A method for inducing the directed differentiation of induced pluripotent stem cells into mesenchymal stromal cells

Abstract

The application discloses a method for inducing directional differentiation of induced pluripotent stem cells into mesenchymal stromal cells and application thereof, and belongs to the technical field of stem cells and regenerative medicine. The method comprises the following steps: under a low-oxygen condition, using a culture medium containing a Wnt agonist and a TGF-beta inhibitor to induce the induced pluripotent stem cells into neural crest cells; under intermittent physical relaxation stimulation, using a culture medium in which the Wnt agonist is removed, the concentration of the TGF-beta inhibitor is reduced, and TGF-beta 1 is newly added to induce the neural crest cells to mature into mesenchymal stromal cells; and sequentially performing tangential flow ultrafiltration, size exclusion chromatography purification and final concentration on a culture supernatant to obtain exosomes. The application realizes short-period high-purity differentiation of iMSCs and large-scale preparation of exosomes with high recovery rate, and application of the exosomes in immune cell regulation and nerve cell repair.

Description

Technical Field

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

[0002] Mesenchymal stromal cells (MSCs) have broad application prospects in cell therapy and regenerative medicine due to their multi-lineage differentiation, tissue repair, and immunomodulatory capabilities. However, traditional tissue-derived MSCs (such as bone marrow and umbilical cord) suffer from limited donor sources, strong intercellular heterogeneity, and large batch-to-batch variability, which severely restrict their standardization and large-scale application.

[0003] Induced pluripotent stem cells (iPSCs) are abundant, have strong expansion capabilities, and are free from ethical controversies, making them a high-quality seed cell for preparing induced mesenchymal stem cells (iMSCs). Currently reported iPSC-to-iMSC differentiation techniques mainly fall into four categories: First, the monolayer spontaneous differentiation method, which is simple to operate but has low differentiation efficiency and cell purity, with an overall culture cycle of 3-4 weeks; second, the monolayer directed differentiation method based on the mesoderm or neural crest pathway, which, while having advantages in efficiency and purity, still requires a differentiation cycle of 3-4 weeks; third, the embryoid differentiation method, which not only has a long cycle of 4-6 weeks but also suffers from poor batch stability and generally low cell purity; and fourth, the organoid / three-dimensional culture derivation method, which has a differentiation cycle of 4-6 weeks and suffers from complex procedures, high preparation costs, and low cell yield. In particular, the organoid differentiation pathway is circuitous and highly dependent on excipients such as matrix gels and hydrogels, further limiting its standardized application and clinical translation.

[0004] In summary, all current mainstream differentiation technologies have their own shortcomings. There is an urgent need in the industry to develop a new targeted differentiation scheme for iPSCs to iMSCs that has a shorter differentiation cycle, a more direct induction pathway, a clearly defined culture system composition, and does not require additional purification culture. Summary of the Invention

[0005] The purpose of this invention is to provide a method and application for the directed differentiation of induced pluripotent stem cells into mesenchymal stromal cells, in order to solve the technical problems of existing methods for differentiating iPSCs into iMSCs, such as indirect differentiation pathways, long cycles, dependence on animal-derived components, and unstable purity and function.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for the directed differentiation of induced pluripotent stem cells into mesenchymal stromal cells, comprising the following stages performed sequentially: Phase 1: Under hypoxic culture conditions, induced pluripotent stem cells were induced to differentiate into neural crest cells using a first culture medium containing Wnt signaling pathway agonists and TGF-β signaling pathway inhibitors. Second stage: Under intermittent physical relaxation stimulation, the neural crest cells were induced to mature into mesenchymal matrix cells using a second culture medium; In this culture medium, compared to the first culture medium, the Wnt signaling pathway agonist was removed, the concentration of the TGF-β signaling pathway inhibitor was reduced, and TGF-β1 was added.

[0007] Furthermore, the low-oxygen culture condition is an oxygen volume fraction of 5%.

[0008] Furthermore, the Wnt signaling pathway agonist is used at a concentration of 1-10 μM in the first culture medium; the TGF-β signaling pathway inhibitor is used at a concentration of 5-20 μM in the first culture medium, and the concentration is reduced to 1-10 μM in the second culture medium; the TGF-β1 is used at a concentration of 0.5-5 ng / mL in the second culture medium.

[0009] Furthermore, the Wnt signaling pathway agonist is CHIR99021; the TGF-β signaling pathway inhibitor is SB431542.

[0010] Furthermore, the cell culture containers in the first stage are coated with fibronectin at a concentration of 10-50 μg / mL; In the second stage, the cell culture vessel was coated with a double layer of collagen I and fibronectin in a 1:1 ratio, with the coating concentration of collagen I being 5-20 μg / mL and the coating concentration of fibronectin being 5-20 μg / mL.

[0011] Furthermore, the first culture medium is CDM medium, with IMDM / F12 as the basic component, and ITS, BSA and lipid concentrate added; The second culture medium was α-MEM, supplemented with 5-15% serum-free substitute KOSR and 1-10 ng / mL bFGF.

[0012] Furthermore, the intermittent physical relaxation stimulation includes: intermittent gas exchange and mechanical stress release of the culture vessel every 24 hours, with each session lasting 5-15 minutes.

[0013] Secondly, the present invention provides an induced mesenchymal matrix cell, characterized in that it is prepared by the above method.

[0014] Thirdly, this invention provides the application of induced mesenchymal stromal cells in the preparation of drugs for treating immune dysfunction diseases and neuronal cell damage diseases. The immune dysfunction diseases include: rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, neuromyelitis optica, autoimmune uveitis, allergic rhinitis, allergic asthma, allergic dermatitis, dry eye syndrome, etc.; the neuronal cell damage diseases include: Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, ischemic stroke, diabetic retinopathy, glaucoma, etc.

[0015] Fourthly, the present invention provides exosomes from induced mesenchymal stem cells (iMSC-Exos), which are obtained by isolation and purification of induced mesenchymal stromal cells.

[0016] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects: The method for directed differentiation of induced pluripotent stem cells (iPSCs) into mesenchymal stromal cells provided by this invention directly follows a two-level linear pathway from iPSCs to NCCs and then to iMSCs, avoiding redundant intermediate states such as the mesoderm and three-dimensional spheroids. This minimizes transcriptional fate conversion steps and reduces differentiation time from the developmental root. Upon completion of NCC induction, the CD271 (NGFR) mesenchymal marker is already stably highly expressed. After transfer to the second-stage culture medium, only minor adjustments to the TGF-β signaling are needed for rapid maturation into standard MSCs, eliminating the need to start the entire mesenchymal gene network from scratch and significantly reducing the time and cost of transcriptional remodeling. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the process for the directed differentiation of induced pluripotent stem cells into mesenchymal stromal cells according to the present invention; Figure 2 This is a diagram illustrating the morphology and phenotype analysis of neural crest cells in this invention. Figure 3 This is a diagram of the morphology of mesenchymal matrix cells in this invention. Figure 4 This is a flow diagram of the mesenchymal matrix cell surface markers detection method of the present invention; Figure 5This is a diagram illustrating the three-lineage differentiation capacity of mesenchymal matrix cells in this invention. Figure 6 This is a graph showing the detection of the immunomodulatory capacity of mesenchymal matrix cells in this invention. Figure 7 This is a diagram showing the expression of genes related to immune regulation in mesenchymal stromal cells according to the present invention. Figure 8 This is a schematic diagram of the iMSC-Exos collection, purification, and quality control process of the present invention; Figure 9 This is a comparison of the recovery rates of iMSC-Exos by tangential flow ultrafiltration & size exclusion chromatography and conventional ultracentrifugation (purification of iMSC-Exos). Figure 10 This is a particle size distribution diagram of NTA in this invention; Figure 11 This is a TEM image of the present invention; Figure 12 The results of nanofluid cytometry detection of exosome biomarkers in this invention; Figure 13 This invention uses Western blot to detect the expression of biomarkers for HFL1, HFL1-Exos, iMSC, and iMSC-Exos. Figure 14 This is a comparison chart of the protein ratio of iMSC-Exos particles purified by the two methods of this invention (quality control of iMSC-Exos). Figure 15 This is a graph showing the verification results of the anti-inflammatory function of iMSC-Exos in this invention; Figure 16 This is a diagram showing the verification results of the neural repair function of the iMSC-Exos of this invention. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. 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. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0020] I. Experimental Materials ; ;

[0021] II. Experimental Methods like Figure 1 As shown, a method for the directed differentiation of induced pluripotent stem cells into mesenchymal stromal cells is presented. This method achieves efficient directed differentiation in an animal-free system through dynamic temporal regulation of two-stage signaling pathways and combined stimulation by the physicochemical properties of the microenvironment. The method includes the following stages performed sequentially: Step 1, First Stage: Under hypoxic culture conditions, induced pluripotent stem cells were induced to differentiate into neural crest cells using a first culture medium containing Wnt signaling pathway agonists and TGF-β signaling pathway inhibitors.

[0022] Step 2, second stage: Under intermittent physical relaxation stimulation, the culture container is opened for 10 minutes every 24 hours, and the neural crest cells are induced to mature into mesenchymal stromal cells using the second culture medium; the second culture medium, compared with the first culture medium, removes the Wnt signaling pathway agonist, reduces the concentration of the TGF-β signaling pathway inhibitor, and adds TGF-β1.

[0023] Specifically, in the first stage, the concentration of the Wnt signaling pathway agonist in the first culture medium is 1-10 μM, and the concentration of the TGF-β signaling pathway inhibitor in the first culture medium is 5-20 μM. As a preferred embodiment, the Wnt signaling pathway agonist is CHIR99021 at a concentration of 3 μM; the TGF-β signaling pathway inhibitor is SB431542 at a concentration of 10 μM in the first culture medium. Within this concentration range, CHIR99021 can effectively activate Wnt signaling, stabilize β-catenin nuclear translocation, thereby activating the neural plate boundary development program in the neuroectoderm and blocking the spontaneous differentiation of iPSCs into the endoderm and mesoderm; SB431542 is a dual SMAD inhibitor, inhibiting TGF / Activin / Nodal signaling and blocking pluripotency maintenance signals, inhibiting the production of contaminated cells such as epidermal and mesodermal cells.

[0024] In the first stage of the culture system construction, the oxygen concentration under hypoxic culture conditions was 5% to simulate the physiological oxygen tension in early embryonic development. The first culture medium was IMDM / F12 basal medium supplemented with ITS, BSA, and lipid concentrate, and the culture vessels were coated with fibronectin at a concentration of 10-50 μg / mL. In this embodiment, the preferred fibronectin coating concentration was 25 μg / mL, which provides a suitable scaffold for cell adhesion and migration, promoting efficient migration of neural crest cells from the edge of iPSC colonies. The first stage of culture lasted 4-8 days, preferably 6 days.

[0025] In the second stage, the concentration of TGF-β signaling pathway inhibitors in the second culture medium was reduced to 1-10 μM, and TGF-β1 was added at a concentration of 0.5-5 ng / mL. In this example, the concentration of SB431542 was specifically reduced to 5 μM, and the concentration of TGF-β1 was 1 ng / mL. Reducing SB431542 from 10 μM to 5 μM, rather than completely withdrawing it, relieved the strong inhibition of mesenchymal-related genes while preserving the basal signaling level required to maintain cell plasticity; while 1 ng / mL of TGF-β1 was sufficient to initiate the SMAD2 / 3-dependent maturation program and promote the expression of markers such as CD73, CD90, and CD105. Similarly, within the range of 0.5-5 ng / mL of TGF-β1, cells were able to complete the phenotypic conversion to mesenchymal stromal cells, with only slight differences in the maturation rate.

[0026] In the second-stage culture system, the second culture medium was α-MEM medium supplemented with 5-15% KOSR and 1-10 ng / mL bFGF. In this embodiment, the KOSR addition ratio was 10%, and the bFGF concentration was 5 ng / mL. The culture vessel was bilayer-coated with collagen I and fibronectin in a 1:1 ratio, wherein the coating concentrations of collagen I and fibronectin were independently 5-20 μg / mL, and in this embodiment, both were preferably 10 μg / mL. This specific matrix combination and concentration ratio provided optimal mechanical support and biochemical signals for the transformation of neural crest cells into mesenchymal stromal cells. The second-stage culture duration was 5-10 days, preferably 8 days, so that the total differentiation cycle was controlled within 12-18 days, preferably 14 days.

[0027] Example 1: NCC Morphology and Phenotypic Analysis Collect Day 6 differentiated cells and digest them into single-cell suspensions using TrypLE or Accutase. Wash twice with PBS and adjust the cell density to 1×10⁶ cells / cells. 6 cells / mL. Take 100 μL of cell suspension (approximately 1 × 10⁻⁶ cells / mL). 5 NCC cells were incubated at 4°C for 30 minutes in the dark with PE-labeled anti-human CD271 (NGFR / p75NTR) antibody. An isotype control was also included. The cells were washed twice with PBS to remove unbound antibodies and resuspended in 300 μL PBS for flow cytometry analysis. A negative threshold was set using the isotype control to analyze the proportion of CD271-positive cells. Under a light microscope, NCC cells migrating from the edge of iPSC colonies appeared as slender spindles. Flow cytometry analysis revealed that 82.1% of the cells were CD271-positive, indicating successful acquisition of high-purity neural crest cells, which are ready for the second stage of NCC differentiation into iMSCs. Figure 2 ).

[0028] Example 2: iMSC (P0 generation) morphology When the cells adhered and grew to a confluence of approximately 70%-80%, their morphology was observed and photographed under a regular optical microscope. iMSC cells showed uniform adherence and growth, exhibiting a whorled or parallel arrangement. Individual cells were elongated spindle-shaped, with high cell density, uniform distribution, and no obvious undifferentiated cell clumps or contaminating cells, demonstrating good overall morphological uniformity. Figure 3 ).

[0029] Example 3: Flow cytometry detection of surface markers in iMSCs (P0 passage) Collect P0 generation iMSC cells and adjust the density to 1×10⁻⁶. 6 cells / mL; fluorescently labeled antibodies: CD73-FITC, CD90-PE, CD105-APC, and negative marker antibodies (CD34-APC, CD45-PE, HLA-DR-APC) were added respectively; incubated at 4°C in the dark for 30 minutes, washed with PBS, and detected by flow cytometry. Gating was performed using isotype controls. Results: The positive rates of CD73 / CD90 / CD105 in the P0 generation iMSCs of this invention were all ≥99.5%, and the total negative markers were <0.5%, fully meeting the identification criteria for MSCs. Figure 4 ).

[0030] Example 4: Trilineal Differentiation Capacity Detection (1) Osteogenic differentiation: iMSCs at 2×10 4 cells / cm 2 Inoculate, and when the confluence reaches 70%, switch to osteogenic induction medium; after 2 weeks of induction, fix with 4% paraformaldehyde and stain with alizarin red (pH 4.2, 20 minutes).

[0031] (2) Adipogenic differentiation: iMSCs at 2×10 4 cells / cm 2 Inoculate and when the confluence reaches 100%, switch to lipid induction medium A; after 3 days, switch to solution B, and after 1 day, switch back to solution A, repeating the cycle for 4 weeks; stain with Oil Red O (0.5% Oil Red O isopropanol solution, 15 minutes).

[0032] (3) Chondrogenic differentiation: 3×10 5 The iMSC cells were centrifuged to form microspheres, which were then added to chondrogenic induction medium. After 3 weeks of culture, the cells were embedded in paraffin, sectioned, and stained with Alcian blue (pH 2.5, 30 minutes). The results indicate that the iMSCs of this invention have significant osteogenic, adipogenic, and chondrogenic differentiation capabilities. Figure 5 ).

[0033] Example 5: Detection of Immune Modulation Capacity iMSC with 2×10 5 Cells / well plated overnight; add PBMC (5×10⁻⁶) 6 Cells / well were co-cultured for 45 hours; then stimulated with PMA (50 ng / mL) + Lonomycin (1 μg / mL) + BFA (10 μg / mL) for 5 hours; CD3 was detected by flow cytometry. + T cell intracellular IFN-γ and TNF-α. The results suggest that the iMSCs of this invention have an inhibition rate of ≥92% against IFN-γ and ≥88% against TNF-α, significantly superior to umbilical cord-derived ucMSCs. Figure 6 ).

[0034] Example 6 IDO and HLA-G Expression detection methods iMSC with 2×10 5 Cells were seeded in wells overnight; IFN-γ (30 ng / mL) was added and the cells were stimulated for 24 hours; total RNA was extracted with Trizol and reverse transcribed into cDNA; quantitative real-time PCR was performed. IDO and HLA-G mRNA expression, in order to GAPDH As an internal reference, the relative expression level was calculated using the 2-ΔΔCt method. Following IFN-γ stimulation, the expression level in the iMSCs of this invention... IDO and HLA-G The mRNA expression level of these cells was significantly higher than that of ucMSCs, and more than 5-fold upregulated compared to the unstimulated group, suggesting that they have high expression of immune regulation-related genes. Figure 7 ).

[0035] Example 7: Collection and Purification of iMSC-Exos 1. iMSC culture and supernatant collection The prepared iMSCs were seeded into cell culture flasks and cultured in exosome-free α-MEM medium (with 10% KOSR added) for 72 hours. The cell culture supernatant was collected, totaling approximately 500 mL.

[0036] 2. Clarification process The collected supernatant was centrifuged sequentially as follows: 300×g for 10 minutes to remove cell debris; 2,000×g for 20 minutes to remove microvesicles; and 10,000×g for 30 minutes to remove large vesicles and apoptotic bodies, resulting in a clear supernatant.

[0037] 3. Tangential flow ultrafiltration A 100 kDa PES membrane pack (polyethersulfone) was installed in the tangential flow ultrafiltration system. Operating parameters: transmembrane pressure 0.8 bar, tangential flow rate 3 m / s. 500 mL of the clarified supernatant was concentrated to 25 mL (20-fold concentration), then washed with 5 times its volume (125 mL) of PBS to remove small proteins and metabolic waste. The concentrate was collected for later use.

[0038] 4. Size exclusion chromatography Take 2.5 mL of the above concentrated solution and load it onto a qEV size size exclusion column (35 nm pore size). Use PBS as the mobile phase and a flow rate of 0.8 mL / min. Discard the first 6 mL of flow-through (containing large particles and some extraneous proteins) and collect the fraction from 7 to 11 mL (exosome enrichment zone).

[0039] 5. Final Concentration The collected fraction was concentrated by centrifugation (4,000 × g, 4 °C) using a 100 kDa ultrafiltration tube to approximately 0.5 mL, with a final exosome concentration of approximately 8 × 10⁻⁶. 11 Particles / mL. After aliquoting, store at -80℃ (iMSC-Exos collection, purification, and quality control procedures are as follows). Figure 8 (As shown).

[0040] Example 8 Comparison of recovery rates of iMSC-Exos by different separation methods Take 1000 mL of serum-free culture medium of induced mesenchymal stromal cells (iMSCs), centrifuge at 300×g for 10 min to remove cell pellet, centrifuge at 2000×g for 20 min to remove cell debris and apoptotic bodies, collect the supernatant and divide it into two portions, and purify iMSC-derived exosomes (iMSC-Exos) by ultracentrifugation and tangential flow ultrafiltration combined with size exclusion chromatography, respectively.

[0041] (1) The control group used the traditional ultracentrifugation method to purify exosomes: the pretreated supernatant was transferred to an ultracentrifuge tube and centrifuged at 10000×g and 4℃ for 30 min to remove large vesicle impurities; the upper clear liquid was taken and ultracentrifuged at 110000×g and 4℃ for 70 min, the supernatant was discarded, the bottom precipitate was resuspended in PBS, and the precipitate was washed again by centrifugation at 110000×g and 4℃ for 70 min. Finally, the iMSC-Exos sample purified by ultracentrifugation was obtained by resuspending with an equal volume of PBS.

[0042] (2) The experimental group used tangential flow ultrafiltration in series with size exclusion chromatography to purify exosomes: ① Clarification treatment: The collected supernatant was centrifuged sequentially at 300×g for 10 minutes, 2000×g for 20 minutes, and 10000×g for 30 minutes to remove cells and large cell debris; ②Tangential flow ultrafiltration: Use a membrane with a molecular weight cutoff of 100 kDa, control the transmembrane pressure at 0.5-1.5 bar, concentrate 20-50 times, and wash with PBS 5-10 times the volume; ③ Size exclusion chromatography: A chromatographic column with a pore size of 35 nm (such as a qEV column) is used, with PBS as the mobile phase, and the exosome enrichment fraction is collected; ④ Final concentration: Further concentrate to 1×10 using an ultrafiltration tube with a molecular weight cutoff of 100 kDa. 11 -1×10 12 Particles / mL.

[0043] (3) Quantitative detection of exosome recovery rate: Nanoparticle tracking analysis (NTA) was used to quantify the total number of exosome particles in the two purified products. Based on the total number of exosome particles in the pretreated original supernatant, the exosome recovery rate of the two purification methods was calculated using the following formula: Recovery rate (%) = (Total number of purified exosome particles ÷ Total number of exosome particles in the original supernatant) × 100% Figure 9 Significance criteria: This means that P < 0.001, indicating a significant difference.

[0044] Example 9: Quality Detection of Exosomes (1) NTA particle size detection method Take 10 μL of the purified exosome sample and dilute it with PBS to a suitable concentration (approximately 1 × 10⁻⁶). 8 Up to 1×10 9 Particles per milliliter). Detection was performed using a nanoparticle tracking analyzer. The detection temperature was set to 25 degrees Celsius, the injection rate to 50 μL per minute, and each sample was collected three times, with each collection lasting 60 seconds. The average particle size, peak particle size, and particle concentration were recorded. The results showed that the average particle size of iMSC-EVs was 82.1 ± 15.3 nm, and the particle concentration was 1.62 × 10⁻⁶. 10 particles / mL ( Figure 10 ).

[0045] (2) TEM morphological observation method Take 10 μL of the purified exosome suspension and add it to a copper mesh support. Let it stand at room temperature for 2 minutes. Blot away excess liquid with filter paper. Add 10 μL of 2% phosphotungstic acid negative staining solution and stain at room temperature for 2 minutes. Blot away excess staining solution with filter paper and allow to air dry. Observe under a transmission electron microscope with an accelerating voltage of 80 to 100 kV and a magnification of 50,000 to 100,000 times, and take typical field-of-view images. The results show typical saucer-like or spherical vesicle structures with a diameter of about 100 nm. Figure 11 ).

[0046] (3) Detection of exosome markers by nanoflow cytometry and Western blot The purified exosomes were filtered through a 0.22 μm filter membrane to remove aggregated particles. The exosome concentration was pre-determined using NTA and adjusted to 1×10⁻⁶ with PBS. 9 Up to 1×10 10 Particles per milliliter.

[0047] Take 100 μL of exosome suspension and add the following fluorescently labeled antibodies: CD9-FITC (5 μL), CD63-PE (5 μL), and CD81-APC (5 μL). Also prepare isotype control antibodies (IgG-FITC, IgG-PE, IgG-APC, etc.). Incubate at 4°C in the dark for 30 minutes.

[0048] After incubation, add 1 mL of PBS and use ultracentrifugation (300,000×g for 30 minutes) to remove unbound antibodies. Discard the supernatant and resuspend the precipitate in 100 μL of PBS.

[0049] Detection was performed using a nanoflow cytometer. A side-scattering (SSC) threshold was set to exclude noise signals, and at least 10,000 exosome particle events were acquired. The positivity rate and mean fluorescence intensity (MFI) of each fluorescence channel were recorded.

[0050] Using isotype controls as a gating mechanism, the positive criterion was a fluorescence intensity exceeding the 99% confidence interval of the isotype control. Results showed that the CD9 positivity rate of iMSC-Exos was 22.9%, the CD63 positivity rate was 21.8%, and the CD81 positivity rate was 55.2%. Figure 12 In addition, Western blot was used to detect the expression of HFL1 and iMSCs and their exosome markers in control cells. Figure 13 ).

[0051] (4) The particle protein ratio was calculated using the BCA method combined with NTA. Figure 14 Significance criteria: represent P <0.01, the difference is significant.

[0052] Example 10: Validation of the anti-inflammatory function of iMSC-Exos An inflammation model of human peripheral blood mononuclear cells (PBMCs) stimulated by LPS was established. Peripheral blood was collected from healthy donors, and PBMCs were separated using Ficoll density gradient centrifugation. Blood was diluted 1:1 with PBS and slowly added to the supernatant of the Ficoll separation medium. The cells were centrifuged at 400×g for 30 minutes, and the white membrane layer was collected. The cells were washed twice with PBS, resuspended in complete culture medium, and the cell density was adjusted to 1×10⁶ cells / mL. 8The experiment was divided into three groups. Control group: only LPS (1 μg / mL) was added for stimulation; iMSC-Exos group: LPS (1 μg / mL) and iMSC-Exos (1×10⁻⁶ cells / mL) were added. 9 Particles / mL); Control exosome group: LPS (1 μg / mL) and control exosomes (1×10⁻⁶) were added. 9 Particles / mL (control exosomes were HFL1 cell exosomes). Cells from each group were seeded into 24-well plates, 1 mL per well, and incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0053] Total RNA was extracted from each group of PBMCs using commercially available Trizol reagent according to the manufacturer's instructions. Trizol was added to the cell pellet for complete lysis, followed by chloroform separation and aspiration of the upper aqueous phase. Total RNA was precipitated with isopropanol, washed with 75% enzyme-free ethanol, dried at room temperature, and reconstituted with RNase-free water. RNA purity and concentration were determined using a Nanodrop 2000 nucleic acid analyzer, maintaining an OD260 / OD280 ratio between 1.8 and 2.1. RNA integrity was verified by agarose gel electrophoresis, and qualified samples were stored at -80°C for later use. An equal volume (1 μg) of qualified total RNA was used to synthesize cDNA using a reverse transcription kit according to the manufacturer's instructions. Real-time quantitative PCR amplification was performed using the SYBR Green quantitative PCR kit, with each group's cDNA serving as a template to amplify pro-inflammatory factors. IL-6 , TNF-α , IL-1β With anti-inflammatory factors IL-10 The internal reference gene is a housekeeping gene that is stably expressed. GAPDH The relative mRNA expression levels of each target gene were calculated using the 2-ΔΔCt method, with the mean gene expression level of the PBS+PBMC control group as the baseline, and the relative mRNA expression levels of each factor were standardized. Significance criteria: ns represents no statistically significant difference; represent P <0.0001, the difference is statistically significant ( Figure 15 ).

[0054] Example 11: Verification of neural repair function of iMSC-Exos A neuronal oxidative stress injury model was established by treating mouse hippocampal neurons (HT-22) with 200 μM H2O2 for 24 hours. HFL1-Exos (1×10⁻⁶) were then used to treat the neurons. 9 Particles / mL) and iMSC-Exos (1×10⁻⁶) 9Cells were treated with EdU (particles / mL) for 48 hours, with a control group cultured in normal complete culture medium (without oxidative damage stimulation). After treatment, cells in each group were incubated with EdU working solution to label proliferating cells; fixed with paraformaldehyde, permeabilized, and then incubated with a fluorescent substrate for color development (EdU-positive proliferating cells showed orange-red fluorescence); all cell nuclei were counterstained with Hoechst 33342 nuclear dye (blue fluorescence); fluorescence images of each group were acquired using a fluorescence microscope, and merged into a single image. Multiple fields of view were randomly selected for photography, and the proportion of EdU-positive cells to total cell nuclei was statistically analyzed. ANOVA was used to analyze differences between groups: ns represents no statistical difference. represent P <0.001, represent P <0.0001.

[0055] EdU results showed that a large number of orange-red EdU-positive proliferating cells were observed in the control group; the number of EdU-positive cells decreased significantly after H2O2 oxidative damage, and cell proliferation was severely inhibited; after HFL1-Exos intervention, cell proliferation did not recover significantly; while the iMSC-Exos treatment group significantly increased the number of EdU-positive cells, effectively reversing the proliferative damage caused by oxidative stress. Figure 16 ).

[0056] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for the directed differentiation of induced pluripotent stem cells into mesenchymal stromal cells, characterized in that, This includes the following stages performed sequentially: Phase 1: Under hypoxic culture conditions, induced pluripotent stem cells were induced to differentiate into neural crest cells using a first culture medium containing Wnt signaling pathway agonists and TGF-β signaling pathway inhibitors. Second stage: Under intermittent physical relaxation stimulation, the neural crest cells were induced to mature into mesenchymal matrix cells using a second culture medium; In this culture medium, compared to the first culture medium, the Wnt signaling pathway agonist was removed, the concentration of the TGF-β signaling pathway inhibitor was reduced, and TGF-β1 was added.

2. The method for directed differentiation of induced pluripotent stem cells into mesenchymal stromal cells according to claim 1, characterized in that, The hypoxic culture conditions were defined as an oxygen volume fraction of 5%.

3. The method for directed differentiation of induced pluripotent stem cells into mesenchymal stromal cells according to claim 1, characterized in that, The Wnt signaling pathway agonist was used at a concentration of 1-10 μM in the first culture medium; the TGF-β signaling pathway inhibitor was used at a concentration of 5-20 μM in the first culture medium, and the concentration was reduced to 1-10 μM in the second culture medium; the TGF-β1 was used at a concentration of 0.5-5 ng / mL in the second culture medium.

4. The method for directed differentiation of induced pluripotent stem cells into mesenchymal stromal cells according to claim 3, characterized in that, The Wnt signaling pathway agonist is CHIR99021; the TGF-β signaling pathway inhibitor is SB431542.

5. The method for directed differentiation of induced pluripotent stem cells into mesenchymal stromal cells according to claim 1, characterized in that, The cell culture containers in the first stage were coated with fibronectin at a concentration of 10-50 μg / mL; In the second stage, the cell culture vessel was coated with a double layer of collagen I and fibronectin in a 1:1 ratio, with the coating concentration of collagen I being 5-20 μg / mL and the coating concentration of fibronectin being 5-20 μg / mL.

6. The method for directed differentiation of induced pluripotent stem cells into mesenchymal stromal cells according to claim 1, characterized in that, The first culture medium is CDM medium, with the basic components being IMDM / F12, and it is supplemented with ITS, BSA and lipid concentrate; The second culture medium was α-MEM, supplemented with 5-15% serum-free substitute KOSR and 1-10 ng / mL bFGF.

7. The method for directed differentiation of induced pluripotent stem cells into mesenchymal stromal cells according to claim 1, characterized in that, The intermittent physical relaxation stimulation includes: intermittent gas exchange and mechanical stress release of the culture vessel every 24 hours, with each session lasting 5-15 minutes.

8. An induced mesenchymal stromal cell, characterized in that, Prepared by the method according to any one of claims 1-7.

9. The use of the induced mesenchymal stromal cells according to claim 8 in the preparation of drugs for treating immune dysfunction and neuronal cell damage.

10. An exosome, characterized in that, The exosomes are obtained by isolation and purification of the induced mesenchymal matrix cells as described in claim 8.

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