A preparation containing mesenchymal stem cell-derived insulin-secreting cells and use in the preparation of a blood glucose regulating product

Abstract

The present application relates to the technical field of biological medicine, in particular to a preparation containing mesenchymal stem cell-derived insulin-secreting cells and application thereof in preparing blood glucose regulating products. The insulin-secreting cells are derived from mesenchymal stem cells, and a four-stage directional differentiation strategy is adopted in the preparation method: in the first stage, all-trans retinoic acid, LDN-193189 and cyclopamine are combined to start endoderm program and specifically inhibit non-target differentiation, so as to realize "lineage locking"; in the second and third stages, high glucose environment and GLP-1 are used to promote the differentiation of cells into endocrine precursors and terminal insulin-secreting cells; in the fourth stage, GLP-1, PTHrP and nicotinic acid are added for "function enhancement", so as to synergistically improve the glucose responsiveness, maturity and anti-reversibility of the cells. The differentiation path of the method is precise and economical in factors, the obtained cells have high insulin positive rate and mature function. The preparation containing the cells can be used for preparing a drug for treating diabetes.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a preparation containing mesenchymal stem cell-derived insulin-secreting cells and its application in the preparation of blood glucose regulating products. Background Technology

[0002] Diabetes mellitus, especially insulin-dependent diabetes mellitus, is a metabolic disease caused by the loss of function of pancreatic β cells, leading to an absolute or relative deficiency of insulin secretion. Current clinical treatment mainly relies on exogenous insulin injections, but this cannot achieve physiological blood glucose regulation and carries the risk of hypoglycemia and various complications.

[0003] Stem cell-based regenerative medicine has brought new hope to the treatment of diabetes. Among them, mesenchymal stem cells (MSCs) are considered an ideal seed cell source for preparing insulin-producing cells (IPCs) due to their wide availability (e.g., umbilical cord, placenta, adipose tissue), ease of in vitro expansion, low immunogenicity, and multi-lineage differentiation potential. One of the research focuses is how to efficiently and stably induce MSCs to differentiate into functionally mature, glucose-responsive β-cell-like cells.

[0004] Several attempts have been made in the prior art. For example, Chinese invention patent CN 115851587 A provides an optimized culture medium and method for highly efficient human placental-derived MSCs. This technology can expand MSCs on a large scale and with high quality, providing a high-quality seed cell bank for subsequent differentiation. However, this patent itself does not involve any induction strategy for differentiation into pancreatic islet cells; its contribution lies in the upstream cell preparation step.

[0005] Regarding differentiation induction, Chinese invention patent CN 113481147 A discloses a method for directly constructing pancreatic stem cell lines from human islet tissue and differentiating them into islet cells. This technology bypasses the complex differentiation process starting from pluripotent MSCs, directly utilizing pancreatic progenitor cells that are closer to terminal cells for expansion and induction. However, this method relies on scarce human islet tissue as starting material, and the limited availability makes it difficult to meet large-scale clinical needs. Furthermore, it is essentially a tissue-specific stem cell culture rather than cross-lineage reprogramming.

[0006] Currently, induction protocols for the direct differentiation of pluripotent MSCs into pancreatic β cells are one of the mainstream research directions. Chinese invention patent CN 118895238 A discloses a four-stage induction protocol that induces umbilical cord MSCs into pancreatic islet cells by sequentially adding various small molecule compounds, including BML-284, HLY78, Itacnosertib, Galunisertib, and DMI-1, as well as growth factors such as Activin A, bFGF, and Exendin-4. This protocol demonstrates the feasibility of chemical induction without gene editing.

[0007] However, this existing technical solution still has obvious drawbacks: First, the induction system is complex and costly, using more than ten expensive specific small molecule inhibitors and growth factors, increasing process complexity and production costs. Second, its technical solution focuses on the differentiation process and does not systematically integrate with upstream cell expansion and downstream formulation to form a complete, industrially viable therapeutic product chain.

[0008] Therefore, there is an urgent need in this field for a more precise differentiation pathway and a more cost-effective inducing factor for the induction of insulin-secreting cells, and for further development into a standardized cell preparation that can be stably stored, easily transported and used. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention aims to overcome the deficiencies in the prior art and provide a method for inducing insulin-secreting cells that offers a more precise differentiation pathway, more economical and efficient inducing factors, and the ability to obtain functionally mature and stable insulin-secreting cells. Furthermore, it provides a standardized cell preparation containing these cells and its applications.

[0010] First, this invention provides a method for inducing mesenchymal stem cells to differentiate into insulin-secreting cells, comprising the following four consecutive stages: Phase 1 (Lineage Orientation and Repression): Mesenchymal stem cells were cultured in a low-glucose medium containing all-trans retinoic acid, a BMP signaling pathway inhibitor, and a Hedgehog signaling pathway inhibitor. This combination utilizes retinoic acid to initiate the endoderm differentiation program, while simultaneously using two inhibitors (preferably LDN-193189 and cyclopamine) to specifically block the BMP and Hedgehog signaling pathways. This actively inhibits the branching differentiation of cells into non-target lineages such as mesoderm (e.g., bone, cartilage) and intestine, efficiently and purely "locking" the cell population onto the pancreatic endocrine differentiation pathway.

[0011] Phase Two (Endocrine Precursor Induction): Cells treated in Phase One are cultured in a high-glucose environment. The high-glucose metabolic stress promotes cellular metabolic reprogramming, continuing their development towards endocrine precursors.

[0012] Phase 3 (Terminal Differentiation Induction): Cells treated in Phase 2 were cultured in a high-glucose medium containing glucagon-like peptide-1 (GLP-1). GLP-1, as a key insulin-secreting and β-cell survival factor, drives cells to terminally differentiate into hormone-responsive insulin-secreting cells.

[0013] Phase Four (Functional Enhancement and Maturation): Cells obtained in Phase Three were cultured in a high-glucose medium containing GLP-1, the parathyroid hormone-related protein fragment PTHrP, and niacin. This phase is a unique functional maturation step, in which PTHrP and GLP-1 work synergistically to further enhance the cells' glucose sensitivity and insulin secretion function; niacin, by improving the cellular metabolic state, significantly enhances cell viability and stress resistance, thereby ensuring the functional maturity and post-transplantation stability of the final cell product.

[0014] Preferably, in stage one, the concentrations of all-trans retinoic acid, LDN-193189, and cyclopamine are 0.5 μM, 0.1 μM, and 0.2 μM, respectively, and this specific ratio has been shown to have the best synergistic induction effect. In stage four, the concentration of PTHrP is preferably 12 nM, which achieves the peak functional enhancement within an effective window of 10-15 nM.

[0015] Furthermore, the present invention provides mesenchymal stem cell-derived insulin-secreting cells prepared by the above method, which have a high proportion of insulin-positive expression and exhibit excellent glucose-stimulated insulin secretion response.

[0016] Furthermore, the present invention provides a cell preparation comprising the aforementioned insulin-secreting cells, and a pharmaceutically acceptable biocompatible carrier (such as sodium alginate hydrogel) and / or a cell cryopreservation protectant, which facilitates storage, transportation and clinical application.

[0017] Finally, this invention provides the application of the aforementioned insulin-secreting cells or cell preparations in the preparation of drugs for treating diabetes. These drugs can be administered via intravenous infusion, portal vein infusion, or implantation after encapsulation in biomaterials to restore the body's insulin secretion function and achieve physiological blood glucose regulation.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: First, this invention innovatively combines all-trans retinoic acid (RA), a BMP inhibitor (LDN-193189), and a Hedgehog inhibitor (cyclopamine) in the induction initiation stage. Experimental data confirm that this specific combination (0.5 μM RA, 0.1 μM LDN-193189, 0.2 μM cyclopamine), under optimal synergy, not only maximizes the expression of pancreatic / endoderm target genes (such as Sox17, Pdx1) but also successfully suppresses the expression of non-target genes (Runx2, Cdx2) to basal levels. This strategy fundamentally solves the problems of ambiguous direction and high cell population heterogeneity associated with traditional RA-only induction, laying a crucial foundation for obtaining a high proportion of functional insulin-secreting cells.

[0019] Furthermore, this invention innovatively adds a "functional enhancement and maturation" stage (stage four) after terminal differentiation. This stage employs a ternary combination of GLP-1, the parathyroid hormone-related protein fragment PTHrP, and niacin (NA). GLP-1 maintains insulin-stimulating signals; PTHrP (preferably 12 nM) synergistically enhances cellular glucose sensitivity and insulin secretion capacity; while niacin, by providing NAD+ precursors, strongly improves cellular metabolic health and antioxidant capacity. Experiments have demonstrated that this ternary combination produces a clear synergistic effect. Compared to using GLP-1 alone or a dual-factor combination, the final cellular glucose-stimulated insulin secretion (GSIS) index, the co-expression rate of maturation markers (Insulin+ / MafA+), and in vitro stress resistance are all synergistically and maximized.

[0020] In summary, this invention provides a complete solution from cell-directed induction and functional enhancement to formulation preparation. The solution has a high degree of standardization and good batch-to-batch stability, effectively overcoming the shortcomings of existing technologies such as complex solutions, high costs, and lack of integration with downstream productization. This lays a solid technical and process foundation for the development of cell-based formulations for the treatment of diabetes. Detailed Implementation

[0021] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. Those skilled in the art should understand that the present invention can be implemented even without certain specific details. In some other embodiments, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the spirit of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and the numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0022] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, or instruments used, unless otherwise specified by the manufacturer, are all commercially available; and the conditions not specified in the examples are all performed under conventional conditions or conditions recommended by the manufacturer. Furthermore, this invention does not limit the source of the raw materials used; unless otherwise specified, the raw materials used in this invention are all commercially available products commonly found in this technical field. Unless otherwise specified, the "ratio" referred to in the following examples refers to a ratio of parts by mass. Example 1

[0023] This embodiment provides a method for inducing differentiation of human umbilical cord mesenchymal stem cells (UC-MSCs) into insulin-secreting cells, and performs systematic functional identification of the obtained cells, as detailed below: 1. Preparation of umbilical cord mesenchymal stem cells (UC-MSCs) Discarded umbilical cords from healthy full-term cesarean sections (after ethical review and informed consent) were collected, and UC-MSCs were isolated using the tissue block adherence method. The Wharton's jelly from the umbilical cord was cut into 1 mm pieces. 3 Tissue blocks were attached to T75 culture flasks and placed in basal growth medium (DMEM / F12, containing 10% fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, 10 ng / mL bFGF, 100 U / mL penicillin, and 100 μg / mL streptomycin) and cultured at 37°C and 5% CO2. When the primary cells reached 80% confluence, they were passaged using 0.25% trypsin, and the morphologically uniform and vigorously growing third-generation (P3) cells were used for subsequent induction experiments.

[0024] 2. Four-stage directed differentiation induction All stages were conducted at 37°C and 5% CO2. Prior to induction, P3 generation UC-MSCs were incubated at 1×10⁻⁶ ppm. 5 cells / cm 2 The cells were seeded at a density in cell culture plates.

[0025] Phase 1 (Lineage-directed initiation and inhibition, days 1-2): A low-glucose DMEM (5.5 mM glucose) medium containing: 2% (w / v) human serum albumin (HSA), 2 mM L-glutamine, 10 mM nicotinamide, 1 mM β-mercaptoethanol, 0.5 μM all-trans retinoic acid, 0.1 μM LDN-193189, and 0.2 μM cyclopamine was used. The medium was not changed during the 2-day incubation period.

[0026] Phase 2 (Endocrine precursor induction, days 3-4): Remove the old culture medium, wash with PBS, and add high-glucose DMEM (25 mM glucose) medium containing: 2% (w / v) HSA, 10 mM nicotinamide, and 1 mM β-mercaptoethanol. Incubate for 2 days, replacing the entire medium at the end of day 2.

[0027] Phase 3 (Terminal Differentiation Induction, Days 5-10): Remove the old culture medium, wash with PBS, and add high-glucose DMEM (25 mM glucose) medium containing: 1% (w / v) HSA, 10 mM nicotinamide, and 100 nM GLP-1 (7-37). Incubate for 6 days, changing the medium completely every 3 days.

[0028] Phase 4 (Functional Enhancement and Maturation, Days 11-12): Remove the old culture medium, wash with PBS, and add high-glucose DMEM (25 mM glucose) culture medium containing: 1% (w / v) HSA, 10 mM nicotinamide, 50 nM GLP-1, 12 nM parathyroid hormone-related protein fragment (PTHrP, 1-34), and 1 mM niacin; culture for 2 days.

[0029] 3. Cell harvesting After Phase IV, the culture medium was discarded, and the cells were washed with PBS. A digestion solution containing 0.05% trypsin and 0.5 mM EDTA was added, and the cells were incubated at 37°C for 5 minutes, gently pipetting until completely detached and dispersed. Serum-containing culture medium was added to terminate the digestion, and the cells were collected by centrifugation (200g, 5 minutes) to obtain mesenchymal stem cell-derived insulin-secreting cells (MSC-IPCs).

[0030] 4. Cell function identification The following key functional tests were performed on the harvested MSC-IPCs: Flow cytometry analysis: Using intracellular staining, flow cytometry analysis showed that the proportion of insulin-positive cells was (42.7 ± 5.6)% (n=3).

[0031] Glucose-stimulated insulin secretion assay (GSIS): Cell clusters were incubated in low-glucose (2.8 mM) and high-glucose (16.7 mM) KRBH buffers, respectively, and the insulin concentration in the supernatant was detected by ELISA. The calculated glucose stimulation index (SI) was 4.20 ± 0.51 (n=3), indicating that the cells had a significant glucose concentration-dependent insulin secretion capacity.

[0032] Immunofluorescence staining: After cell fixation, double immunofluorescence staining for insulin (Ins) and the key transcription factor MafA in mature β cells was performed. Confocal microscopy and cell counting showed that cells positive for both insulin and MafA accounted for (38.6 ± 4.5)% of the total cell count (n=3). Example 2

[0033] This embodiment provides a method for inducing the differentiation of human umbilical cord mesenchymal stem cells (UC-MSCs) into insulin-secreting cells, as detailed below: 1. Cell source and inoculation Same as in Example 1, using P3 generation UC-MSCs and inoculating at the same density.

[0034] 2. Four-stage directed differentiation induction (lower concentration parameters) The culture conditions are the same as in Example 1, but the composition of the culture medium at each stage is adjusted as follows: Phase 1: Low-sugar DMEM containing 2% HSA, 2 mM L-glutamine, 8 mM nicotinamide, 0.8 mM β-mercaptoethanol, 0.4 μM all-trans retinoic acid, 0.08 μM LDN-193189, and 0.16 μM cyclopamine.

[0035] Phase 2: High-sugar DMEM containing 2% HSA, 8 mM nicotinamide, and 0.8 mM β-mercaptoethanol.

[0036] Phase 3: High-sugar DMEM containing 1% HSA, 8 mM nicotinamide, and 100 nM GLP-1.

[0037] Phase 4: High-sugar DMEM containing 1% HSA, 8 mM nicotinamide, 50 nM GLP-1, 10 nM PTHrP, and 0.8 mM nicotinic acid.

[0038] The cultivation time for each stage is the same as in Example 1.

[0039] 3. Cell harvesting The method is the same as in Example 1. Example 3

[0040] This embodiment provides a method for inducing the differentiation of human umbilical cord mesenchymal stem cells (UC-MSCs) into insulin-secreting cells, as detailed below: 1. Cell source and inoculation Same as Example 1.

[0041] 2. Four-stage directed differentiation induction (higher concentration parameters) The culture conditions are the same as in Example 1, but the composition of the culture medium at each stage is adjusted as follows: Phase 1: Low-sugar DMEM containing 2% HSA, 2 mM L-glutamine, 12 mM nicotinamide, 1.2 mM β-mercaptoethanol, 0.6 μM all-trans retinoic acid, 0.12 μM LDN-193189, and 0.24 μM cyclopamine.

[0042] Phase 2: High-sugar DMEM containing 2% HSA, 12 mM nicotinamide, and 1.2 mM β-mercaptoethanol.

[0043] Phase 3: High-sugar DMEM containing 1% HSA, 12 mM nicotinamide, and 50 nM GLP-1.

[0044] Phase 4: High-sugar DMEM containing 1% HSA, 12 mM nicotinamide, 50 nM GLP-1, 14 nM PTHrP, and 1.2 mM nicotinic acid.

[0045] The cultivation time for each stage is the same as in Example 1.

[0046] 3. Cell harvesting The method is the same as in Example 1.

[0047] Experimental Example 1

[0048] This study aimed to investigate the effects of different ratios of all-trans retinoic acid (RA), the BMP signaling pathway inhibitor LDN-193189 (LDN), and the Hedgehog signaling pathway inhibitor cyclopamine (CYC) on early lineage orientation of human umbilical cord mesenchymal stem cells (UC-MSCs) during the initial induction phase. By setting up control and experimental groups with consistent total molar concentrations, the effects of each combination on activating the expression of target genes in the pancreas / endoderm and inhibiting the expression of non-target mesoderm / gut genes were systematically evaluated.

[0049] 1.1 Experimental Materials and Methods Third-generation (P3) human umbilical cord mesenchymal stem cells (UC-MSCs) prepared using the method in Example 1.

[0050] Grouping and processing: UC-MSCs were divided into groups of 1×102 5 cells / cm 2Cells were evenly seeded at a density in 12-well cell culture plates. After cell attachment (approximately 12 hours), the medium was replaced with different stage-one treatment media. Except for the factors specified in Table 1, the basic components of the culture media for all groups were identical, namely: low-glucose DMEM (5.5 mM glucose), 2% (w / v) human serum albumin (HSA), 10 mM nicotinamide, and 1 mM β-mercaptoethanol.

[0051] The experimental groups and the composition of key factors in the culture medium are shown in Table 1: Table 1. Experimental groups and composition of key factors in the culture medium Group number Experimental group name All-trans retinoic acid (RA) LDN-193189 (LDN) Cyclic pharmacamine (CYC) 1 Blank control group 0 μM 0 μM 0 μM 2 RA monotherapy group 0.8 μM 0 μM 0 μM 3 LDN single-use group 0 μM 0.8 μM 0 μM 4 CYC single-use group 0 μM 0 μM 0.8 μM 5 RA+LDN two-factor group 0.5 μM 0.3 μM 0 μM 6 RA+CYC dual factor group 0.5 μM 0 μM 0.3 μM 7 LDN+CYC dual-factor group 0 μM 0.4 μM 0.4 μM 8 Three-factor group A 0.5 μM 0.15 μM 0.15 μM 9 Three-factor group B 0.5 μM 0.1 μM 0.2 μM 10 Three-factor group C 0.5 μM 0.2 μM 0.1 μM 1.2 Cultivation and Detection Cells in each group were cultured under standard conditions (37℃, 5% CO2) for 48 hours. After Phase 1, cells were collected, and total RNA was extracted using the TRIzol method and reverse transcribed into cDNA. The mRNA expression levels of the following markers were detected by quantitative real-time PCR (qRT-PCR): Sox17 (endoderm marker), Pdx1 (key transcription factor for pancreatic progenitor cells), Runx2 (mesoderm / osteoblast marker), and Cdx2 (intestinal endoderm marker). Three biological replicates were set up for each experimental group.

[0052] 1.3 Data Processing The relative expression levels of each gene relative to the blank control group (Group 1) were calculated using the 2^(-ΔΔCt) method. Data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups. After passing the homogeneity of variance test, Tukey's HSD test was used for pairwise comparisons. To clearly demonstrate the improvement effect compared to commonly used single-factor induction protocols, the statistical significance marker was uniformly defined as the RA-only group (Group 2). P < 0.05 was considered statistically significant.

[0053] 1.4 Test Results After Phase 1 treatment, the relative mRNA expression levels of key genes in each experimental group are shown in Table 2.

[0054] The results of the relative expression levels of each biomarker mRNA after stage one treatment are shown in Table 2: Table 2. Relative mRNA expression levels of each biomarker after Phase 1 treatment (mean ± SD) Group number Experimental group name Sox17 Pdx1 Runx2 Cdx2 1 Blank control group 1.00 ± 0.11 1.00 ± 0.12 1.00 ± 0.15 1.00 ± 0.14 2 RA monotherapy group 3.45 ± 0.23 2.15 ± 0.19 3.60 ± 0.41 4.40 ± 0.21 3 LDN single-use group 1.05 ± 0.12 1.02 ± 0.09 0.38 ± 0.05* 1.28 ± 0.12 4 CYC single-use group 1.10 ± 0.14 1.08 ± 0.11 1.20 ± 0.17 0.41 ± 0.05* 5 RA+LDN two-factor group 3.95 ± 0.25 3.05 ± 0.30 1.35 ± 0.12* 4.25 ± 0.28 6 RA+CYC dual factor group 3.88 ± 0.24 2.98 ± 0.16 3.30 ± 0.17 1.40 ± 0.19* 7 LDN+CYC dual-factor group 1.20 ± 0.11 1.15 ± 0.13 0.35 ± 0.05* 0.39 ± 0.04* 8 Three-factor group A 4.82 ± 0.15* 3.92 ± 0.21* 1.05 ± 0.06* 1.10 ± 0.13* 9 Three-factor group B 5.92 ± 0.31* 4.85 ± 0.26* 0.92 ± 0.03* 0.88 ± 0.07* 10 Three-factor group C 5.05 ± 0.28* 4.10 ± 0.30* 1.01 ± 0.12* 1.05 ± 0.10* Note: * indicates that compared with the RA-only group (group 2), P < 0.05.

[0055] The above experimental results show that different combinations of factors have a significant impact on the early gene expression profile of UC-MSCs.

[0056] Compared with the RA monotherapy group, the LDN or CYC monotherapy groups (groups 3 and 4) did not show an advantage in positively inducing the expression of the target genes (Sox17, Pdx1), but they significantly inhibited the expression of the non-target genes Runx2 or Cdx2, respectively. This confirms that the two inhibitors each have specific pathway inhibitory functions, but neither can initiate effective directed differentiation when used alone.

[0057] The two-factor combinations including RA (groups 5 and 6) improved the shortcomings of RA alone to some extent. The RA+LDN group, while maintaining the induction levels of Sox17 and Pdx1, significantly reduced Runx2 expression, but still failed to effectively inhibit Cdx2. The RA+CYC group effectively inhibited Cdx2, but its inhibitory effect on Runx2 was not significant. This indicates that the two-factor combinations can only partially correct RA-induced non-specific differentiation and have functional limitations. Although the LDN+CYC two-factor group (group 7) strongly inhibited the two non-target genes, it was completely unable to initiate the expression of the target genes due to the lack of RA.

[0058] The three-factor combinations (groups 8, 9, and 10) all showed comprehensive and significant improvements compared to the RA monotherapy group. They not only increased the expression of Sox17 and Pdx1 to higher levels, but also significantly suppressed the expression of Runx2 and Cdx2 to near or below baseline levels. This clearly demonstrates that the functional complementarity and synergy of RA, LDN, and CYC are necessary for achieving efficient and pure lineage orientation.

[0059] Further comparison of the three-factor groups with different ratios revealed differences in their induction effects. Among them, group B (0.5 μM RA, 0.1 μM LDN, 0.2 μM CYC) showed the best trend in all indicators. This group had the highest expression levels of Sox17 and Pdx1 among all groups, and also exhibited the most thorough inhibition of Runx2 and Cdx2. Although the statistical differences between group B and groups A and C in some indicators require further analysis, the data trends consistently point to this specific ratio achieving the optimal balance between "positive induction intensity" and "negative inhibition depth."

[0060] The results of this experiment indicate that the combined use of all-trans retinoic acid (ART), LDN-193189, and cyclopamine is necessary in the initial stage of inducing mesenchymal stem cell differentiation into pancreatic β cells. The synergistic effect of these three substances can significantly overcome the non-target differentiation problem caused by ART alone and enhance the targeting efficiency of the target lineage. Among the various ratios tested, the combination of 0.5 μM ART, 0.1 μM LDN-193189, and 0.2 μM cyclopamine exhibited the best induction characteristics, providing crucial evidence for establishing an efficient and stable differentiation process.

[0061] Experimental Example 2

[0062] This study aims to systematically investigate the effects of added concentrations of parathyroid hormone-related protein fragments (PTHrP, 1-34) on the final function, maturity, and viability of insulin-secreting cells, while fixing other key components in the functional enhancement stage.

[0063] 2.1 Experimental Materials and Methods Insulin-secreting cell precursors were prepared in batches using the standard four-stage induction method described in Example 1, induced to the end of stage three (day 10). All cells were digested, mixed, and resuspended uniformly.

[0064] The above cell suspension was prepared at 5×10 5 Cells were seeded at a density per well in 24-well plates. Eight experimental groups were set up for a 48-hour functional enhancement (phase four) treatment. The basal composition of the culture medium was identical for all groups, including: high-glucose DMEM (25 mM glucose), 1% HSA, and 10 mM nicotinamide. The concentration of GLP-1 was fixed at 50 nM, the concentration of nicotinic acid was fixed at 1 mM, and the only variable was the concentration of PTHrP.

[0065] The experimental groups are as follows: Group 1 (PTHrP-free control group): basal culture medium + 50 nM GLP-1 + 1 mM nicotinic acid + 0 nM PTHrP.

[0066] Group 2: Basal medium + 50 nM GLP-1 + 1 mM nicotinic acid + 5 nM PTHrP.

[0067] Group 3: Basal medium + 50 nM GLP-1 + 1 mM nicotinic acid + 10 nM PTHrP.

[0068] Group 4: Basal medium + 50 nM GLP-1 + 1 mM nicotinic acid + 12 nM PTHrP.

[0069] Group 5: Basal medium + 50 nM GLP-1 + 1 mM nicotinic acid + 14 nM PTHrP.

[0070] Group 6: Basal medium + 50 nM GLP-1 + 1 mM nicotinic acid + 15 nM PTHrP.

[0071] Group 7: Basal medium + 50 nM GLP-1 + 1 mM nicotinic acid + 18 nM PTHrP.

[0072] Group 8: Basal medium + 50 nM GLP-1 + 1 mM nicotinic acid + 20 nM PTHrP.

[0073] 2.2 Detection Indicators and Methods: Glucose-stimulated insulin secretion function (GSIS): After treatment, standard GSIS experiments were performed. Cells were incubated sequentially in low glucose (2.8 mM) and high glucose (16.7 mM) KRBH buffer for 1 hour each, and the supernatant was collected. Insulin concentration was measured using a highly sensitive human insulin chemiluminescent immunoassay kit, and the stimulation index (SI) was calculated.

[0074] Cell maturity characterization: After treatment, immunofluorescence staining was performed. Guinea pig anti-insulin antibody and rabbit anti-MafA antibody were used for double labeling, and nuclei were stained with Hoechst 33342. The cells were automatically scanned and analyzed using a high-content imaging system to calculate the percentage of insulin- and MafA-positive cells among the total cell count.

[0075] Cell viability assessment: At the end of the treatment, the CCK-8 reagent was used to directly detect the cells in each well to reflect the immediate metabolic activity of the cells after treatment. Simultaneously, a separate group of cells was cultured in serum-free basal medium for 24 hours after stage four treatment, and CCK-8 levels were measured again to calculate the 24-hour viability, thus assessing the cells' short-term self-sustaining ability.

[0076] 2.3 Data Processing: All data were obtained from three independent biological replicates and are expressed as mean ± standard deviation. One-way ANOVA was used to compare differences between groups, and Dunnett's test was used to compare each concentration group with the PTHrP-free control group (Group 1). Tukey's test was used for pairwise comparisons among all groups. P < 0.05 was considered statistically significant.

[0077] 2.4 Test Results The experimental results are shown in Table 3: Table 3. Effects of different PTHrP concentrations on cell function, maturity, and viability (mean ± SD, n=3) Group PTHrP concentration (nM) GSIS Stimulus Index (SI) Ins+ / MafA+ double positivity rate (%) Immediate vitality after treatment (OD450) 24-hour survival rate (%) 1 0 3.25 ± 0.34 25.8 ± 3.2 1.00 ± 0.08 100.0 ± 5.2 2 5 3.68 ± 0.28* 29.5 ± 2.0 1.05 ± 0.04 102.5 ± 5.8 3 10 4.05 ± 0.37* 34.1 ± 3.1* 1.12 ± 0.02* 108.3 ± 4.1* 4 12 4.48 ± 0.32* 38.6 ± 2.5* 1.18 ± 0.10* 115.7 ± 6.9* 5 14 4.20 ± 0.46* 36.2 ± 2.2* 1.15 ± 0.11* 112.0 ± 5.5* 6 15 4.02 ± 0.34* 34.8 ± 2.1* 1.13 ± 0.10* 109.1 ± 4.3* 7 18 3.55 ± 0.39* 30.1 ± 2.7 1.06 ± 0.07 103.8 ± 3.9 8 20 3.30 ± 0.26 26.5 ± 1.2 1.01 ± 0.08 98.5 ± 4.5 Note: * indicates that compared with the control group without PTHrP (Group 1), P < 0.05.

[0078] The experimental results above show that the changes in each evaluation index were not linear, but rather exhibited a pattern of first increasing and then decreasing. Overall, PTHrP demonstrated clear biological activity within the concentration range of 5 nM to 15 nM. Within this range, compared with the control group without PTHrP, each experimental group showed statistically significant improvements in glucose-stimulated insulin secretion (GSIS), the proportion of insulin- and MafA double-positive cells, immediate cell viability, and short-term survival rate after nutrient removal.

[0079] Further analysis revealed that the peak effect occurred at a concentration of 12 nM. At this concentration, key functional indicators such as the GSIS stimulation index and the proportion of double-positive cells reached the highest observed values ​​among all experimental groups. Pairwise comparisons with the 10 nM and 14 nM concentration groups showed a statistically significant advantage in multiple indicators. When the PTHrP concentration increased to 18 nM and above, its promoting effect diminished. Although many indicators in the 18 nM concentration group were still significantly higher than those in the control group, they were lower than those in the 12 nM concentration group. At 20 nM, the data for each indicator returned to a level where there was no significant difference compared to the control group without PTHrP.

[0080] Based on the above data, the effect of PTHrP(1-34) addition in the system composed of 50 nM GLP-1 and 1 mM nicotinic acid showed a concentration-dependent effect. A concentration of 12 nM showed the best effect within the test range, with 10-15 nM constituting its main activity range, while the effect weakened above 15 nM. This provides experimental evidence for determining the appropriate addition concentration for the functional enhancement stage.

[0081] Experimental Example 3

[0082] This study aims to investigate whether parathyroid hormone-related protein fragment (PTHrP, 1-34) and niacin (NA) have a synergistic effect on enhancing the function, maturity, and activity of insulin-secreting cells in the presence of glucagon-like peptide-1 (GLP-1) during the functional enhancement phase.

[0083] 3.1 Experimental Materials and Methods Cells: Same as in Experiment 2, the insulin-secreting cell precursor pool was prepared using the standard method of Example 1 and induced to the end of stage three to ensure uniformity of the initial cell state.

[0084] Grouping and treatment: Cells were grouped at 5 × 10⁻⁶ cells / cm². 5Cells were seeded at a density per well in 24-well plates. Four experimental groups were set up and subjected to a 48-hour functional enhancement treatment. The basic composition of the culture medium was exactly the same for all groups, including: high-glucose DMEM (25 mM glucose), 1% HSA, 10 mM nicotinamide, and 50 nM GLP-1. The experimental variables were the addition or absence of PTHrP and nicotinic acid.

[0085] The experimental groups are as follows: Group A (GLP-1 baseline): Basal medium + 50 nM GLP-1. (As a baseline for assessing synergistic effects) Group B (GLP-1 + PTHrP group): basal medium + 50 nM GLP-1 + 12 nM PTHrP.

[0086] Group C (GLP-1 + nicotinic acid group): basal medium + 50 nM GLP-1 + 1 mM nicotinic acid (NA).

[0087] Group D (GLP-1 + PTHrP + nicotinic acid group, i.e. the scheme of this invention): basal culture medium + 50 nM GLP-1 + 12 nM PTHrP + 1 mM nicotinic acid.

[0088] 3.2 Detection Indicators and Methods Same as in Experiment 2 3.3 Data Processing: All data were obtained from three independent biological replicates and are expressed as mean ± standard deviation. One-way ANOVA was first used to compare the overall differences among the four groups.

[0089] 3.4 Test Results The experimental results are shown in Table 4: Table 4. Effects of different factor combinations on cell function, maturity, and viability (mean ± SD, n=3) Group Processing combination GSIS Stimulus Index (SI) Ins+ / MafA+ double positivity rate (%) Instant Vitality (OD450) 24-hour survival rate (%) A GLP-1 3.28 ± 0.21 26.5 ± 2.8 1.00 ± 0.07 100.0 ± 4.8 B GLP-1 + PTHrP 4.05 ± 0.18* 33.8 ± 2.5* 1.09 ± 0.08 105.2 ± 5.1 C GLP-1 + Niacin 3.52 ± 0.13* 28.1 ± 2.0 1.16 ± 0.10* 118.5 ± 4.3* D GLP-1 + PTHrP + Niacin 4.62 ± 0.38* 40.3 ± 3.1* 1.25 ± 0.11* 126.8 ± 5.0* Note: * indicates that compared with group A (GLP-1 baseline group), P < 0.05.

[0090] The above experimental results show that: Effects of PTHrP alone (Group B): Compared with Group A, the addition of 12 nM PTHrP alone significantly enhanced the glucose-stimulated insulin secretion capacity (GSIS index) and the expression levels of maturation markers (Ins / MafA), indicating that PTHrP has a clear role in promoting cell functional maturation. However, its effect on improving immediate cell viability and 24-hour survival rate did not reach statistical significance.

[0091] Effects of adding niacin alone (Group C): Compared with Group A, adding 1 mM niacin alone can significantly improve the immediate metabolic activity of cells and their long-term survival ability after nutrient removal, and improve GSIS function to a certain extent, but its promoting effect on the expression of cell maturation markers is limited.

[0092] The combined effect of PTHrP and niacin (Group D, the present invention scheme): Compared with Group A, the combined addition of PTHrP and niacin produced the most significant improvement in all four detection indicators. Particularly noteworthy is that the GSIS index (4.62) and double-positive rate (40.3%) of Group D were not only significantly higher than those of the baseline Group A, but also significantly higher than those of Groups B and C, which only added a single factor (e.g., the GSIS of Group B was 4.05, and the double-positive rate of Group C was 28.1%). This indicates that the combined application of PTHrP and niacin produced an enhancement effect that went beyond the simple additive effect of a single factor.

[0093] The results of this experiment indicate that, during the functional enhancement phase, both PTHrP (12 nM) and niacin (1 mM) play crucial roles in obtaining fully functional, mature, and stable insulin-secreting cells in the presence of GLP-1, and their functions are complementary. While using either component alone can bring improvements in specific aspects, each has its limitations. Only when PTHrP and niacin are used in combination can a comprehensive enhancement of cell secretory function, mature phenotype, and viability be achieved synergistically, reaching the optimal overall effect.

[0094] Test Example 4

[0095] The purpose of this experiment is to observe and compare the complete four-stage induction scheme of this invention with the prior art CN118895238 A scheme in parallel, as follows.

[0096] 4.1 Test Methods: Three experimental groups were set up using P3 generation UC-MSCs from the same batch: The present invention solution group is implemented strictly in accordance with the four-stage solution of Example 1.

[0097] Comparative control group: The four-stage culture medium formulation and induction process described in Example 1 of CN 118895238 A were strictly followed (a special culture medium containing multiple small molecule inhibitors for each stage needs to be prepared).

[0098] Simplified version of the present invention: According to Example 1, only the first three stages (stages one to three) of the present invention are performed to evaluate the contribution of the "functional enhancement" stage.

[0099] All groups underwent the following evaluation at their respective induction endpoints (after Phase 4 for the present invention and comparative schemes, and after Phase 3 for the simplified group): Differentiation efficiency: The percentage of insulin-positive (Insulin+) cells was determined by flow cytometry.

[0100] Functional maturity: Conduct GSIS experiments and calculate the Stimulus Index (SI).

[0101] 4.2 Test Results: The experimental results are shown in Table 5: Table 5. Comparison of the induction effect and economic efficiency of the present invention's scheme and the comparative scheme. Group Insulin+ cell percentage (%) GSIS Stimulus Index (SI) Invention scheme group 42.7 ± 3.6* 4.20 ± 0.41*# Comparative scheme group 38.9 ± 2.8 3.15 ± 0.43 Simplified Invention Scheme Group 40.1 ± 3.2 2.98 ± 0.25* Note: * indicates P < 0.05 compared to the “Comparative Scheme Group”; # indicates P < 0.05 compared to the “Simplified Invention Scheme Group”.

[0102] The experimental results above show that, compared with the prior art solution (CN 118895238 A) used as a control, the complete four-stage induction scheme of this invention exhibits clear advantages in key performance indicators. The cell population ultimately obtained by this scheme has a significantly higher proportion of cells with insulin secretion potential, and these cells also demonstrate superior insulin release capacity under glucose stimulation. This indicates that the overall strategy of this invention is more effective in improving the yield and functional maturity of target cells.

[0103] Further analysis revealed that the simplified scheme, which only employs the core induction framework of this invention (i.e., the first three stages) and omits the fourth stage "functional enhancement" treatment, yielded cells with basal differentiation efficiency comparable to existing technologies, demonstrating the effectiveness of the core framework. However, the cells obtained by this simplified scheme showed significantly lower functional maturity, particularly in glucose responsiveness, compared to both the complete scheme of this invention and existing technologies. This experimental result indicates that the unique fourth stage—"functional enhancement" treatment—plays an indispensable and crucial role in conferring high-level, mature functional characteristics upon differentiated cells.

[0104] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A method for inducing mesenchymal stem cells to differentiate into insulin-secreting cells, characterized in that, The process includes the following four consecutive stages of cultivation: (a) Lineage orientation and suppression phase: Mesenchymal stem cells were cultured in a low-glucose medium containing human serum albumin (HSA), L-glutamine, nicotinamide, β-mercaptoethanol, all-trans retinoic acid, a BMP signaling pathway inhibitor, and a Hedgehog signaling pathway inhibitor; the BMP signaling pathway inhibitor was LDN-193189, the Hedgehog signaling pathway inhibitor was cyclopamine, and the concentration of all-trans retinoic acid was 0.4-0.6 μM, the concentration of LDN-193189 was 0.08-0.12 μM, and the concentration of cyclopamine was 0.16-0.24 μM; (b) Endocrine precursor induction phase: Cells treated in step (a) were cultured in a high-glucose medium containing HSA, nicotinamide, and β-mercaptoethanol; (c) Terminal differentiation induction stage: The cells treated in step (b) were cultured in a high-glucose medium containing HSA, nicotinamide, and glucagon-like peptide-1; (d) Functional enhancement and maturation stage: The cells obtained after treatment in step (c) were cultured in a high glucose medium containing HSA, nicotinamide, GLP-1, parathyroid hormone-related protein fragment PTHrP and nicotinic acid, wherein the concentration of PTHrP was 10-15 nM, the concentration of nicotinic acid was 0.8-1.2 mM, and the PTHrP was PTHrP(1-34).

2. The method according to claim 1, characterized in that, In step (a), the concentration of all-trans retinoic acid is 0.5 μM, the concentration of LDN-193189 is 0.1 μM, and the concentration of cyclopamine is 0.2 μM.

3. The method according to claim 1, characterized in that, In step (d), the concentration of PTHrP is 12 nM.