Method for inducing human adipose-derived stem cells to differentiate into insulin-secreting cells in vitro and application of method

Abstract

The invention relates to the technical field of regenerative medicine and cell therapy, in particular to a method for inducing mesenchymal stem cells to differentiate into insulin-secreting cells and application of the method. According to the method, a culture system is regulated and controlled by stages, the glucose concentration is respectively regulated in different induction stages, an NAD + precursor substance, a fatty acid composition and an antioxidant factor are added, and a three-dimensional microcarrier culture mode is combined, so that the cell metabolism state, the oxidation-reduction balance and the space structure are dynamically regulated and controlled. Under the condition of not depending on recombinant protein inducing factors such as Activin A and Wnt3a, the efficient directional differentiation of the mesenchymal stem cells to the insulin-secreting cells is realized. By adopting the method disclosed by the invention, the differentiation efficiency can be improved, the induction period can be shortened, the mature beta-like cells with a stable glucose stimulation dependent insulin secretion function can be obtained, and the method is suitable for induced differentiation and related research application of the human adipose-derived mesenchymal stem cells.

Description

Technical Field

[0001] This invention relates to the fields of regenerative medicine and cell therapy, specifically to a method for inducing human adipose stem cells to differentiate into insulin-secreting cells in vitro and its application. Background Technology

[0002] Diabetes mellitus is one of the most prevalent chronic metabolic diseases worldwide, characterized by a reduction in the number or dysfunction of pancreatic β-cells, leading to an absolute or relative deficiency of insulin. While insulin injections can control blood sugar, they cannot mimic the physiological rhythm of insulin secretion and carry the risk of hypoglycemia. Islet transplantation can restore endogenous glucose regulation, but it is limited by severe donor shortages, immune rejection, and the risks of long-term immunosuppressant use. Therefore, in vitro induction of functional insulin-secreting cells from stem cells has become a highly promising alternative strategy.

[0003] Mesenchymal stem cells (MSCs) are widely studied for the in vitro induction of insulin-secreting cells due to their abundant sources, low immunogenicity, strong in vitro expansion capacity, and lack of tumorigenic risk. Among them, MSCs derived from human adipose tissue are considered ideal autologous cell sources because they are readily available, abundant, and exhibit good cryopreservation stability. However, current methods for inducing MSCs into insulin-secreting cells often employ alternating low-glucose and high-glucose culture media, the addition of inducing factors such as nicotinamide, β-mercaptoethanol, GLP-1, and Activin A, and a multi-stage culture process to simulate pancreatic development. Although these methods can induce cells to express some islet-related markers, they still suffer from significant drawbacks such as long induction cycles, unstable efficiency, insufficient induced cell functionality, and a lack of systematic culture medium design.

[0004] Numerous reports on "islet-like cells" primarily focus on the expression of islet-related markers, exhibiting limited or unstable insulin secretion capacity under glucose stimulation, failing to meet practical application requirements. The root cause lies in the fact that existing technologies often employ empirical addition of inducing factors, neglecting the metabolic state required for pancreatic β-cell maturation and paying insufficient attention to key factors such as the NAD⁺ / NADH ratio, fatty acid-glucose synergistic metabolism, and endoplasmic reticulum stress regulation. Furthermore, existing patents largely concentrate on induction methods, lacking protection for the system design and functional division of the culture medium composition itself, making it easily circumvented. Therefore, there is an urgent need for a culture medium system with a rationally designed composition, a clearly defined mechanism, and the ability to be protected as an independent product, to achieve efficient induction of mesenchymal stem cells into functional insulin-secreting cells. Summary of the Invention

[0005] The purpose of this invention is to provide a culture medium based on a multi-dimensional synergistic induction system of metabolic reprogramming, signaling pathway regulation, and endoplasmic reticulum homeostasis maintenance, which precisely regulates intracellular NAD+. +This invention significantly improves differentiation efficiency, shortens the induction period, and yields mature β-like cells with stable glucose-stimulated insulin secretion (GSIS) without relying on recombinant protein inducing factors such as Activin A and Wnt3a, by adjusting NADH redox state, exogenous fatty acid composition ratio, dynamic glucose supply concentration, and antioxidant stress level. This invention is particularly suitable for mesenchymal stem cells derived from human adipose tissue and can be used as a standalone kit for preclinical studies and drug screening in diabetic cell replacement therapy.

[0006] To address the aforementioned technical problems, this invention provides a method for inducing mesenchymal stem cells to differentiate into insulin-secreting cells, comprising the following steps: The first step, cell seeding and expansion, involves seeding mesenchymal stem cells into a culture system containing basal medium for adherent culture, and culturing them at 37°C and 5% CO2 for 1–3 days until the cell confluence reaches 70–90%. The second step, the metabolic pre-programming stage, involves placing the cells obtained in the first step into a low-concentration glucose DMEM medium containing low-adhesion microcarriers. The microcarriers are kept in suspension by slow continuous shaking or intermittent gentle shaking for 1–3 days. The third step, the lineage-directed differentiation stage, involves placing the cells obtained in the second step in a medium-concentration glucose DMEM medium containing medium-adhesive microcarriers. The microcarriers are kept in suspension by slow continuous shaking or intermittent gentle shaking for 1–3 days. The fourth step, the terminal functional maturation stage, involves placing the cells obtained in the third step in a high-concentration glucose DMEM medium containing highly adhesive microcarriers. The microcarriers are kept in suspension by slow continuous shaking or intermittent gentle shaking for 4–8 days to obtain insulin-secreting cells.

[0007] The second step culture medium also contains a basic inducer and NAD⁺ precursor.

[0008] The third step culture medium also contains a basic inducer, NAD⁺ precursor, and a fatty acid composition.

[0009] The fourth step culture medium also contains a basic inducer, NAD⁺ precursor, fatty acid composition, and an antioxidant-endoplasmic reticulum homeostasis regulator.

[0010] The fatty acid composition comprises oleic acid and palmitic acid, wherein the molar ratio of oleic acid to palmitic acid is 3:1 to 6:1.

[0011] The mesenchymal stem cells mentioned above are human adipose-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, or umbilical cord mesenchymal stem cells.

[0012] In the second step, the low-adhesion microcarriers are selected from polystyrene microspheres or Alginate microspheres with a particle size of 50–200 μm.

[0013] In the third step, the medium-adhesive microcarriers have a particle size of 100–250 μm and are coated with a small amount of collagen or gelatin on their surface.

[0014] In the third step, the highly adhesive ECM enriched microcarriers have a particle size of 150–300 μm and their surface contains collagen I / IV, HA, or laminin.

[0015] The present invention also provides the application of the above method in the preparation of insulin-secreting cells.

[0016] Beneficial effects of the present invention Compared with the prior art, the present invention has the following outstanding advantages: First, the induction efficiency is significantly improved. Through precise regulation of metabolic preprogramming and lineage-directed differentiation, the differentiation cycle can be shortened to 10–12 days, and the proportion of PDX1⁺ / NKX6.1⁺ double-positive progenitor cells is much higher than that of traditional methods.

[0017] Second, pancreatic islet function was significantly enhanced. The obtained cells possessed the real ability to stimulate insulin secretion with glucose, the GSIS ratio remained stable between 2.8 and 3.6, and the secretion of C-peptide increased at least twice under high glucose concentration conditions, approaching the level of human primary islets.

[0018] Third, cell survival rate was significantly improved. Through terminal protection with rhodioloside or taurine, the cell survival rate in the terminal stage was significantly higher than that in the unprotected group, ensuring the abundance of functional cell populations. Detailed Implementation

[0019] This invention constructs a multi-dimensional synergistic metabolism-signal-homeostasis regulation network, which forms a positive feedback loop through precise time windows and concentration ratios, jointly promoting the efficient transformation of human adipose-derived mesenchymal stem cells (hADSCs) into functional insulin-secreting cells (IPCs).

[0020] Mechanism of metabolic regulation of NAD⁺ / NADH. The function of pancreatic β-cells is highly dependent on ATP produced by mitochondrial oxidative phosphorylation, and the efficiency of ATP production is directly regulated by the intracellular redox state of NAD⁺ / NADH. NAD⁺ is not only a key coenzyme in the tricarboxylic acid cycle and electron transport chain, but also an essential substrate for the deacetylase SIRT1. SIRT1 regulates PGC-1α through deacetylation, the latter being a major regulator of mitochondrial biosynthesis. In aging or diabetes, decreased NAD⁺ levels lead to reduced SIRT1 activity, increased PGC-1α acetylation and inactivation, and a decline in mitochondrial number and function, ultimately resulting in impaired glucose-stimulated insulin secretion.

[0021] This invention introduces the NAD⁺ precursor NMN in the early induction stage (metabolic preprogramming phase), which can be efficiently converted into NAD⁺ via the nicotinamide phosphoribosyltransferase (NAMPT) pathway, significantly increasing intracellular NAD⁺ reserves and the NAD⁺ / NADH ratio. The elevated NAD⁺ levels activate SIRT1, thereby promoting PGC-1α deacetylation and nuclear translocation, and upregulating the expression of genes related to mitochondrial DNA replication, respiratory chain complex assembly, and fatty acid β-oxidation. This metabolic reprogramming process lays the energy foundation for the subsequent high-energy-consuming synthesis and secretion of insulin.

[0022] Mechanism of synergistic induction by fatty acids. Fatty acids are important energy substrates for β cells, but different types of fatty acids have drastically different effects on cell fate. Saturated fatty acids such as palmitic acid are prone to lipotoxicity at high concentrations, leading to endoplasmic reticulum stress, reactive oxygen species accumulation, and apoptosis; while monounsaturated fatty acids such as oleic acid have a protective effect, promoting triglyceride synthesis to isolate free palmitic acid and activating peroxisome proliferator-activated receptors, thereby upregulating transcriptional regulatory pathways related to PDX1 and MAFA and enhancing the expression of key pancreatic transcription factors.

[0023] The key breakthrough of this invention lies in discovering and defining the optimal molar ratio of oleic acid to palmitic acid within the range of 3:1 to 6:1. At this ratio, oleic acid not only serves as an energy substrate for β-oxidation but also acts as an acyl donor to esterify palmitic acid into neutral lipid droplets for storage, effectively avoiding lipotoxicity caused by the accumulation of free palmitic acid. Simultaneously, the presence of an appropriate amount of palmitic acid enhances cell membrane fluidity and promotes the correct localization and function of the glucose transporter GLUT2 on the plasma membrane. This specific fatty acid combination mimics the physiological lipid composition of the pancreatic developmental microenvironment, providing precise metabolic signals for hADSCs and guiding their directional differentiation into the pancreatic lineage. Furthermore, human adipose-derived mesenchymal stem cells, due to their high expression of the fatty acid transporter CD36, exhibit significantly higher oleic acid uptake efficiency than bone marrow or umbilical cord-derived mesenchymal stem cells, thus demonstrating a more sensitive and efficient response to this fatty acid combination.

[0024] Mechanism of Glucose Gradient Metabolic Training. Glucose is not only the energy source for β cells but also a key signaling molecule for their functional maturation. The development of true β cells involves a gradual transition from a low-glucose embryonic environment to a high-glucose adult environment. Based on this, this invention designs a three-stage glucose concentration increment strategy: the first stage (5.5 mM-6.0 mM) simulates the low-glucose state of early embryonic development, inhibiting mTOR pathway activity and promoting autophagy to clear abnormal mitochondria, creating a "clean" starting point for subsequent metabolic reprogramming; the second stage (11.0 mM-12.0 mM) moderately activates glucokinase (GCK) and glycolysis pathways, initiating the expression of pancreatic progenitor cell markers such as PDX1; the third stage (25.0 mM-26.0 mM) provides strong glucose stimulation, training the cells' glucose transport, glycolysis rate, and mitochondrial respiratory response threshold, ultimately establishing a stable metabolic-secretion coupling mechanism. If a constant high glucose concentration is used throughout, cells exhibit problems such as sluggish glucose sensing, high secretory heterogeneity, and functional instability due to a lack of adaptive training.

[0025] Antioxidant-endoplasmic reticulum homeostasis regulation mechanism. During terminal differentiation, cells need to synthesize a large amount of insulin precursor protein in a short period of time. This process places a huge burden on the endoplasmic reticulum folding system, easily inducing endoplasmic reticulum stress. At the same time, mitochondrial respiration is enhanced under high glucose stimulation, inevitably producing excessive reactive oxygen species. If left untreated, these two stresses will synergistically activate the apoptosis pathway, leading to the death of a large number of functional cells.

[0026] This invention introduces rhodioloside or taurine in the third stage of culture. Rhodioloside works through a dual mechanism: on the one hand, it activates the nuclear factor E2-related factor 2 (Nrf2) pathway, upregulating the expression of antioxidant enzymes such as heme oxygenase-1 and superoxide dismutase 2, effectively scavenging reactive oxygen species; on the other hand, it inhibits the PERK-eIF2α-ATF4-CHOP endoplasmic reticulum stress-induced apoptosis pathway, significantly reducing Caspase-3 activation levels, thereby greatly improving the survival rate and functional stability of terminal cells.

[0027] Taurine provides protection through different mechanisms: as an organic osmolarity regulator, it maintains cell volume stability in a high glucose environment; by weakly chelating free cytoplasmic calcium ions, it prevents mitochondrial damage caused by calcium overload; at the same time, it promotes glutathione synthesis, enhances overall antioxidant capacity, and optimizes the redox environment of the endoplasmic reticulum lumen to support the correct folding of insulin precursors.

[0028] This invention does not require the use of Activin A during core induction and can still yield functionally mature insulin-secreting cells even with significantly reduced GLP-1 usage or without adding GLP-1. NMN enhances NAD. +After reaching a certain level, enhanced SIRT1 activity can inhibit SMAD7 expression, thereby relieving the negative regulation of TGF-β receptor and indirectly enhancing SMAD2 / 3 phosphorylation, partially mimicking the endodermal-inducible effect of Activin A. Simultaneously, oleic acid activation of PPARδ can promote CREB phosphorylation, enhance insulin gene transcription, and partially compensate for the role of the GLP-1 / cAMP pathway.

[0029] Current methods for inducing mesenchymal stem cells into insulin-secreting cells mostly rely on two-dimensional adherent culture or a single three-dimensional culture condition to complete the entire induction process. However, islet development and β-cell maturation do not occur in a static or singular microenvironment, but are accompanied by continuous changes in cell metabolic state, cell-cell interaction patterns, and cell-matrix adhesion. Two-dimensional adherent culture forces cells to maintain a high degree of adhesion and spread in the early stages of induction, which can easily solidify the mesenchymal phenotype and inhibit the initiation of endocrine lineage-related signaling pathways. On the other hand, a single three-dimensional culture condition remains unchanged throughout the induction process, making it difficult to simultaneously meet the high cell-cell contact required in the early stages of induction and the stable ECM support environment required in the terminal maturation stage. Therefore, a single culture mode cannot simultaneously meet the differentiated spatial microenvironment requirements of different stages in islet-like cell development at the mechanistic level.

[0030] This invention reveals that the regulatory effect of metabolic reprogramming factors (including nicotinamide, NMN, fatty acids, and dynamic glucose supply) on the differentiation direction of mesenchymal stem cells is significantly dependent on the three-dimensional spatial structure and adhesion state of the cells. Microcarriers with different adhesion properties can construct differentiated cellular mechanical microenvironments during induction, thereby synergistically regulating metabolism to achieve phased and directed differentiation.

[0031] In the metabolic preprogramming stage, a three-dimensional suspension culture environment is constructed using low-adhesion inert microcarriers. These microcarriers are preferably polystyrene low-adhesion microcarriers without ECM coating, such as Cytodex®3 or equivalent products. These microcarriers provide only limited physical support and do not induce significant cell-matrix adhesion, allowing cells to form loose three-dimensional aggregates primarily through cell-cell contact. Under these low-adhesion conditions, cytoskeleton tension is reduced, and mechanosensitive signals such as YAP / TAZ are suppressed, which is beneficial for NAD+. + The activation of metabolic pathways and the remodeling of mitochondrial function drive the transformation of cells from a metabolic mode dominated by glycolysis to a metabolic state characteristic of endocrine cells.

[0032] As the induction progresses into the lineage orientation phase, a simple low-adhesion suspension state is insufficient to maintain stable expression of endocrine transcription factor networks. This invention introduces moderately adhesive microcarriers to provide adequate cell-matrix signaling while maintaining the three-dimensional aggregated structure. These moderately adhesive microcarriers are preferably polystyrene microcarriers coated with gelatin or type I collagen, such as Cytodex®1 or equivalent products. These microcarriers enhance the cell's response to fatty acid signals and moderate-to-high glucose environments, promoting the sustained expression of pancreatic endocrine-related transcription factors (such as PDX1 and NKX6.1), thereby achieving stable orientation towards the pancreatic endocrine lineage.

[0033] During terminal differentiation, the establishment of insulin secretion function depends not only on the insulin gene expression level but also on the stability of islet-like structures and the spatial organization of intercellular signals. This invention further employs highly adhesive ECM-enriched microcarriers, preferably collagen or hyaluronic acid-based three-dimensional porous microcarriers, such as Cultispher®-G or equivalent products. These microcarriers provide extracellular matrix support within the cell population, promoting cell polarity establishment and spatial rearrangement of secretion-related organelles, thereby enhancing the synchronous response of the cell population to glucose stimulation. Simultaneously, ECM-mediated adhesion signaling significantly reduces stress and apoptosis levels during terminal differentiation, increasing the proportion of functional insulin-secreting cells and population coherence.

[0034] In contrast, continuously suspended systems lacking ECM support, or those introducing highly adhesive microcarriers at an early stage, struggle to simultaneously achieve metabolic reprogramming efficiency, lineage stability, and end-function maturity. The following examples illustrate the implementation of the present invention in detail, thereby providing a full understanding of how the invention uses technical means to solve technical problems and achieve technical effects, and enabling its implementation.

[0035] All experiments in this invention used the same batch of human adipose-derived stem cells (hADSCs). The cells were obtained from 5 healthy adult donors (aged 30–45 years) and were isolated from liposuction waste adipose tissue by type I collagenase (Sigma-Aldrich, C0130) digestion at 37°C for 60 min. The cells were then expanded in vitro to the 4th generation (P4).

[0036] Phenotypic identification by flow cytometry: The CD73-PE (Abcam, ab23989), CD90-FITC (Abcam, ab11416), CD105-APC (Abcam, ab11407), CD34-FITC (Abcam, ab81289), and CD45-PE (Abcam, ab28153) antibodies were detected. The positive rates of CD73⁺ / CD90⁺ / CD105⁺ were all greater than 95%, and the negative rates of CD34⁻ / CD45⁻ were all less than 2%, which met the definition criteria of mesenchymal stem cells by the International Society for Cell Therapy (ISCT).

[0037] Functional assessment parameters included: glucose-stimulated insulin secretion ratio (GSIS), C-peptide secretion (pg / mL), percentage of PDX1⁺ / NKX6.1⁺ double-positive cells (%), cell viability (%), intracellular NAD⁺ / NADH ratio, and relative expression of cleaved caspase-3 protein. All experiments were repeated three times, and data are expressed as mean ± standard deviation.

[0038] Example 1

[0039] Human adipose-derived mesenchymal stem cells were used at a rate of 1×10⁻⁶ 6 Cells were seeded at a density of [number] cells / mL in T75 culture flasks and added to DMEM / F12 basal medium (BIOFIL, DME101500), supplemented with 2% (v / v) human serum albumin (Ambroxol, P100722393) and 2 mM L-glutamine (Gibco, A4000225301). The flasks were incubated statically at 37°C, 5% CO2, and saturated humidity for 2 days. After cell attachment, the medium was replaced with fresh medium, and the culture continued until cell confluence reached 90%. Subsequently, a three-stage differentiation induction process was performed: Phase 1 Metabolic Preprogramming: The original culture medium was aspirated, and the cells were gently washed once with pre-warmed PBS. Then, pre-prepared low-concentration glucose DMEM medium (BIOFIL, glucose concentration 5.5 mM) containing low-adhesion microcarriers was added. The microcarriers were polystyrene low-adhesion microcarriers Cytodex®3 (particle size 50–150 μm, Sigma-Aldrich, GE17-0485-03), with a microcarrier concentration of 2 mg / mL. The medium was supplemented with 10 mM nicotinamide (Sigma-Aldrich, N0636), 1 mM β-mercaptoethanol (Sigma-Aldrich, M6250), and 250 μM β-nicotinamide mononucleotide (NMN) (MedChemExpress, HY-13428), for a total volume of 15 mL. The cells were suspended in suspension using a low-speed shaker or static intermittent gentle shaking method to allow the cells to form loose three-dimensional aggregates on the microcarrier surface without complete spreading. The culture was then placed at 37°C and 5% humidity. Incubate in a CO2 incubator for 2 days.

[0040] Second-stage lineage orientation: Allow the microcarriers to settle, discard the supernatant culture medium, and gently wash once with preheated PBS to remove residual first-stage culture medium. Then add pre-prepared medium-concentration glucose DMEM culture medium (self-prepared, based on high-concentration DMEM glucose culture medium diluted to a final glucose concentration of 11.0 mM) containing moderately adhesive microcarriers. The microcarriers are polystyrene microcarriers Cytodex® 1 (particle size 130–220 μm, Sigma-Aldrich, GE17-0448-02) coated with gelatin or type I collagen, at a dosage of 3 mg / mL. The culture medium is supplemented with 10 mM nicotinamide, 1 mM β-mercaptoethanol, 250 μM NMN, 150 μM oleic acid (Sigma-Aldrich, O1008), and 30 μM palmitic acid (Sigma-Aldrich, P9767). Oleic acid and palmitic acid were dissolved in 0.1 M NaOH beforehand, and then mixed with 1% BSA solution to form a fatty acid-BSA complex. The final BSA concentration was 0.4% (w / v, g / mL) to ensure water solubility and bioavailability. The total volume was 15 mL. The cells were cultured in suspension under low-speed shaking or intermittent gentle shaking conditions. During this stage, the cells formed a stable three-dimensional multicellular aggregation structure on the surface of the microcarrier, promoting directional differentiation into the pancreatic endocrine lineage. The cells were cultured for 2 days.

[0041] The third stage, final maturation: The culture medium was removed and washed with PBS in the same manner as described above. Then, pre-prepared high-concentration glucose DMEM medium (Gibco, glucose concentration 25.0 mM) containing highly adhesive ECM enriched microcarriers was added. The microcarriers were hyaluronic acid / collagen composite microcarriers or collagen-coated microcarriers Cultispher®-G (particle size 150–300 μm, Sigma-Aldrich, M9418), at a concentration of 4 mg / mL. The medium was supplemented with 10 mM nicotinamide, 1 mM β-mercaptoethanol, 250 μM NMN, 150 μM oleic acid, 30 μM palmitic acid (added as a fatty acid-BSA complex in the same manner as above), and 50 μM rhodioloside (Sigma-Aldrich, S7917). The total volume was 15 mL. The microcarriers were kept in suspension by slow continuous shaking or intermittent gentle shaking for 6 days, during which the medium was replaced with fresh medium every 48 hours.

[0042] After induction, cells were collected using Accutase cell digestion solution (Sigma-Aldrich, A6964), counted, and then subjected to functional assessment.

[0043] The GSIS detection method is as follows: Take 1×10 5 Cells were seeded per well in 24-well plates and starved for 2 hours (Krebs-Ringer buffer, 2.8 mM glucose), followed by stimulation for 1 hour with 2.8 mM (low glucose) and 25 mM (high glucose) Krebs-Ringer buffer, respectively. Supernatants were collected, and secretion levels were measured using a human insulin ELISA kit (Mercodia, 10-1113-01) and a human C-peptide ELISA kit (Mercodia, 10-1137-01). GSIS = high glucose insulin secretion / low glucose insulin secretion. The double positivity rate of PDX1 and NKX6.1 was detected by flow cytometry (antibodies: Abcam ab47381 + Abcamab192399). Cell viability was determined using trypan blue staining. The NAD⁺ / NADH ratio was determined using the NAD / NADH Quantification Kit (Sigma-Aldrich, MAK037).

[0044] The relative expression level of Cleaved Caspase-3 protein was detected by Western blotting: Cells after induced differentiation were collected, and total protein was extracted using RIPA lysis buffer (containing protease inhibitors). Protein concentration was determined by the BCA method. 30 μg of total protein was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, and blocked with 5% skim milk for 1 h. Cleaved Caspase-3 primary antibody (Cell Signaling Technology, 9664S, diluted 1:1000) was added and incubated overnight at 4°C. HRP-labeled secondary antibody (1:5000) was incubated at room temperature for 1 h. The cells were developed using a chemiluminescence assay kit, and the band grayscale values ​​were analyzed using ImageJ software. GAPDH (Cell Signaling Technology, 5174S) was used as an internal control to calculate the relative expression level of Cleaved Caspase-3 protein.

[0045] Cellular functional heterogeneity assessment: Single-cell flow cytometry was used to analyze the fluorescence intensity of PDX1, NKX6.1 and insulin cells, and the coefficient of variation (CV) was calculated for quantification.

[0046] The results showed that the GSIS ratio was 3.6 ± 0.2, C-peptide secretion reached 880 ± 45 pg / mL under high glucose conditions, the proportion of PDX1⁺ / NKX6.1⁺ double-positive cells was 51.3 ± 3.1%, the cell viability was 98.2 ± 0.8%, and the NAD⁺ / NADH ratio was 2.9 ± 0.2. These results indicate that the obtained cells possess true and stable glucose-responsive insulin secretion function, approaching the level of human primary pancreatic islets.

[0047] Example 2

[0048] Except for replacing the antioxidant rhodioloside in the third stage with taurine (Sigma-Aldrich, T8691), the other operating steps are completely consistent with those in Example 1.

[0049] The results showed that the GSIS ratio was 2.8 ± 0.2, the C-peptide secretion under high glucose conditions was 720 ± 38 pg / mL, the proportion of PDX1⁺ / NKX6.1⁺ double-positive cells was 45.7 ± 2.9%, and the cell viability was 95.4 ± 1.1%. These results demonstrate that taurine can effectively replace the cell-protective function of GLP-1, and that functional insulin-secreting cells with therapeutic potential can still be obtained even after complete removal of the expensive recombinant protein.

[0050] NAD + Effects of NADH on metabolic regulation Based on the scheme of Example 1, only the addition of NMN was adjusted, and the rest was the same as in Example 1. The following comparison group was set up.

[0051] Comparative Example 1-1: NMN was removed, and the remaining components and procedures were exactly the same as in Example 1; Comparative Examples 1-2: NMN concentration reduced to 25 μM, added throughout the process; Comparative Examples 1-3: NMN concentration increased to 1000 μM, added throughout the process; Comparative Examples 1-4: 250 μM NMN was only added in the second stage with a lineage-directed approach, and no NMN was added at other times; Comparative Examples 1-5: 250 μM NMN was added only during the final maturation stage in the third phase, and no NMN was added at other times.

[0052] The test results are shown in Table 1.

[0053] Table 1: Test Results Group GSIS ratio NAD⁺ / NADH ratio PDX1⁺ / NKX6.1⁺ Double Positive Rate (%) Example 1 3.6 ± 0.2 2.9 ± 0.2 51.3 ± 3.1 Comparative Example 1-1 1.8 ± 0.1 1.6 ± 0.1 19.5 ± 1.8 Comparative Examples 1-2 2.1 ± 0.2 1.8 ± 0.1 25.4 ± 2.0 Comparative Examples 1-3 2.3 ± 0.2 2.0 ± 0.2 27.8 ± 2.3 Comparative Examples 1-4 2.9 ± 0.2 2.3 ± 0.2 42.3 ± 2.7 Comparative Examples 1-5 2.7 ± 0.2 2.1 ± 0.2 40.1 ± 2.5 The results showed that the GSIS ratio of Comparative Example 1-1 was only 1.8 ± 0.1, which was significantly lower than that of Example 1 (3.6 ± 0.2) (p < 0.001). At the same time, the double positivity rate of PDX1⁺ / NKX6.1⁺ was only 19.5%, indicating that without NAD⁺ supplementation, cells could not effectively initiate the pancreatic lineage orientation and functional maturation program, proving that the NAD⁺ metabolic reprogramming module is indispensable.

[0054] Although Comparative Examples 1-2 showed some improvement, their GSIS was only 2.1, indicating that the concentration was insufficient to fully activate the SIRT1-PGC-1α pathway. Comparative Examples 1-3 were actually weaker than the 250 μM group, possibly because high concentrations of NMN triggered NAD⁺ metabolic feedback inhibition or non-specific effects, proving that 250 μM was the optimal concentration.

[0055] Timing-specific validation: The GSIS of Comparative Examples 1-4 and 1-5 were 2.9 and 2.7, respectively. Although they were better than the group without NMN, they were significantly lower than the group with NMN throughout the entire process (3.6), indicating that NAD⁺ support needs to be maintained throughout the entire induction process—in the early stage for metabolic preprogramming and mitochondrial proliferation, and in the later stage for maintaining high-energy-consuming insulin synthesis and secretion.

[0056] Effect of the molar ratio of oleic acid to palmitic acid With the total fatty acid concentration fixed at 180 μM, based on Example 1, only the molar ratio of oleic acid to palmitic acid was adjusted, and the test results are shown in Table 2.

[0057] Table 2: Test Results Group OA:PA molar ratio GSIS ratio Cell viability (%) Cleaved Caspase-3 expression Example 1 5:1 3.6 ± 0.2 98.2 ± 0.8 0.4 ± 0.1 Example 3 4:1 3.5 ± 0.2 97.9 ± 0.9 0.5 ± 0.1 Example 4 6:1 3.5 ± 0.2 98.0 ± 0.7 0.5 ± 0.1 Comparative Example 2-1 1:1 1.2 ± 0.1 65.3 ± 3.2 3.5 ± 0.4 Comparative Example 2-2 10:1 2.0 ± 0.2 92.1 ± 1.5 1.2 ± 0.2 Comparative Examples 2-3 1:0 2.3 ± 0.2 93.5 ± 1.3 1.0 ± 0.1 Comparative Examples 2-4 0:1 0.8 ± 0.1 42.7 ± 2.8 5.8 ± 0.6 The results showed that the cell viability of Comparative Example 2-1 and Comparative Example 2-4 was only 65.3% and 42.7%, respectively, and the expression of Cleaved Caspase-3 was significantly increased, confirming that the accumulation of free palmitic acid triggered a strong apoptotic signal and had clear lipotoxicity.

[0058] Although the survival rates of Comparative Examples 2-2 and 2-3 were acceptable (>92%), their GSIS ratios were only 2.0–2.3, significantly lower than the 3.6 of Example 1. This indicates that an appropriate amount of palmitic acid is crucial for maintaining cell membrane fluidity, GLUT2 localization, and downstream signal transduction, and that complete removal of PA is detrimental to functional maturation.

[0059] Only when the OA:PA ratio is in the range of 3:1 to 6:1, GSIS ≥ 3.5 and survival rate > 97%, with 5:1 being the peak performance point. This ratio provides sufficient OA to activate the PPARδ pathway, while retaining an appropriate amount of PA to support membrane structure and signal transduction, and avoiding lipotoxicity.

[0060] Effects of glucose gradient culture Based on the scheme in Example 1, only the glucose concentration strategy was adjusted, and the following experimental groups were set up: Comparative Example 3-1: Two-stage glucose, with low glucose concentration (5.5 mM) in the first and second stages, and high glucose concentration (25 mM) maintained in the third stage; Comparative Example 3-2: Constant high concentration of glucose (25 mM) throughout; Comparative Example 3-3: Constant low concentration of glucose (5.5 mM) throughout.

[0061] The test results are shown in Table 3.

[0062] Table 3: Test Results Group GSIS ratio Insulin secretion response delay time (minutes) Functional heterogeneity (CV%, based on single-cell GSIS) Example 1 3.6 ± 0.2 8.2 ± 0.5 18.3 ± 1.2 Comparative Example 3-1 2.4 ± 0.2 15.6 ± 1.1 29.7 ± 2.0 Comparative Example 3-2 1.6 ± 0.1 >30 (Platform not yet reached) 38.2 ± 2.5 Comparative Example 3-3 0.9 ± 0.1 No response — Comparative Example 3-2 cells were exposed to a high-glucose environment for a long time, and their glucose transport and metabolic pathways were in a saturated state, losing their dynamic response ability. Their GSIS was only 1.6, and the response delay was more than 30 minutes, making it impossible to establish effective secretion coupling.

[0063] In Example 1, the 11.1 mM glucose stage moderately activated glucokinase (GCK) and glycolysis pathways, initiating PDX1 expression and upregulating GLUT2 membrane localization, laying the metabolic and signaling foundation for subsequent high glucose stimulation. Comparative Example 3-1, which skipped this stage, resulted in a 33% decrease in GSIS and a nearly 100% increase in response delay.

[0064] The functional heterogeneity of the three-stage regimen (CV = 18.3%) was significantly lower than that of the two-stage regimen (29.7%) and the constant high glucose regimen (38.2%), indicating that the cell population function is more homogeneous, which is beneficial to the safety and predictability of clinical application.

[0065] The insulin secretion response time of Example 3-1 (8.2 minutes) was close to the physiological range of human primary pancreas (5–10 minutes), while the response time of other groups was significantly delayed.

[0066] Effects of antioxidant-endoplasmic reticulum homeostasis regulation Based on the scheme of Example 1, only the antioxidant in the third stage was adjusted, and the following comparative examples were set up.

[0067] Comparative Example 4-1: No antioxidants; Comparative Example 4-2: 5 mM N-acetylcysteine ​​(NAC); Comparative Example 4-3: 100 μM Vitamin C (VitC); Comparative Example 4-4: High stress conditions (PA concentration increased to 50 μM, no GLP-1, no antioxidant).

[0068] The test results are shown in Table 4.

[0069] Table 4: Test Results Group antioxidants GSIS ratio Cell viability (%) Example 1 Rhodioloside (50 μM) 3.6 ± 0.2 98.2 ± 0.8 Example 2 Taurine (2 mM) 2.8 ± 0.2 95.4 ± 1.1 Comparative Example 4-1 none 3.0 ± 0.2 92.0 ± 1.3 Comparative Example 4-2 NAC (5 mM) 2.4 ± 0.2 85.3 ± 1.8 Comparative Example 4-3 VitC (100 μM) 2.1 ± 0.2 83.7 ± 1.6 Comparative Example 4-4 High stress without protection 1.0 ± 0.1 58.2 ± 2.5 Comparative Example 4-1 had a GSIS of 3.0, while Example 1 improved to 3.6, demonstrating that the module not only maintains cell survival but also further optimizes the insulin synthesis and secretion pathway by reducing oxidative damage and ER stress, thereby enhancing its function.

[0070] Rhodioloside and taurine are significantly more effective than traditional antioxidants NAC and Vitamin C (GSIS difference > 1.0) because they have multiple mechanisms: rhodioloside activates the Nrf2 pathway and inhibits the PERK-CHOP axis; taurine regulates osmotic pressure, chelates calcium ions, and promotes glutathione synthesis. Both of them surpass the single effect of simply scavenging ROS.

[0071] Example 2 (taurine) achieved GSIS=2.8 under GLP-1-free conditions, which was significantly better than the unprotected group, confirming that a zero recombinant protein induction system can be constructed.

[0072] Under high stress conditions (Comparative Examples 4-4), the survival rate of the unprotected group was only 58.2%. This indicates that under the dual stress conditions of fatty acid loading and high glucose concentration, the antioxidant-endoplasmic reticulum homeostasis regulation module of the present invention plays a crucial role in maintaining the survival of functional cells.

[0073] Influence of three-dimensional suspended microcarrier induction system Based on the scheme of Example 1, the following comparative example is set up.

[0074] Comparative Example 5-1: Differentiation was induced using a traditional two-dimensional adherent culture method. Human adipose-derived mesenchymal stem cells were cultured at a rate of 1×10⁻⁶. 6 Cells were seeded at a density of 100 cells / mL in T75 culture flasks and added to DMEM / F12 basal medium supplemented with 2% (v / v) human serum albumin and 2 mM L-glutamine. The cells were cultured at 37°C and 5% CO2. Once the cells adhered and reached 80–90% confluence, they were directly induced to differentiate in stages in the original culture flasks.

[0075] Phase 1 metabolic preprogramming: Discard the original culture medium, wash once with PBS, add low-concentration glucose DMEM medium (5.5 mM glucose concentration), which contains 10 mM nicotinamide, 1 mM β-mercaptoethanol and 250 μM β-nicotinamide mononucleotide, and culture for 2 days under two-dimensional adherent conditions.

[0076] Second stage lineage orientation: The medium was changed to medium-concentration glucose DMEM (final glucose concentration 11.0 mM), with the addition of 10 mM nicotinamide, 1 mM β-mercaptoethanol, 250 μM NMN, 150 μM oleic acid and 30 μM palmitic acid, and cultured for another 2 days under two-dimensional adherent conditions.

[0077] The third stage, final maturation, involved replacing the medium with high-concentration glucose DMEM (25.0 mM glucose), supplemented with 10 mM nicotinamide, 1 mM β-mercaptoethanol, 150 μM oleic acid, 30 μM palmitic acid, and 50 μM rhodioloside. The medium was cultured under two-dimensional adherent conditions for 6 days, with the medium being replaced every 48 hours during this period.

[0078] After induction, the cells were subjected to functional and phenotypic tests using the same method as in the previous examples.

[0079] Comparative Example 5-2: Based on the scheme of Example 1, the polystyrene microcarrier Cytodex®1 microcarrier was used in different stages.

[0080] The test results are shown in Table 5.

[0081] Table 5: Test Results Group GSIS ratio Double positivity rate (%) of PDX1⁺ / NKX6.1⁺ Cleaved Caspase-3 expression Example 1 3.6 ± 0.2 51.3 ± 3.1 0.4 ± 0.1 Comparative Example 5-1 1.5 ± 0.2 21.8 ± 2.4 1.1 ± 0.2 Comparative Example 5-2 2.4 ± 0.2 36.7 ± 2.8 0.7 ± 0.1 Experimental results showed that, compared with the two-dimensional adhesion induction method of Comparative Example 1, Example 1 of the present invention exhibited significant advantages in both functional and phenotypic maturity. The GSIS value of Example 1 was significantly higher than that of Comparative Example 5-1; simultaneously, the proportion of PDX1⁺ / NKX6.1⁺ double-positive cells was significantly higher than that in the two-dimensional adhesion case, indicating that staged three-dimensional culture significantly promoted the synergistic expression of key pancreatic endocrine transcription factors. Furthermore, the relative expression level of Cleaved Caspase-3 was significantly reduced in Example 1, indicating a significant decrease in apoptosis levels.

[0082] Compared to Comparative Example 5-2, although non-staged three-dimensional culture improved cell function and survival to some extent, its GSIS value and PDX1⁺ / NKX6.1⁺ double positivity rate were still significantly lower than those in Example 1 of this invention, and the apoptosis level was higher. This indicates that using only a single three-dimensional microcarrier is insufficient to achieve full maturation of islet-like cells. Introducing microcarriers with different adhesion properties and ECM compositions at different induction stages to dynamically regulate cell spatial structure, mechanical state, and metabolic environment is a key technical means to achieve highly functional insulin-secreting cells.

[0083] All of the foregoing primary implementations of this intellectual property right do not limit other forms of implementation of this new product and / or new method. Those skilled in the art will utilize this important information to modify the foregoing to achieve similar implementations. However, all modifications or alterations based on this new product invention are reserved rights.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for inducing mesenchymal stem cells to differentiate into insulin-secreting cells, characterized in that, Includes the following steps: The first step, cell seeding and expansion, involves seeding mesenchymal stem cells into a culture system containing basal medium for adherent culture for 1–3 days, until the cell confluence reaches 70–90%. The second step, the metabolic pre-programming stage, involves placing the cells obtained in the first step into a low-concentration glucose DMEM medium containing low-adhesion microcarriers. The microcarriers are kept in suspension by slow continuous shaking or intermittent gentle shaking for 1–3 days. The third step, the lineage-directed differentiation stage, involves placing the cells obtained in the second step in a medium-concentration glucose DMEM medium containing medium-adhesive microcarriers. The microcarriers are kept in suspension by slow continuous shaking or intermittent gentle shaking for 1–3 days. The fourth step, the terminal functional maturation stage, involves placing the cells obtained in the third step in a high-concentration glucose DMEM medium containing highly adhesive microcarriers. The microcarriers are kept in suspension by slow continuous shaking or intermittent gentle shaking for 4–8 days to obtain insulin-secreting cells.

2. The method for inducing mesenchymal stem cells to differentiate into insulin-secreting cells as described in claim 1, characterized in that: The second step culture medium also contains a basic inducer and NAD⁺ precursor.

3. The method for inducing mesenchymal stem cells to differentiate into insulin-secreting cells as described in claim 1 or 2, characterized in that: The third step culture medium also contains a basic inducer, NAD⁺ precursor, and a fatty acid composition.

4. The method for inducing mesenchymal stem cells to differentiate into insulin-secreting cells as described in claim 1 or 2, characterized in that: The fourth step culture medium also contains a basic inducer, NAD⁺ precursor, fatty acid composition, and an antioxidant-endoplasmic reticulum homeostasis regulator.

5. The method for inducing mesenchymal stem cells to differentiate into insulin-secreting cells as described in claim 3 or 4, characterized in that: The fatty acid composition comprises oleic acid and palmitic acid, wherein the molar ratio of oleic acid to palmitic acid is 3:1 to 6:

1.

6. The method for inducing mesenchymal stem cells to differentiate into insulin-secreting cells as described in claim 1 or 2, characterized in that: The mesenchymal stem cells mentioned are human adipose-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, or umbilical cord mesenchymal stem cells.

7. The method for inducing mesenchymal stem cells to differentiate into insulin-secreting cells as described in claim 1 or 2, characterized in that: The low-adhesion microcarriers in the second step are selected from polystyrene microspheres or Alginate microspheres, with a particle size of 50–200 μm.

8. The method for inducing mesenchymal stem cells to differentiate into insulin-secreting cells as described in claim 1 or 2, characterized in that: The medium-adhesive microcarriers in the third step have a particle size of 100–250 μm and are coated with a small amount of collagen or gelatin on their surface.

9. The method for inducing mesenchymal stem cells to differentiate into insulin-secreting cells as described in claim 1 or 2, characterized in that: In the third step, highly adhesive ECM enriched microcarriers with a particle size of 150–300 μm and a surface containing collagen I / IV, HA, or laminin.

10. The use of the method according to any one of claims 1–9 in the preparation of insulin-secreting cells.