2 weeks);

[0040] 2. Prepare rat autologous iPSC-derived transplanted chips according to the methods in Examples 1-4;

[0041] 3. The chip was implanted into the pancreatic body of rats laparoscopically, with two chips (total cell count 2 × 10⁻⁶) transplanted per rat. 6 indivual);

[0042] 4. Monitoring Results:

[0043] Two weeks post-transplant: blood glucose dropped to 12.3±1.5mmol / L, and fasting insulin was 15.2±2.1μU / mL (preoperative <5μU / mL);

[0044] Six weeks post-transplantation: blood glucose decreased to 6.8±0.7mmol / L, insulin was 28.5±3.2μU / mL, and the glucose tolerance test showed that insulin secretion was in a physiological response;

[0045] Eight weeks post-transplantation: 80% of the scaffold has degraded, and immunohistochemistry shows the presence of insulin at the transplant site. + The cells do not exhibit an immune rejection response.

Claims

1. A pancreatic β-cell precursor cell transplantation chip for diabetes treatment, characterized in that, include: (a) Functional unit: pancreatic β cell precursor cells obtained by directed differentiation of human induced pluripotent stem cells (iPSCs), wherein the precursor cells simultaneously express Pdx1, Nkx6.1 and NeuroD1 markers and have the potential to differentiate into mature insulin-secreting β cells; (b) Carrier unit: a tissue-compatible three-dimensional scaffold encapsulating the β-cell precursor cells, the scaffold being a porous structure with a pore size of 80-300 μm, made of a biodegradable biomaterial selected from polycaprolactone (PCL), hyaluronic acid, chitosan or composites thereof, and the scaffold being completely degradable in vivo within 6-12 weeks.

2. The transplanted chip according to claim 1, characterized in that, The human iPSCs are derived from the patient's own somatic cells through reprogramming, and the somatic cells are selected from peripheral blood mononuclear cells, skin fibroblasts, or oral mucosal cells.

3. The transplanted chip according to claim 1, characterized in that, During the induction and differentiation of the β-cell precursor cells, the cells sequentially undergo the endoderm differentiation stage, the primitive intestinal differentiation stage, the pancreatic fate determination stage, and the β-cell precursor shaping stage, with specific inducing factor combinations added at each stage.

4. A method for preparing a pancreatic β-cell precursor cell transplantation chip according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Obtaining autologous iPSCs: Autologous somatic cells from patients are collected and iPSCs are obtained through reprogramming of four factors: Oct4, Sox2, Klf4 and c-Myc. After being identified as qualified by pluripotency markers (OCT4, SOX2, NANOG), they are ready for use. (2) Induction of differentiation of β-cell precursor cells: ① Endoderm induction: iPSCs were seeded into Matrigel-coated culture plates and cultured for 2-3 days in RPMI / B27 medium containing Activin A (50-150 ng / mL) and Wnt3a (20-50 ng / mL); ② Differentiation of primitive intestinal segments: Change to a culture medium containing FGF7 (10-30 ng / mL) and Retinoic Acid (0.5-2 μM) and culture for 3-4 days; ③ Pancreas fate determination: Add KGF (20-60 ng / mL) and DAPT (5-15 μM), and culture for 5-7 days; ④ β-cell precursor shaping: Culture in a medium containing nicotinamide (5-15 mM), Exendin-4 (10-50 nM) and T3 (10-50 nM) for 8-12 days to obtain β-cell precursor cells; (3) Preparation of three-dimensional scaffold: The biomaterial described in claim 1 is used to make a three-dimensional scaffold with a pore size of 80-300μm and a porosity of 70%-90% by electrospinning or 3D printing technology, and then sterilized. (4) Chip assembly: The β-cell precursor cells obtained in step (2) are assembled at 5 × 10⁻⁶ cells per cell. 5 -2×10 6 pcs / cm 2 The cells were seeded at a density on the scaffold from step (3) and cultured in DMEM / F12 medium containing 10% autologous serum for 24-48 hours to allow the cells to attach and proliferate evenly, forming a transplanted chip.

5. The method according to claim 4, characterized in that, The β-cell precursor cells obtained in step (2) were analyzed by flow cytometry, and Pdx1 was found to be present. + Nkx6.1 + Double positivity rate ≥95%, NeuroD1 + Positive rate ≥ 90%.

6. The method according to claim 4, characterized in that, In step (3), the degradation rate of the three-dimensional scaffold can be controlled by the material ratio. When the mass ratio of PCL to hyaluronic acid is (3:1)-(1:1), the degradation cycle is 8-10 weeks.

7. The use of the pancreatic β-cell precursor cell transplantation chip according to any one of claims 1-3 in the preparation of an implantable medical device for treating type I or type II diabetes.

8. A method for treating diabetes, characterized in that, Includes the following steps: (1) Prepare a patient-derived pancreatic β-cell precursor cell transplantation chip according to the method of claim 4; (2) The chip is implanted into the pancreatic parenchyma of the patient through laparoscopic minimally invasive surgery. 1-3 chips are transplanted per case, with a total cell count of 1×10⁻⁶. 7 -5×10 7 indivual; (3) Postoperative monitoring: Blood glucose levels and insulin secretion were monitored 1-12 weeks after transplantation. The β-cell precursor cells in the chip differentiated into mature β-cells under the induction of the in vivo microenvironment, realizing the physiological secretion of insulin and improving pancreatic function.

9. The method according to claim 8, characterized in that, In step (2), the chip is implanted in the pancreatic body or tail region, and no immunosuppressants are needed after transplantation.

10. The method according to claim 8, characterized in that, Three to six weeks after transplantation, patients' fasting insulin levels increased by ≥50% compared to pre-operative levels, and glycated hemoglobin (HbA1c) decreased by ≥1.5% compared to pre-operative levels.

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