A method of inducing differentiation of iPSCs into mesenchymal stromal cells

Abstract

The application discloses a method for inducing iPSC to differentiate into mesenchymal stromal cells, comprising the following steps: S1, iPSC expansion culture; S2, three-dimensional suspension culture; S3, mesoderm induction; S4, preparation of mesenchymal stromal cells. The method realizes efficient and stable conversion from iPSC to iMSC through the combination strategy of two-dimensional expansion, three-dimensional induction of mesoderm differentiation and two-dimensional differentiation into iMSC; the positive rates of MSC surface markers CD90, CD73, CD13, CD29 and CD44 cells of the obtained iMSC are not less than 90%, the positive rates of negative CD34, CD45 and HLA-DAR cells are less than 10%, the iMSC has in-vitro three-lineage differentiation ability and PBMC proliferation inhibition function, and meets the quality standard of clinical-grade cell treatment products; and the overall scheme has good repeatability and batch stability.

Description

Technical Field

[0001] This invention belongs to the field of stem cell regenerative medicine technology, specifically relating to a method for inducing iPSCs to differentiate into mesenchymal stromal cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) possess multipotent differentiation potential, immunomodulatory functions, and paracrine activity, demonstrating broad clinical application prospects in cell therapy, bone / cartilage repair, intervention for autoimmune diseases, and tissue engineering. Traditionally, MSCs are obtained from bone marrow, adipose tissue, and umbilical cord. However, these sources have limitations: limited donor availability, poor phenotypic and functional consistency between different batches, making it difficult to meet the needs of large-scale clinical production. Furthermore, the isolation process typically relies on culture systems containing animal serum or animal-derived components, posing safety risks such as significant batch-to-batch variations and potential pathogen transmission, thus restricting their standardized application in clinical-grade products.

[0003] Induced pluripotent stem cells (iPSCs) are considered an important way to solve the problem of limited MSC donor sources due to their unlimited proliferation capacity and multi-lineage differentiation potential. iPSC-derived mesenchymal stromal cells (iMSCs) can be produced in batches and in a uniform manner, providing a feasible solution for the large-scale preparation of clinical-grade cell therapy products.

[0004] Currently reported methods for differentiating iPSCs into MSCs mainly suffer from the following technical problems: First, existing differentiation protocols mostly involve mesodermal induction under two-dimensional adherent conditions, limiting the culture scale and resulting in low yields, making it difficult to meet the needs of large-scale production. Second, existing protocols generally use culture systems containing serum or animal-derived components, leading to complex compositions, large batch-to-batch variability, and the risk of animal-derived contamination, which does not meet the quality control requirements for clinical-grade production. Third, some protocols have long induction cycles, which may cause cells to undergo multiple passages and experience functional decline. Therefore, establishing a method for differentiating iPSCs into iMSCs with a clearly defined chemical composition throughout the process, free of animal-derived components, and balancing large-scale yields and differentiation efficiency is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for inducing iPSCs to differentiate into mesenchymal stromal cells.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for inducing iPSCs to differentiate into mesenchymal stromal cells includes the following steps: S1. iPSC amplification culture: iPSC cells were placed in T25 for two-dimensional adherent amplification culture. The adherent cells were digested into a single-cell suspension using a cell dissociation agent. The cells were then passaged 3 to 9 times the culture area to obtain amplified iPSCs. S2, Three-dimensional suspension culture: The amplified iPSCs obtained in step S1 are digested into a single-cell suspension using protease and then subjected to three-dimensional suspension culture in a medium containing ROCK inhibitor to allow the cells to aggregate and form embryoids; S3, Mesodermal Induction: Discard the culture supernatant containing ROCK inhibitor, add mesodermal induction medium to the embryoids from step S2, and culture at 37°C and 5% CO2 to induce iPSCs to differentiate into mesodermal cells; S4. Preparation of mesenchymal stromal cells: The mesodermal cell spheroids obtained in step S3 were transferred to a two-dimensional culture vessel coated with extracellular matrix proteins and cultured sequentially in MSC induction medium A and MSC induction medium B, followed by (2~4)×10⁻⁶ cells / mL. 4 cells / cm 2 The cells were passaged at a high density, every 3-4 days, and mesenchymal matrix cells were obtained after 2-3 passages.

[0007] Preferably, the cell dissociation agent in step S1 is ReleSR digestion solution.

[0008] Preferably, the three-dimensional suspension culture in step S2 is performed in one of the following ways: (a) The single-cell suspension was prepared at 1×10 6 ~4×10 6 Cells / well were seeded at a density of 1,000 cells / well into a culture plate with a microporous array structure, cultured overnight in a medium containing ROCK inhibitor to form embryoids, and then the embryoids were transferred to a three-dimensional suspension bioreactor for culture. (b) The single-cell suspension was diluted with 1×10⁻⁶ 5 ~2×10 5 Cells were directly seeded into a three-dimensional suspension bioreactor at a density of 100 cells / mL, cultured overnight in a medium containing ROCK inhibitor, and after standing, the supernatant was discarded and replaced with mesotherapy induction medium for further culture.

[0009] Preferably, the ROCK inhibitor in step S2 is Y27632, with a final concentration of 5-10 μM; the protease is one of Accutase, TrypLUS, and TrypLE.

[0010] Preferably, the final concentrations of each component in the mesodermal induction medium in step S3 are: 1~6 μM CHIR99021, 5~60 ng / ml bFGF, and 5~60 ng / ml BMP-4.

[0011] Preferably, the induction differentiation time in step S3 is 2-3 days, and the proportion of TBXT-positive cells in the mesodermal cells obtained after induction is not less than 90%.

[0012] Preferably, the MS induction medium A in step S4 is obtained by adding the following components to the complete iPSC medium: 5~10μM SB-431542, 5~50ng / mL BMP-4, 5~50ng / mL bFGF; the MS induction medium B is obtained by adding the following components to the MSC basal medium: 2.5μM SB-431542, 5~50ng / mL BMP-4, 5~50ng / mL bFGF, 5~50ng / mL PDGF-BB, 50~300μM sodium vitamin C, 5~50ng / mL EGF, 0.1~1.0% HSA, 0.5~1.0× ITS-X, 0.5~1.0× GlutaMAX, 0.5~1.0× NEAA.

[0013] Preferably, the extracellular matrix protein in step S4 is selected from one or more of Laminin-521, Laminin-511, Fibronectin, and Vitronectin, and is cultured in MSC induction medium A for 2 days and in MSC induction medium B for 6-14 days.

[0014] The present invention also protects a mesenchymal stromal cell prepared by the method described above, wherein the mesenchymal stromal cell meets the following phenotypic criteria: the positive rates of CD90, CD73, CD13, CD29, and CD44 cells are all not less than 90%; and the positive rates of negative CD34, CD45, and HLA-DAR cells are all less than 10%.

[0015] The present invention also protects the application of the mesenchymal stromal cells described above in cell therapy, bone / cartilage repair, treatment of autoimmune diseases or tissue engineering, characterized in that the mesenchymal stromal cells have the ability to differentiate into adipogenic, chondrogenic and osteogenic lineages in vitro, and have an inhibitory effect on the proliferation of peripheral blood mononuclear cells.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The method for inducing iPSC differentiation into mesenchymal stromal cells provided by the present invention provides a method for differentiating iPSC into iMSCs using a culture system with clear chemical composition, no serum and no animal-derived components throughout the process. Through a combination strategy of two-dimensional amplification, three-dimensional induction of mesoderm differentiation and two-dimensional differentiation into iMSCs, the efficient and stable transformation from iPSC to iMSC is achieved. The positive rate of the MSC surface markers CD90, CD73, CD13, CD29 and CD44 cells of the obtained iMSCs is not less than 90%, and the positive rate of negative CD34, CD45 and HLA-DAR cells is less than 10%. At the same time, it has the ability to differentiate into three lineages in vitro and the function of inhibiting the proliferation of PBMCs, which meets the quality standards of clinical-grade cell therapy products. The overall scheme has good reproducibility and batch-to-batch stability.

[0017] (2) The method for inducing iPSCs to differentiate into mesenchymal stromal cells provided by the present invention adopts a three-dimensional suspension culture method in the mesodermal induction stage, so that iPSCs aggregate to form embryoids under suspension conditions and simultaneously receive mesodermal induction signals. Compared with the traditional two-dimensional adherent induction method, three-dimensional suspension culture can make full use of the longitudinal space of the bioreactor, significantly increase the cell processing volume per unit volume, thereby reducing the cost of large-scale production. Under specific mesodermal induction culture medium conditions, the proportion of TBXT positive mesodermal cells obtained after 2-3 days of induction can reach more than 90%, and the induction efficiency meets the requirements of subsequent differentiation steps for the purity of mesodermal cells. A specific combination of CHIR99021 (Wnt signaling pathway agonist), bFGF and BMP-4 is used as a mesodermal induction factor. In a serum-free culture medium with clear chemical composition, the Wnt, TGF-β / Nodal and BMP signaling pathways are synergistically activated to effectively promote the conversion of iPSCs to mesodermal fate. This combination of factors can achieve efficient mesodermal induction without animal-derived components, avoiding the quality risks of introducing heterologous proteins. At the same time, the concentration range of each factor is clearly defined, which is conducive to the standardized management of process parameters.

[0018] (3) The method for inducing iPSC differentiation into mesenchymal stromal cells provided by this invention employs a sequential induction strategy of MSC induction medium A and MSC induction medium B during the iMSC induction stage: Medium A uses SB-431542 to inhibit the TGF-β / Activin signaling pathway while providing BMP-4 and bFGF to guide mesodermal cells to initiate the transformation towards mesenchyme; Medium B further introduces PDGF-BB, EGF, sodium vitamin C, and HSA on the basis of Medium A to provide a suitable biochemical environment for the maturation and expansion of iMSCs. The sequential use of the two-stage mediums finely regulates the induction and differentiation process of mesoderm into iMSCs in stages. Compared with a single medium scheme, this helps to improve the phenotypic purity of the final product MSCs, shorten the overall induction cycle, and reduce the risk of cell function decline due to long-term culture. Attached Figure Description

[0019] Figure 1 The iPSC cell morphology of this invention; Figure 2 The EB sphere shape is shown in Embodiment 1 of the present invention; Figure 3 This is the morphology at the end of mesodermal induction in Example 1 of the present invention; Figure 4 The iMSC cell morphology is shown in Example 1 of this invention; Figure 5 The image shows the UMAP results of single-cell sequencing identification of mesenchymal stromal cells prepared in Embodiment 1 of this invention. Figure 6 The results of the three lineage differentiation of mesenchymal stromal cells prepared in Example 1 of the present invention are shown, where a is the osteogenic result, b is the chondrogenic result, and c is the adipogenic result. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Unless otherwise specified, all chemical reagents and materials in this invention are purchased from the market or synthesized from raw materials purchased from the market.

[0022] In this invention, the iPSC complete culture medium was purchased from Ajinomoto Co., Ltd., Japan, and is a commercial StemFit Basic 03 culture medium kit; the MSC basal culture medium was purchased from Suzhou Youyi Biotechnology Co., Ltd., and is a Stem Cell 01 culture medium kit; ITS-X, GlutaMAX, and NEAA were all added in concentrated form, and the concentration was indicated by "×" to represent the multiplier relationship. 1× indicates the use at the recommended working concentration (i.e., 100× concentrate added to the culture medium at a volume ratio of 1:100), 0.5× indicates the use at 1 / 2 of the recommended working concentration (i.e., 100× concentrate added to the culture medium at a volume ratio of 1:200), and so on. All of the above supplements are conventional commercial products in the field of cell culture, and those skilled in the art can select the appropriate specifications of products based on the above concentration definitions.

[0023] Example 1 A method for inducing iPSCs to differentiate into mesenchymal stromal cells includes the following steps: S1. iPSC Expansion Culture: After thawing the frozen iPSC cells, they were seeded into T25 culture flasks coated with matrix gel and placed in a 37°C, 5% CO2 incubator. The culture was carried out daily with complete iPSC culture medium (with clear chemical composition, serum-free, and free of animal-derived components). When the cell confluence reached 80%~90%, the adherent cells were digested into a single-cell suspension using a non-enzymatic cell dissociation reagent (ReleSR). The suspension was then passaged at a ratio of 6 times the culture bottom area to T175 culture flasks. The culture was continued with daily medium changes. When the cell confluence reached 80%~90% again, expanded iPSCs were obtained, and the next stage began. S2, Three-dimensional suspension culture: Expanded iPSCs were digested into single-cell suspensions using Accutase, and after counting, cultured at 2×10⁶ cells per cell. 6 Cells were seeded at a density of 10 cells / well into Aggrewell 400 microplates. Y27632 was added to the culture medium to a final concentration of 10 μM. The plates were then incubated overnight at 37°C with 5% CO2 to allow the cells to aggregate in the microplates and form uniform embryoid bodies (EB spheres). The next day, all EB spheres were collected and transferred to a three-dimensional suspension bioreactor (ABLE reactor). The supernatant containing Y27632 was discarded, and mesotherapy induction medium was added for further culture. S3. Mesodermal induction: Mesodermal induction medium composition (final concentration): 3 μM CHIR99021, 20 ng / mL bFGF, 20 ng / mL BMP-4, dissolved in serum-free basal medium with well-defined chemical composition. EB spheres were cultured in the above medium at 37°C and 5% CO2 for 2 days. After induction, flow cytometry showed that the proportion of TBXT-positive cells reached 92%, confirming that the mesodermal induction effect met expectations, and proceeding to the next stage. S4. Preparation of mesenchymal stromal cells: All obtained mesodermal cell spheroids were transferred to T225 culture flasks coated with Laminin-521. MSC induction medium A (iPSC complete medium supplemented with: 7 μM SB-431542, 20 ng / mL BMP-4, 20 ng / mL bFGF) was added, and the cells were cultured at 37℃ in a 5% CO2 incubator for 2 days. Medium A was discarded, and MSC induction medium B (MSC basal medium with chemically defined components supplemented with: 2.5 μM SB-431542, 20 ng / mL BMP-4, 20 ng / mL bFGF, 20 ng / mL PDGF-BB, 100 μM sodium vitamin C, 20 ng / mL EGF, 0.5% HSA, 1.0× ITS-X, 1.0× GlutaMAX, 1.0× NEAA) was added, and the cells were cultured again. From day 4 of culture, the cells were injected at a rate of 3×10⁻⁶. 4 cells / cm 2 The cells were passaged at a density of 3 days, for a total of 2 passages, to obtain mesenchymal matrix cells.

[0024] The generally accepted characteristics of iPSC cells are: cell clonal cluster growth, clear clonal margins, high nucleocytoplasmic ratio, homogeneous morphology, and close contact between cells within the clone, exhibiting typical iPSC cell characteristics, such as... Figure 1 As shown, it meets the feature requirements.

[0025] from Figure 2 As can be seen, the cells grow in suspension as spheres, and the spheres are of uniform size. The better the uniformity, the more beneficial it is to the uniformity of the final differentiated product.

[0026] from Figure 3 As can be seen, the uniformity of the spheres after mesodermal induction is still relatively good. The better the uniformity, the more conducive it is to the uniformity of the final product after differentiation.

[0027] MSC cells are characterized by adherent growth, fibroblast-like growth, and spindle-shaped or irregularly triangular forms. The cell morphology obtained through differentiation in this invention is as follows: Figure 4 As shown, the cells conform to the characteristics of MSCs and have good cell homogeneity.

[0028] Example 2 A method for inducing iPSCs to differentiate into mesenchymal stromal cells includes the following steps: S1. iPSC Expansion Culture: After thawing the frozen iPSC cells, they were seeded into T25 culture flasks coated with matrix gel and placed in a 37°C, 5% CO2 incubator. The culture was carried out daily with iPSC complete medium (with clear chemical composition, serum-free, and free of animal-derived components). When the cell confluence reached 80%~90%, the adherent cells were digested into a single-cell suspension using a non-enzymatic cell dissociation reagent (ReleSR). The suspension was then passaged at a ratio of 3 times the culture bottom area to T75 culture flasks and cultured daily with the medium changed again. When the cell confluence reached 80%~90% again, expanded iPSCs were obtained, and the next stage began. S2, Three-dimensional suspension culture: Expanded iPSCs were digested into single-cell suspensions using Accutase, and after counting, cultured at 1×10⁻⁶ cells per cell. 5 Cells were directly seeded at a density of 100 cells / mL into a three-dimensional suspension bioreactor (ABLE shake flask), and iPSC complete medium was added. Y27632 was added to a final concentration of 5 μM. The cells were incubated overnight at 37°C and 5% CO2. The next day, after standing for 5 minutes, the culture supernatant was aspirated and discarded. Mesodermal induction medium was added and the cells were cultured overnight to allow the cells to aggregate under suspension conditions to form embryoids and simultaneously initiate the mesodermal induction program. S3. Mesodermal induction: Mesodermal induction medium composition (final concentration): 6 μM CHIR99021, 10 ng / mL bFGF, 10 ng / mL BMP-4, dissolved in serum-free basal medium with well-defined chemical composition. The embryoids were cultured in the above medium at 37°C and 5% CO2 until the total induction time reached 3 days. After induction, the proportion of TBXT-positive cells was detected by flow cytometry and reached 91%, confirming that the mesodermal induction effect met expectations. S4. Preparation of mesenchymal stromal cells: All obtained mesodermal cell spheroids were transferred to T175 culture flasks coated with Laminin-521. MSC induction medium A (iPSC complete medium supplemented with: 5 μM SB-431542, 10 ng / mL BMP-4, 10 ng / mL bFGF) was added, and the cells were cultured for 2 days. Then, the cells were replaced with MSC induction medium B (chemically defined MSC basal medium supplemented with: 2.5 μM SB-431542, 10 ng / mL BMP-4, 10 ng / mL bFGF, 10 ng / mL PDGF-BB, 150 μM sodium vitamin C, 10 ng / mL EGF, 0.3% HSA, 0.5 × ITS-X, 0.5 × GlutaMAX, 0.5 × NEAA) and cultured until day 14. From day 5 onwards, the cells were cultured at a rate of 2 × 10⁻⁶. 4 cells / cm 2 The cells were passaged at a density of 3 times, every 4 days, to obtain mesenchymal matrix cells.

[0029] Example 3 A method for inducing iPSCs to differentiate into mesenchymal stromal cells includes the following steps: S1. iPSC Expansion Culture: After thawing the frozen iPSC cells, they were seeded into T25 culture flasks coated with matrix gel and placed in a 37°C, 5% CO2 incubator. The culture was carried out daily with complete iPSC culture medium (with clear chemical composition, serum-free, and free of animal-derived components). When the cell confluence reached 80%~90%, the adherent cells were digested into a single-cell suspension using a non-enzymatic cell dissociation reagent (ReleSR). The suspension was then passaged at a ratio of 9 times the culture bottom area and expanded into T225 culture flasks. The culture was continued with daily medium changes. When the cell confluence reached 80%~90% again, expanded iPSCs were obtained, and the next stage began. S2, Three-dimensional suspension culture: Amplified iPSCs were digested into single-cell suspensions using Accutase at a concentration of 1×10⁻⁶ cells / cells. 6 Cells were seeded at a density of 100 cells / well into Aggrewell 400 microplates, and Y27632 was added to a final concentration of 5 μM. The plates were cultured overnight to form embryoids. The next day, EB spheres were collected and transferred to a three-dimensional suspension bioreactor. The supernatant containing Y27632 was discarded, and mesotherapy induction medium was added. S3. Mesodermal induction: Mesodermal induction medium composition (final concentration): 1 μM CHIR99021, 5 ng / mL bFGF, 5 ng / mL BMP-4. The embryoids were cultured in the above medium at 37°C and 5% CO2 for 3 days. After induction, the proportion of TBXT-positive cells was detected by flow cytometry to be 90%, confirming that the mesodermal induction effect met expectations, and proceeding to the next stage. S4. Preparation of mesenchymal stromal cells: All obtained mesodermal cell spheroids were transferred to T225 culture flasks coated with Laminin-521. MSC induction medium A (iPSC complete medium supplemented with: 10 μM SB-431542, 50 ng / mL BMP-4, 50 ng / mL bFGF) was added, and the cells were cultured at 37℃ in a 5% CO2 incubator for 2 days. Medium A was discarded, and MSC induction medium B (MSC basal medium with chemically defined components supplemented with: 2.5 μM SB-431542, 50 ng / mL BMP-4, 50 ng / mL bFGF, 50 ng / mL PDGF-BB, 200 μM sodium vitamin C, 50 ng / mL EGF, 1.0% HSA, 1.0× ITS-X, 1.0× GlutaMAX, 1.0× NEAA) was added. The cells were cultured for 10 days. From day 4 onwards, the cells were injected at a rate of 4×10⁻⁶. 4 cells / cm 2The cells were passaged at a density of 3 days, for a total of 2 passages, to obtain mesenchymal matrix cells.

[0030] Flow cytometry detection method for induced iMSC cells 1. Prepare the required number of cells (usually 0.5~1×10⁶ cells per test tube). 6 Aliquot the cells into 1.5 mL centrifuge tubes. Centrifuge at 300 g for 5 minutes and carefully discard the supernatant. Resuspend the cells once with pre-chilled staining buffer (calcium- and magnesium-free D-PBS solution containing 1% goat serum), centrifuge again, and discard the supernatant. After discarding the supernatant, resuspend the cell pellet in 100 μL of pre-chilled staining buffer. Add the following ingredients to the cell suspension according to the table below: Gently vortex to mix. After incubating at 4°C in the dark for 30 minutes, add 1 mL of pre-chilled staining buffer, centrifuge at 300g for 5 minutes, discard the supernatant, and repeat the washing process twice. Resuspend the cells in 200-300 μL of flow cytometry staining buffer and immediately analyze using a flow cytometer. The test results are shown in Table 1 below.

[0031] Table 1. Flow cytometry results of induced iMSC cells As can be seen from Table 1 above, the identification of the surface markers of differentiated cells in this invention is as follows: CD13 (aminopeptidase N / APN): It is involved in cell adhesion and migration. Activation of CD13 can enhance the monolayer adhesion of human mesenchymal stem cells, regulate the angiogenesis mechanism, and may participate in the regeneration process of skeletal muscle, and has potential anti-inflammatory effects.

[0032] CD29 (integrin β1 chain): mediates the adhesion of MSCs to the extracellular matrix, which is the basis of homing ability, and at the same time regulates osteogenic / chondrogenic differentiation through the PI3K / Akt pathway.

[0033] CD44 (hyaluronic acid receptor): Mediates cell adhesion and migration: By binding to hyaluronic acid, type I and type IV collagen, it mediates cell-cell and cell-matrix adhesion. This interaction is key to cell migration ability and directly affects the homing ability of MSCs, i.e., whether MSCs can successfully reach the site of injury. Regulates stem cell behavior: CD44 is an important component of the stem cell microenvironment, capable of integrating signals and regulating key behaviors such as stem cell self-renewal. Promotes tissue repair: After transplantation of exogenous MSCs, increased CD44 expression and enhanced migration ability of endogenous MSCs can be observed, thereby promoting tissue healing (such as fractures).

[0034] CD73 (5'-nucleotidase): hydrolyzes AMP to produce adenosine, a core molecule in immune regulation—inhibiting T cell activation, reducing the release of inflammatory factors, and promoting angiogenesis. The 2025 standard requires a clearly defined positive rate (usually ≥95%), and its expression level is directly related to clinical anti-inflammatory effects.

[0035] CD90 (Thy-1 antigen): Maintains the stemness of MSCs, inhibits premature differentiation through the MAPK pathway, and enhances cell adhesion and homing.

[0036] CD105 (endothelial glycoprotein): regulates the TGF-β signaling pathway, directing osteogenic / chondrogenic differentiation (inhibiting adipogenic differentiation), and is a key quality control indicator for bone repair MSC preparations. The positive rate is related to cell source and in vitro cell culture time.

[0037] HLA-ABC (MHC-I class molecule): Expression of HLA-ABC gives MSCs the potential to be recognized by the immune system. HLA-ABC molecules are ligands of inhibitory receptors on the surface of natural killer (NK) cells, and expression of HLA-ABC helps MSCs evade killing by NK cells in the immune system.

[0038] CD45 (Common Leukocyte Antigen): A unique marker for all hematopoietic cell lineages (T cells, B cells, NK cells, monocytes, and granulocytes). Significance: A negative CD45 result is the "gold standard" for ruling out hematopoietic contamination.

[0039] CD31 (platelet endothelial cell adhesion molecule): Expressing cells: vascular endothelial cells, platelet-specific marker. Significance: A negative result is key to ruling out endothelial cell contamination.

[0040] CD34 (marker for hematopoietic stem / progenitor cells): This is a marker specific to hematopoietic stem / progenitor cells and endothelial progenitor cells. Significance: A negative result is crucial for ruling out contamination by hematopoietic stem / progenitor cells or endothelial progenitor cells.

[0041] HLA-DR (MHC class II molecule): A marker specific to dendritic cells, macrophages, and activated T cells. Significance: A negative result is a core characteristic of "low immunogenicity" in MSCs.

[0042] CD106 (VCAM-1): Its low expression in the resting state of MSCs is not functionally representative. CD106 is only functionally upregulated under inflammatory stimulation and is the most important adhesive immunosuppressive molecule on the surface of MSCs.

[0043] HLA-G (non-classical MHC-I): It is expressed at extremely low levels on MSCs, but its expression can be induced after entering the body and being exposed to a hypoxic environment or an anti-inflammatory microenvironment such as IL-10. It is the strongest known innate immune tolerance molecule in the human body.

[0044] Methods for detecting the proliferation inhibition rate of PBMCs in mesenchymal stromal cells: 1. Add 0.5-1 ml of Laminin-521 coating buffer diluted with DPBS (containing calcium and magnesium) to each well of a 24-well plate at a concentration of 5 μg / mL. Coat overnight at 2-8°C or 1-2 hours in advance at 37°C. After removing the coating buffer, add 1 ml of a 7×10⁻⁶ ppm solution to each well. 4 MSC cell suspensions of 10 cells / mL were placed in a 37°C 5% CO2 incubator and cultured until the cells adhered to the culture vessel before proceeding to the next step of the experiment.

[0045] 2. Add 5 μL of CFSE (1000 μmol / mL) to 1 mL of PBMC suspension and incubate at 37°C in the dark for 10 min. Mix the cells every two minutes. After the reaction, add RPMI 1640 complete medium containing 10% FBS and incubate at 37°C for 5 min. Centrifuge at 300g for 5 min, remove the supernatant, and wash twice with complete medium.

[0046] 3. Resuspend the cells in RPMI 1640 complete medium containing PHA-L at a final concentration of 10 μg / mL, and adjust the concentration to 1 × 10⁻⁶. 6 Cells / mL were added to 24-well plates seeded with MSCs, and labeled PBMCs were added at the MSC to PBMC ratio. Each well was supplemented with 1.5 mL of RPMI 1640 complete medium. A PHA-L-stimulated PBMC group (without MSCs) was set up as a negative control; a PBMC-only culture group (without PHA-L stimulation) was also set up as a blank control. Cells were cultured for 6 days. After co-culture, cells were aspirated into 96-well plates, centrifuged at 300g for 5 minutes, and the supernatant was discarded. 1 mL of staining buffer was added, and the cells were centrifuged at 300g for 5 minutes, and the supernatant was discarded. The washing was repeated twice. 7-AAD was diluted 1:1000 with staining buffer, and 150 μL of diluted 7-AAD was added to each well. The mixture was then analyzed by flow cytometry.

[0047] The formula for calculating the inhibition rate is: Inhibition rate % = (Control group - Experimental group) / Control group Methods for detecting the secretion inhibition rate of IL17A, TNFα, and INFgamma: 1. Add 0.5-1 ml of Laminin-521 coating buffer diluted with DPBS (containing calcium and magnesium) to each well of a 24-well plate at a concentration of 5 μg / mL. Coat overnight at 2-8°C or 1-2 hours in advance at 37°C. After removing the coating buffer, add 1 ml of a 7×10⁻⁶ ppm solution to each well. 4MSC cell suspensions of 10 cells / mL were placed in a 37°C 5% CO2 incubator and cultured until the cells adhered to the culture vessel before proceeding to the next step of the experiment.

[0048] 2. Resuscitate PBMCs and dilute them to 3.5 × 10⁻⁶ using RPMI 1640 (containing 10% FBS). 5 Cells / mL, Experimental group: 1 mL / well was added to each iMSC well, followed by IL-2 at a final concentration of 40 IU / mL and PHA-L at a final concentration of 2.5 μg / mL. Blank group: 1 mL of PBMC was added to each well without iMSC, followed by IL-2 at a final concentration of 40 IU / mL and PHA-L at a final concentration of 2.5 μg / mL. The cells were incubated at 37℃ in a CO2 incubator for 5 days. After incubation, the cells were centrifuged at 500g for 5 min, and the cell supernatant was collected as the test sample. The test samples were analyzed using commercial IL17A, TNFα, and INFgamma assay kits. Inhibition rate % = (control group - experimental group) / control group. The test results are shown in Table 2 below.

[0049] Table 2. Immunosuppression test results of mesenchymal stromal cells in this invention. As shown in Table 2 above, one of the recognized biological characteristics of MSCs is their immunosuppressive function. PBMCs, or peripheral blood mononuclear cells (T cells, B cells, NK cells, and monocytes), are the main types of immune cells in the human body. When iMSCs are co-cultured with PBMCs, the iMSCs inhibit the proliferation of the PBMC immune cell population. IL17A, IFNgamma, and TNFα cytokines are cytokines secreted during an immune response; all of these were inhibited in this study, further confirming the immunosuppressive function of MSCs at the cytokine level.

[0050] from Figure 5 As can be seen, single-cell sequencing provides a deep level of identity verification for cell populations: it can analyze the entire transcriptome and precisely distinguish MSCs from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), etc., through gene expression profiling. It can simultaneously detect transcripts from all cells, thereby identifying non-MSC cells with specific gene expression profiles mixed in with the sample. The single-cell identification results obtained in this invention are MSCs, with very few endothelial and neural cells. The gene expression profiling further confirms the identity of the differentiated product as MSCs.

[0051] Trilineage differentiation (osteogenic, chondrogenic, and adipogenic) functional testing is one of the core pillars of mesenchymal stem cell (MSC) identification, serving as functional evidence for their recognition as stem cells and their potential for therapeutic use. (See attached image) Figure 6Figures a, b, and c in the diagram represent the detection results of MSCs obtained in this invention. From the perspective of differentiation function, they further demonstrate that the MSCs obtained in this invention possess the generally accepted typical functional characteristics of MSCs. Figure a shows osteogenic detection: after culture with the osteogenic kit, Alizarin Red S staining showed that calcium nodules were stained orange-red to dark red. Figure b shows chondrogenic detection: after culture with the chondrogenic kit, Alcian Blue staining showed that chondrocytes and matrix were stained blue. Figure c shows adipogenic detection: after culture with the adipogenic kit, Oil Red O staining showed that fat droplets were stained bright red.

[0052] The above description is a further detailed explanation of the present invention in conjunction with specific implementation examples. It should not be considered that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all of these should be considered to fall within the protection scope of the present invention.

[0053] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for inducing iPSCs to differentiate into mesenchymal stromal cells, characterized in that, Includes the following steps: S1. iPSC cells were placed in T25 for two-dimensional adherent expansion culture. The adherent cells were digested into a single-cell suspension using a cell dissociation agent. The cells were then passaged 3 to 9 times the culture area to obtain expanded iPSCs. S2. The amplified iPSCs obtained in step S1 are digested into a single-cell suspension using protease and then subjected to three-dimensional suspension culture in a medium containing ROCK inhibitor to allow the cells to aggregate and form embryoids. S3. Discard the culture supernatant containing ROCK inhibitor, add mesodermal induction medium to the embryoids from step S2, and culture at 37°C and 5% CO2 to induce iPSCs to differentiate into mesodermal cells. S4. Transfer the mesodermal cell spheroids obtained in step S3 to a two-dimensional culture vessel coated with extracellular matrix proteins, and culture them sequentially with MSC induction medium A and MSC induction medium B, followed by (2~4)×10⁻⁶ culture. 4 cells / cm 2 The cells were passaged at a high density, every 3-4 days, and mesenchymal matrix cells were obtained after 2-3 passages.

2. The method according to claim 1, characterized in that, The cell dissociation agent mentioned in step S1 is ReleSR digestion solution.

3. The method according to claim 1, characterized in that, The three-dimensional suspension culture described in step S2 is performed in one of the following ways: (a) The single-cell suspension was prepared at 1×10 6 ~4×10 6 Cells / well were seeded at a density of 1,000 cells / well into a culture plate with a microporous array structure, cultured overnight in a medium containing ROCK inhibitor to form embryoids, and then the embryoids were transferred to a three-dimensional suspension bioreactor for culture. (b) The single-cell suspension was diluted with 1×10⁻⁶ 5 ~2×10 5 Cells were directly seeded into a three-dimensional suspension bioreactor at a density of 100 cells / mL, cultured overnight in a medium containing ROCK inhibitor, and after standing, the supernatant was discarded and replaced with mesotherapy induction medium for further culture.

4. The method according to claim 1, characterized in that, The ROCK inhibitor mentioned in step S2 is Y27632, with a final concentration of 5~10 μM; the protease is Accutase.

5. The method according to claim 1, characterized in that, The final concentrations of each component in the mesodermal induction medium described in step S3 are: 1~6 μM CHIR99021, 5~60 ng / ml bFGF, and 5~60 ng / ml BMP-4.

6. The method according to claim 1, characterized in that, The induction differentiation time in step S3 is 2-3 days, and the proportion of TBXT-positive cells in the mesodermal cells obtained after induction is not less than 90%.

7. The method according to claim 1, characterized in that, The MS induction medium A mentioned in step S4 is obtained by adding the following components to the complete iPSC medium: 5~10μM SB-431542, 5~50ng / mL BMP-4, 5~50ng / mL bFGF; the MS induction medium B is obtained by adding the following components to the MSC basal medium: 2.5μM SB-431542, 5~50ng / mL BMP-4, 5~50ng / mL bFGF, 5~50ng / mL PDGF-BB, 50~300μM sodium vitamin C, 5~50ng / mL EGF, 0.1~1.0wt% HSA, 0.5~1.0× ITS-X, 0.5~1.0× GlutaMAX, 0.5~1.0× NEAA.

8. The method according to claim 1, characterized in that, The extracellular matrix protein mentioned in step S4 is selected from one or more of Laminin-521, Laminin-511, Fibronectin, and Vitronectin; the culture time in MSC induction medium A is 2 days, and the culture time in MSC induction medium B is 6-14 days.

9. A mesenchymal matrix cell prepared by the method according to any one of claims 1-8, characterized in that, The mesenchymal stromal cells meet the following phenotypic criteria: the positive rates of CD90, CD73, CD13, CD29, and CD44 cells are all not less than 90%; the positive rates of negative CD34, CD45, and HLA-DAR cells are all less than 10%.

10. The application of the mesenchymal stromal cells as described in claim 9 in cell therapy, bone / cartilage repair, treatment of autoimmune diseases, or tissue engineering, characterized in that, The mesenchymal stromal cells described herein possess the ability to differentiate into adipogenic, chondrogenic, and osteogenic lineages in vitro, and have an inhibitory effect on the proliferation of peripheral blood mononuclear cells.

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