Preparation method of pancreatic beta cells derived from stem cells

Abstract

The invention provides an efficient and repeatable stem cell-derived pancreatic beta cell preparation method, Wnt and Notch signal channels are regulated and controlled in stages, a culture environment is optimized in combination with bionic matrix stiffness, and differentiation of pluripotent stem cells to functional pancreatic beta cells is promoted. The method specifically comprises the following steps: 1, stage signal regulation: activating a Wnt pathway at the early stage of differentiation to promote endoderm induction, and then inhibiting a Notch pathway to enhance endocrine precursor cell formation; 2, bionic matrix rigidity optimization: adopting hydrogel (-1-5kPa) matched with natural pancreas islet rigidity to simulate an in-vivo microenvironment, and improving beta cell maturity and functions; 3, glucose responsiveness optimization: through later-stage low-glucose / high-glucose alternate culture, the glucose sensitivity of beta cells is enhanced, so that the insulin secretion amount is close to the primary cell level. According to the method, the yield and the function of the beta cells are remarkably improved, and a reliable cell source is provided for diabetes cell therapy.

Description

Technical Field

[0001] This invention belongs to the fields of regenerative medicine and cell engineering, specifically relating to a method for inducing pluripotent stem cells (iPSCs / ESCs) to efficiently differentiate into glucose-responsive pancreatic β-cell clusters by utilizing staged regulation of the Wnt and Notch signaling pathways combined with biomimetic matrix stiffness. The β-cell clusters obtained by this method have insulin secretion function close to that of native pancreatic β-cells and can be used for diabetes treatment, drug screening, and disease model research. Background Technology

[0002] Diabetes is a global health problem, and pancreatic β-cell dysfunction is a major cause of type 1 diabetes and some types of type 2 diabetes. Currently, while islet transplantation can restore glycemic regulation in some patients, donor scarcity and immune rejection limit its application. Stem cell-differentiated pancreatic β-cells are a potential alternative, but existing methods suffer from low differentiation efficiency and immature function.

[0003] In existing technologies, pancreatic β-cell differentiation largely depends on combinations of growth factors (such as Activin A and FGF), but the temporal regulation of the biomechanical microenvironment and key signaling pathways has not been sufficiently optimized, leading to limited cell function. Furthermore, sustained activation of the Notch pathway inhibits endocrine cell fate, while the dynamic regulation of the Wnt pathway is crucial for pancreatic progenitor cell formation. Therefore, a highly efficient differentiation strategy combining biophysical and biochemical signals is urgently needed.

[0004] Early differentiation methods relied on staged induction with high concentrations of growth factors, resulting in β-cells with generally insufficient glucose-stimulated insulin secretion (GSIS) function. Subsequent studies confirmed that Wnt activation enhances endoderm induction and Notch inhibition promotes endocrine differentiation, but insufficient precision in the temporal control of signaling pathways led to fluctuations in differentiation efficiency. Recent advances in biomechanics have revealed that ECM stiffness is a key physical factor regulating β-cell maturation; however, current techniques still treat biochemical signals and the mechanical microenvironment separately. Therefore, a synergistic optimization strategy integrating biomimetic matrix stiffness and precise staged signaling pathway regulation is urgently needed to overcome the technical bottleneck of efficient differentiation of stem cells into functional pancreatic β-cells. Summary of the Invention

[0005] This invention aims to establish a highly efficient and stable stem cell differentiation system for functional pancreatic β-cells. It overcomes the functional bottlenecks of existing technologies by employing a phased, temporal-sequential strategy of regulating the Wnt / Notch signaling pathway in conjunction with biomimetic matrix stiffness (1-5 kPa). Specifically, it activates the Wnt pathway during the pancreatic precursor induction stage to enhance endoderm-directed differentiation, and precisely inhibits the Notch pathway during the endocrine differentiation stage to remove its inhibition of endocrine cell fate. Simultaneously, it utilizes a hydrogel matrix with islet tissue-specific stiffness to simulate the natural microenvironment, activating the Piezo1 isotropic channel through mechanotransduction signals to drive the maturation of glucose-responsive insulin secretion in β-cells. Ultimately, it yields three-dimensional cell clusters with glucose concentration-dependent secretion kinetics (reproducing biphasic secretion characteristics), insulin secretion levels (basal secretion 0.5-2 ng / islet / h, 3-8 times increase after glucose stimulation), and a glucose stimulation index (≥5) close to those of primary islets, providing a clinical-grade cell source for diabetic cell therapy. The following is a detailed explanation of the technical solution and its advantages.

[0006] 1. Staged signaling pathway regulation

[0007] Phase 1 (Endoderm induction, 0-3 days): Activation of the Wnt pathway (CHIR99021) + ActivinA promotes the formation of a fixed endoderm.

[0008] Phase 2 (pancreatic progenitor cells, 4-7 days): Wnt activation was removed, and FGF10 and retinoic acid (RA) were added to induce pancreatic fate.

[0009] Phase 3 (endocrine progenitor cells, 8-12 days): Inhibition of the Notch pathway (DAPT), promotion of Neurogenin 3 (Ngn3) expression, and driving endocrine differentiation.

[0010] Phase 4 (β-cell maturation, 13-20 days): Add insulin-like growth factor (IGF-1) + Exendin-4, combined with glucose fluctuation culture (5mM / 20mM alternating), to enhance functional maturation.

[0011] Cell morphology changes need to be strictly monitored at each stage (e.g., epithelial-like in stage 1 → clustered aggregation in stage 3), and culture time should be dynamically adjusted by biomarker expression.

[0012] 1.1 Stage 1: Endoderm Induction (0-3 days)

[0013] Objective: To promote the differentiation of pluripotent stem cells into the defined endoderm.

[0014] Key regulatory factors:

[0015] Wnt pathway activation: Adding CHIR99021 (3μM) temporarily activates Wnt / β-catenin signaling, promoting the expression of endoderm genes (such as SOX17 and FOXA2).

[0016] TGF-β superfamily signaling: Combined with ActivinA (100 ng / mL), it mimics the Nodal signaling in embryonic development and enhances the directional differentiation of the endoderm.

[0017] Culture medium: Serum-free RPMI medium was used to avoid interference from serum components.

[0018] Detection indicators: On day 3, SOX17 / FOXA2 expression was detected by qPCR or immunofluorescence (positive rate should be >90%). Phase 2: Pancreatic progenitor cell induction (days 4-7)

[0019] Objective: To drive the differentiation of endoderm cells into pancreatic progenitor cells (PDX1+NKX6.1+).

[0020] Key regulatory factors:

[0021] Wnt signal removal: Discontinue CHIR99021 to avoid continuous activation and suppression of pancreatic fate.

[0022] FGF10 (50 ng / mL): Promotes pancreatic spore formation and maintains precursor cell proliferation.

[0023] Retinoic acid (RA, 2 μM): mimics the embryonic foregut development environment and induces PDX1 expression.

[0024] Culture medium: Switch to DMEM / F12 medium containing 1% B27.

[0025] Detection indicator: PDX1 / NKX6.1 co-expression was detected on day 7 (target >70%).

[0026] Phase 3: Induction of endocrine precursor cells (8-12 days)

[0027] Objective: To inhibit the Notch pathway and promote the directed differentiation of the endocrine lineage (Ngn3+).

[0028] Key regulatory factors:

[0029] Notch pathway inhibition: Adding the γ-secretase inhibitor DAPT (10 μM) blocked Notch signaling and relieved the inhibition of Ngn3.

[0030] Low-glucose environment (5mM glucose): mimics the physiological conditions of embryonic endocrine development.

[0031] Culture medium: DMEM / F12 containing NEAA (non-essential amino acids) and 0.5% B27.

[0032] Detection indicators: Ngn3 and NeuroD1 expression were detected on day 12 (positive rate >60%).

[0033] 1.4 Stage 4: β-cell functional maturation (13-20 days)

[0034] Objective: To obtain glucose-responsive β cells (C-peptide + MAFA+).

[0035] Key regulatory factors:

[0036] IGF-1 (50 ng / mL): Promotes β-cell survival and functional maturation.

[0037] Exendin-4 (50 nM): A GLP-1 receptor agonist that enhances insulin synthesis.

[0038] Glucose fluctuation stimulation: Alternating between low-glucose (5mM) and high-glucose (20mM) culture media (switching every 24 hours) to simulate blood glucose fluctuations in vivo.

[0039] Culture medium: DMEM / F12 containing 10 mM nicotinamide and 1% B27.

[0040] Testing indicators:

[0041] On day 20, function was assessed using the GSIS (glucose-stimulated insulin secretion assay), with a target insulin secretion level ≥15 μIU / 10^6 cells / hour.

[0042] Immunostaining was used to detect the colocalization of C-peptide / MAFA (target >80%).

[0043] 2. Optimization of biomimetic matrix stiffness

[0044] Collagen / alginate hydrogel is used, with stiffness adjusted to 1-5 kPa (matching natural pancreatic islets).

[0045] Stiffness was controlled by the concentration of crosslinking agent (such as EDC / NHS) and verified by atomic force microscopy (AFM).

[0046] 2.1 Effect of matrix stiffness on pancreatic β-cell differentiation

[0047] Physiological basis: The elastic modulus of natural pancreatic islet tissue is about 1–5 kPa, and this mechanical environment can promote the functional maturation of β cells.

[0048] Core function:

[0049] Stiffness affects cell fate: an overly soft matrix (<1 kPa) is not conducive to cell aggregation and polarization, while an overly hard matrix (>10 kPa) is prone to fibrosis.

[0050] Mechanotransduction: The expression of PDX1 and NKX6.1 is affected by regulating YAP / TAZ nuclear localization via the integrin-focal adhesion kinase (FAK) pathway. All hydrogels must be sterilized (by gamma ray or ethanol immersion) and equilibrated with PBS for 24 hours before use to avoid residual cross-linking agent toxicity.

[0051] 2.2 Preparation and Optimization of Biomimetic Hydrogels

[0052] Material selection:

[0053] Type I collagen (main component): provides RGD binding sites for natural ECM, supporting cell adhesion.

[0054] Sodium alginate (auxiliary ingredient): via Ca 2+ Cross-linking regulates stiffness and prevents collagenase degradation.

[0055] Stiffness control methods:

[0056] Crosslinking agent concentration:

[0057] The EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide) ratio is adjusted (0.5–2 mM EDC corresponds to 1–5 kPa).

[0058] AFM verification: The actual stiffness is measured using an atomic force microscope to ensure that the error from the target value (e.g., 3 kPa) is <10%.

[0059] 2.3 Synergistic Application of Matrix Stiffness and Differentiation Stage

[0060] Stages 1–2 (Endoderm → Pancreatic precursor):

[0061] Stiffness range: 3–5 kPa, simulating the mechanical environment of the foregut mesoderm, promoting the expansion of PDX1+ cells.

[0062] Culture method: Cells were seeded on a Transwell membrane pre-coated with hydrogel to ensure three-dimensional contact.

[0063] Stages 3–4 (Endocrine precursors → β-cell maturation):

[0064] Stiffness adjustment: Reduced to 1–3 kPa to simulate the soft microenvironment in the later stages of pancreatic islet development, thereby enhancing C-peptide secretion.

[0065] Dynamic adaptation: If cells aggregate into clusters (diameter > 100 μm), local gelation is required to maintain mechanical uniformity.

[0066] 2.4 Functional Verification and Parameter Optimization

[0067] Evaluation indicators:

[0068] Morphology: β cell cluster diameter (target 50–150 μm), edge smoothness (avoid fibrosis).

[0069] Molecular level: Expression level of FAK phosphorylation (p-FAKY397) (quantitative by Western Blot).

[0070] Functional testing: Compared with rigid culture dishes (~1 GPa), the GSIS response on a 3 kPa substrate was improved by 2–3 times.

[0071] Optimization strategy:

[0072] If insulin secretion is insufficient, the stiffness can be fine-tuned to 2.5 kPa and the stage 4 culture period extended by 2–3 days.

[0073] If cell aggregation is poor, increase collagen concentration (from 2 mg / mL to 3 mg / mL) or add laminin (1 μg / cm). 2 ).

[0074] 3. Cell function assessment

[0075] Glucose-stimulated insulin secretion (GSIS): After high glucose stimulation, insulin secretion reaches 80%-90% of that in native cells.

[0076] Biomarker expression: PDX1, NKX6.1, and C-peptide positivity rates >80%. All functional assays require the inclusion of positive controls (human primary pancreatic islet cells) and negative controls (undifferentiated stem cells).

[0077] 3.1 Morphological and biomarker detection

[0078] 3.1.1 Cell morphology observation

[0079] Aggregate morphology: Mature β cell clusters should be regular spherical, 50-150 μm in diameter, with clear edges.

[0080] Cell polarity: The polarization distribution of intracellular insulin vesicles was observed using transmission electron microscopy.

[0081] 3.1.2 Immunofluorescence staining

[0082] Core biomarker detection:

[0083] Early biomarkers: PDX1 (pancreatic precursor), NKX6.1 (pancreatic progenitor cells)

[0084] Maturation markers: C-peptide, MAFA, insulin

[0085] Colocalization analysis: Confocal microscopy was used to analyze the proportion of PDX1+ / NKX6.1+ double-positive cells (target >80%). 3.2 Gene expression analysis

[0086] 3.2.1 qPCR detection

[0087] Key genes:

[0088] Early genes: SOX17, FOXA2 (endoderm)

[0089] Differentiation genes: NGN3, NEUROD1 (endocrine precursor)

[0090] Functional genes: INS, GCK (glucose sensing)

[0091] Reference genes: GAPDH or β-actin as internal controls

[0092] 3.2.2 Single-cell RNA sequencing

[0093] Analysis content:

[0094] Cellular heterogeneity assessment

[0095] The proportion of non-target cell types (such as α cells) is controlled (<5%).

[0096] Mature β cell characteristic gene expression profile

[0097] 3.3 Functional Testing

[0098] 3.3.1 Glucose-stimulated insulin secretion (GSIS) experiment

[0099] Experimental procedure:

[0100] 1. Balance with low sugar (2.8mM) for 1 hour

[0101] 2. High glucose (16.7mM) stimulation for 1 hour

[0102] 3. Potassium ion (30mM KCl) stimulation confirms calcium-dependent secretion.

[0103] Detection method: Insulin content is measured by ELISA or chemiluminescence immunoassay.

[0104] Qualification Standard:

[0105] Stimulation index (high glucose / low glucose secretion ratio) ≥2

[0106] Absolute secretion volume ≥15 μIU / 10^6 cells / hour

[0107] 3.3.2 Calcium Ion Imaging

[0108] Experimental methods:

[0109] Marked with Fluo-4AM dye

[0110] Confocal microscopy records calcium oscillations under glucose stimulation in real time.

[0111] Evaluation indicators:

[0112] Oscillation frequency (target: 2-4 times / minute)

[0113] Oscillation amplitude (ΔF / F0 ≥ 20%)

[0114] 3.4 Long-term functional stability assessment

[0115] 3.4.1 In vitro culture stability

[0116] Test method: Culture continuously for 4 weeks, with GSIS detection performed weekly.

[0117] Acceptable standard: Insulin secretion remains at more than 70% of the initial value in week 4. 3.4.2 In vivo functional verification

[0118] Animal model: STZ-induced diabetic immunodeficient mice

[0119] Transplantation protocol: Subcapsular transplantation of 2 × 10^6 β-cell clusters

[0120] Evaluation indicators:

[0121] Fasting blood glucose recovery time (target: <14 days)

[0122] Glucose tolerance test (AUC decrease >40% 4 weeks post-transplantation)

[0123] 3.5 Quality Control Standards

[0124] 3.5.1 Purity Standard

[0125] β-cell percentage: C-peptide positive cells >80%

[0126] Non-target cells: α cells (glucagon-positive) <5%, δ cells <3% 3.5.2 Functional criteria

[0127] Basal insulin secretion: <5 μIU / 10^6 cells / hour at 5 mM glucose; Glucose responsiveness: ≥15 μIU secreted after stimulation with 16.7 mM glucose.

[0128] 3.5.3 Safety Testing

[0129] Karyotype analysis confirmed no chromosomal abnormalities.

[0130] Tumorigenicity test: No teratoma formation was observed in nude mice after subcutaneous transplantation for 3 months.

[0131] 3.6 Batch Consistency Assessment

[0132] 3.6.1 Comparison of multiple batches

[0133] The difference in biomarker expression among three consecutive batches of cell products was <15%.

[0134] The coefficient of variation of the GSIS stimulation index is <10%.

[0135] 3.6.2 Cryopreservation Recovery Test

[0136] Survival rate after resuscitation >85%

[0137] Post-recovery GSIS functional retention rate >90%

[0138] originality

[0139] 1. Precise timing control technology for signal paths

[0140] This paper proposes a time-series switching strategy of Wnt activation (stage 1) → Notch inhibition (stage 3) for the first time, which breaks through the efficiency bottleneck caused by traditional continuous activation / inhibition.

[0141] Innovative discovery phase 2: Removal of Wnt signaling can prevent abnormal proliferation of pancreatic progenitor cells (reducing non-target cells by 30%). 2. Bionic mechanical microenvironment dynamic adaptation system.

[0142] Unique "3kPa stiffness matrix + stage-dependent stiffness adjustment" technology:

[0143] Phases 1-2 used 4 kPa to promote precursor amplification

[0144] Phase 3-4: Reduced to 2kPa for enhanced functional maturity

[0145] For the first time globally, dynamic coupling regulation of matrix stiffness and differentiation stage has been achieved.

[0146] 3. Glucose Fluctuation Training Program

[0147] Develop "12-hour alternating high and low sugar" "A unique cultivation method that enhances β-cell glucose sensitivity by 2.1 times. This innovative discovery shows that the training can downregulate the Wnt pathway to prevent dedifferentiation (patented proprietary technology)."

[0148] 4. Three-dimensional aggregate quality control system

[0149] A three-dimensional parametric model of "diameter-stiffness-vascularization" was established to ensure a post-transplant survival rate of >90%.

[0150] A pioneering quantification standard for aggregate edge smoothness (patent-specific evaluation index)

[0151] Beneficial effects

[0152] 1. Breakthrough improvement in differentiation efficiency

[0153] β-cell purity was increased from 50-60% in existing technologies to 82±5% (C-peptide + cell ratio).

[0154] The proportion of non-target α cells should be controlled to <5% (compared to 15-20% in traditional methods).

[0155] 2. Its function is similar to that of a progenitor cell.

[0156] The glucose stimulation index reached 2.8 ± 0.3 (3.0-3.5 for primary pancreatic islets).

[0157] Calcium oscillation characteristics:

[0158] Frequency: 2.4 times / minute (originally 2.5 times / minute)

[0159] Synchronization coefficient: 0.71 (originally 0.75)

[0160] 3. Advantages in clinical translation

[0161] The survival rate of cryopreserved tissue after thawing is 93% (compared to <70% using traditional methods).

[0162] Therapeutic effects in animal models:

[0163] Blood sugar levels returned to normal in 12.3 days (40% shorter than existing technologies).

[0164] The therapeutic effect was maintained for >60 days (compared to 35-45 days in the control group).

[0165] 4. Feasibility of standardized production

[0166] Batch-to-batch coefficient of variation <8% (industry standard >20%)

[0167] A single batch can produce up to 1×10^9 functional β cells (meeting clinical transplantation needs).

[0168] 5. Security breakthrough

[0169] Rigorous tumorigenicity testing:

[0170] No teratoma formation was observed during the 3-month observation period.

[0171] Genome stability verification (no mutations found in whole-exome sequencing) Attached Figure Description

[0172] Figure 1 Flowchart of the phased regulation of stem cell differentiation into pancreatic β cells.

[0173] Figure 2 Schematic diagram of biomimetic matrix stiffness control and application.

[0174] Figure 3 The effect of glucose fluctuation training on β-cell function.

[0175] Figure 4 Comparison of treatment effects in animal models.

[0176] Figure 5 Summary of comparative data under different experimental conditions. Detailed Implementation

[0177] Example 1: Wnt / Notch Timing Control Optimization

[0178] 1. Human pluripotent stem cells (hESCs) were seeded on Matrigel coated plates and maintained using mTeSR1 medium.

[0179] 2. Stage 1: Cultured in RPMI medium containing CHIR99021 (3μM) + Activin A (100ng / mL) for 3 days.

[0180] 3. Phase 2: Culture in DMEM / F12 medium containing FGF10 (50 ng / mL) + RA (2 μM) for 4 days.

[0181] 4. Stage 3: Add DAPT (10 μM) to inhibit Notch and incubate for 5 days.

[0182] 5. Phase 4: IGF-1 (50 ng / mL) + Exendin-4 (50 nM) cultured for 7 days, with glucose fluctuation stimulation for the last 3 days.

[0183] Example 2: The Influence of Bionic Matrix Stiffness

[0184] 1. Prepare hydrogels with different stiffnesses (1 kPa, 3 kPa, 5 kPa).

[0185] 2. β cells differentiated on a 3 kPa substrate showed the highest insulin secretion (2-fold increase in GSIS response vs. conventional culture).

[0186] Example 3: Functional Verification

[0187] ELISA results showed that differentiated β cells secreted 15-18 μIU / 10^6 cells / hour of insulin when stimulated with 20 mM glucose (close to 20 μIU for native pancreatic β cells).

[0188] Immunofluorescence confirmed that the percentage of PDX1+ / NKX6.1+ cells was >85%.

[0189] Example 4: Comparison of the effects of different matrix stiffness on β-cell maturation

[0190] 4.1 Experimental Design

[0191] Preparation of collagen / alginate hydrogels with stiffnesses of 1 kPa, 3 kPa, and 5 kPa

[0192] The same stem cell line was divided into three groups and seeded into different substrates.

[0193] The same differentiation scheme was used (as in Example 1).

[0194] 4.2 Key Findings

[0195] In the 3kPa group: the proportion of PDX1+ / NKX6.1+ cells reached 88±3%; the GSIS stimulation index was 2.8±0.3.

[0196] 1 kPa group: Poor cell aggregation, and decreased expression of functional markers by 15-20%.

[0197] The 5 kPa group showed an increased proportion of α cells (12 ± 2%).

[0198] 4.3 Conclusion

[0199] A stiffness of 3 kPa is closest to the mechanical environment of natural pancreatic islets.

[0200] Both excessively hard and excessively soft matrix can affect β-cell purity and function.

[0201] Example 5: Notch Suppression Duration Optimization Experiment

[0202] 5.1 Experimental Procedure

[0203] Set the gradient for the duration of Notch inhibitor DAPT treatment:

[0204] Control group: No inhibition throughout the entire process

[0205] Group A: 8-10 days (2 days)

[0206] Group B: 8-12 days (4 days)

[0207] Group C: 8-14 days (6 days)

[0208] 5.2 Results Analysis: Group B (4-day suppression):

[0209] The peak expression level of Ngn3 increased by 2.1 times.

[0210] The highest yield of β cells was observed (78±5%).

[0211] Longer inhibition (Group C) led to increased apoptosis.

[0212] 5.3 Application Recommendations

[0213] The optimal Notch inhibition window is day 8-12 of differentiation.

[0214] Inhibitor concentration needs to be adjusted in conjunction with cell density monitoring.

[0215] Example 6: Verification of the effect of dynamic glucose training

[0216] 6.1 Training Program

[0217] Experimental group: During the late differentiation stage (days 17-20), patients alternated every 12 hours.

[0218] 5mM glucose medium

[0219] 20mM glucose medium

[0220] Control group: Cultured at a constant 11mM glucose level

[0221] 6.2 Functionality Enhancement

[0222] Experimental group:

[0223] Increased expression of glucose sensing-related genes (GLUT2, GCK) led to a 1.8-2.5-fold improvement in calcium oscillation synchrony (correlation coefficient increased from 0.3 to 0.7).

[0224] Control group:

[0225] It exhibits persistently high basal secretion (loss of glucose sensitivity).

[0226] 6.3 Mechanism Analysis

[0227] Transcriptome analysis showed:

[0228] Glucose fluctuation training upregulates the insulin synthesis pathway

[0229] Downregulate Wnt / β-catenin signaling (to prevent dedifferentiation)

[0230] Example 7: Functional retention test after cryopreservation and thawing 7.1 Cryopreservation protocol

[0231] Use a cryopreservation solution containing 10% DMSO + 20% FBS.

[0232] Programmed cooling: -1℃ / minute to -80℃ followed by liquid nitrogen storage. 7.2 Resuscitation assessment

[0233] Survival rate: 87±4% (trypan blue exclusion method)

[0234] Function recovery:

[0235] 24 hours post-resuscitation: GSIS functional recovery 82±6%

[0236] 72 hours after thawing: Fully restored to pre-freezing levels. 7.3 Optimization directions.

[0237] Adding trehalose (50mM) can further improve the survival rate to 93%. It is recommended to pre-culture for 48 hours after resuscitation before transplantation.

[0238] Example 8: The effect of different cell densities on differentiation

[0239] 8.1 Density Gradient Experiment

[0240] Inoculation density: 0.5 × 10⁻⁶ 5 1×10 5 2×10 5 cells / cm 2 Monitoring cell proliferation and differentiation efficiency at each stage

[0241] 8.2 Determination of Optimal Density

[0242] 1×10 5 cells / cm 2 Group:

[0243] Moderate cell contact leads to optimal concentration of autocrine factors.

[0244] The highest yield of β cells was observed (75±4%).

[0245] Excessive density leads to the formation of an oxygen-deficient core region.

[0246] Example 9: Animal Model Treatment Validation

[0247] 9.1 Transplantation Experiment

[0248] NOD / SCID diabetic mouse model (blood glucose >300 mg / dL)

[0249] Transplantation dose: 1000, 2000, 4000 IEQ / mouse

[0250] 9.2 Treatment efficacy

[0251] 2000IEQ Group:

[0252] Time to normalize blood glucose: 12.3 ± 2.1 days

[0253] Duration > 60 days

[0254] Histological findings:

[0255] Good vascularization (CD31+ area 15±3%)

[0256] No teratoma formation

[0257] This invention achieves efficient and functionally mature stem cell-derived pancreatic β-cell preparation by integrating temporal regulation of signaling pathways and optimization of biomimetic matrix, providing a new strategy for diabetes treatment.

Claims

1. A method for preparing pancreatic β cells derived from stem cells, characterized in that, Includes the following steps: Phased regulation of the Wnt and Notch signaling pathways; A biomimetic matrix stiffness culture system is adopted; Enhance β-cell functional maturation through glucose fluctuation training.

2. The method according to claim 1, characterized in that, The phased regulation of the Wnt and Notch signaling pathways includes: Phase 1 (0-3 days): activating the Wnt pathway and inducing endoderm differentiation in conjunction with TGF-β signaling; Phase 2 (4-7 days): Remove Wnt activation, add FGF10 and retinoic acid to promote pancreatic progenitor cell formation; Phase 3 (8-12 days): Inhibition of the Notch pathway to drive endocrine differentiation; Phase 4 (13-20 days): IGF-1 and Exendin-4 are used to promote β-cell maturation.

3. The method according to claim 2, characterized in that: Phase 1 used CHIR99021 (3 μM) and Activin A (100 ng / mL); Phase 3 used the Notch inhibitor DAPT (10 μM); Phase 4 involves alternating high and low glucose cultures (5mM and 20mM glucose are switched every 12 hours).

4. The method according to claim 1, characterized in that, The biomimetic matrix stiffness culture system is as follows: Collagen / alginate hydrogel, stiffness 1-5 kPa; In stages 1-2, a 3-5 kPa matrix was used to promote precursor amplification; Phase 3-4 reduces the pressure to 1-3 kPa to enhance functional maturity.

5. The method according to claim 4, characterized in that: The stiffness of the matrix is ​​controlled by crosslinking with EDC / NHS; The stiffness error was verified to be <10% using atomic force microscopy (AFM).

6. The method according to claim 1, characterized in that, The glucose fluctuation training includes: Periodic glucose concentration switching is performed during the later stages of differentiation (17-20 days); After each high glucose (20 mM) stimulation, the culture was restored to low glucose (5 mM).

7. The pancreatic β-cell clusters prepared by the method according to any one of claims 1-6, characterized in that: Diameter 50-150μm, edge smoothness >90%; The proportion of C-peptide-positive cells is ≥80%; Glucose stimulation index ≥2.

5.

8. A cell preparation for the treatment of diabetes, characterized in that: It includes the pancreatic β-cell clusters as described in claim 7; Survival rate after cryopreservation and thawing ≥85%; After transplantation, the blood glucose level in diabetic model animals can return to normal within ≤14 days.

9. A dedicated culture system for the method described in claims 1-6, characterized in that... include: Multi-channel, stiffness-adjustable culture device; Programmable glucose concentration switching module; Real-time cell function monitoring sensor.

10. The culture system according to claim 9, characterized in that: The culture device can dynamically adjust the substrate stiffness (adjustable from 1-5 kPa); The glucose switching module supports automatic timed fluid replacement; The monitoring sensor can detect insulin secretion in real time.

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