Mesenchymal stromal cell-based drug and use thereof in treatment of acute cerebral infarction

Abstract

The present invention relates to the technical field of cell therapy. Specifically disclosed are a mesenchymal stromal cell-based drug and a use thereof in the treatment of acute cerebral infarction. The present invention provides a cell-based drug, comprising mesenchymal stromal cells and a pharmaceutically acceptable excipient. Compared with stem cells, the mesenchymal stromal cells have a negative expression level of mRNA encoding self-renewal and differentiation in the mesenchymal stromal cells. The mesenchymal stromal cells account for 90% or more of the cell-based drug. The cell-based drug exhibits the characteristics and mechanism of action of stromal cells, homes to the neovasculature formed by endothelial cells at the injury site within an effective time window, plays a role in promoting / maintaining the neovasculature and remodeling the microenvironment of the injury site, fully ensures the safety and effectiveness of disease treatment, and provides a novel method for the diagnosis and treatment of related diseases.

Description

Mesenchymal stromal cell drugs and their application in the treatment of acute stroke

[0001] Cross-referencing

[0002] This application claims priority to Chinese Patent Application No. 202411513300.X, filed on October 28, 2024, entitled “Mesenchymal stromal cell drugs and their application in the treatment of acute stroke”, the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] This invention relates to the field of cell therapy technology, and more specifically, to mesenchymal stromal cell drugs and their application in the treatment of acute stroke. Background Technology

[0004] In recent years, cell therapy, represented by mesenchymal stromal cells (MSCs), has shown great potential in promoting the regeneration and repair of damaged tissues. MSCs have been extensively studied in clinical trials. Published clinical research results indicate that MSCs have a good safety profile. However, several clinical studies to date have revealed a significant problem: the unstable efficacy of MSCs in treating stroke, which severely limits their widespread clinical application.

[0005] Current methods for identifying the identity and purity of mesenchymal stromal cells (MSCs) primarily rely on the minimum identification criteria for MSCs proposed by the International Society for Cell Therapy (ISCT) in 2006. These criteria utilize flow cytometry to detect the expression of key proteins on the MSC membrane surface at the molecular level. These include the routinely expressed CD73, CD90, and CD105, which are defined by ISCT as the minimum standard markers for MSCs; the absence of expression of CD45, CD34, CD14, and CD19, antigens that distinguish them from hematopoietic stem cells; and the absence of expression of CD11b, CD79a, and HLA-DR23. However, these criteria are not unique to MSCs. This creates a significant risk that other cell types (especially stem cells) may be contaminated in in vitro expanded and cultured MSCs, making them undetectable. This severely impacts the understanding of the therapeutic mechanism of MSCs, the control of safety, and the assurance of efficacy. Summary of the Invention

[0006] One of the objectives of this invention is to provide a cell drug with significantly reduced heterogeneous cell components, more safe and effective treatment, and its application.

[0007] The present invention provides a cell-based drug comprising mesenchymal stromal cells and pharmaceutically acceptable excipients, wherein the expression levels of mRNA encoding self-renewal and differentiation in the mesenchymal stromal cells are negative compared to those in stem cells;

[0008] The mesenchymal matrix cells account for more than 90% of the content of the cell drug.

[0009] This invention provides a cell drug with a high proportion of mesenchymal stromal cells that are negative for self-renewal genes and / or differentiation genes, which significantly reduces the proportion of heterogeneous cells and fully ensures the safety and effectiveness of disease treatment.

[0010] Mesenchymal stromal cells can be induced to migrate to the injury site, where they recruit monocytes and progenitor cells, inhibit the proliferation of peripheral blood lymphocytes, and, induced by signals from the injury site, migrate to the injury site via the newly formed vascular network. There, they parasecrete growth factors and, together with pericytes, promote the functional maturation of immature blood vessels.

[0011] This invention establishes standards based on the identity of stromal cells and the expected therapeutic efficacy indicators, reflecting the scientific nature of quality standards and representing a more scientific and precise advancement in MSC treatment standards. This represents a revolutionary upgrade in the quality system. Furthermore, by utilizing large datasets from single-cell whole transcriptomics, this invention uncovers more characteristics of product safety, efficacy, and quality control that are undetectable by conventional testing, effectively distinguishing bone marrow mesenchymal stem cells from other stem cells, thus demonstrating the precision of quality standards.

[0012] In the cell-based drug of the present invention, the expression levels of mRNAs encoding SOX2, DPPA4, and MYCN in the mesenchymal stromal cells are negative compared to those in stem cells.

[0013] In the cell-based drug of the present invention, the mesenchymal matrix cells home to the neovascularization formed by endothelial cells, promoting the following events:

[0014] a. Promotes the formation of new blood vessels, and / or

[0015] b. Maintain the structure of the newly formed vascular network.

[0016] In the cell-based drug of the present invention, the mesenchymal matrix cells promote an increase in the number of branching points, the number of branch trunks, the total length of branch trunks, and the number of vascular rings in new blood vessels.

[0017] In the cell-based drug of the present invention, the mesenchymal matrix cells maintain the stability of the number of branch points, the number of branch trunks, the total length of branch trunks, and the number of vascular rings in the neovascular network.

[0018] In the cell-based drug of the present invention, the mesenchymal stromal cells home to the site of injury in an effective number, promoting at least one of the following events:

[0019] a. Reduce serum inflammatory factor levels;

[0020] b. Increase the level of nerve growth factor;

[0021] c. Reduces apoptosis of nerve cells surrounding the infarct;

[0022] d. Increase the number of CD31-positive microvessels in the peri-infarct area.

[0023] In the cell-based drug of the present invention, the mesenchymal stromal cells are isolated from one or more of adipose tissue, umbilical cord, placenta, or bone marrow, and undergo at least two passages in the cell culture.

[0024] The present invention also provides the use of mesenchymal stromal cells in the preparation of drugs for the treatment of acute stroke, wherein the expression level of mRNA encoding self-renewal and differentiation in the mesenchymal stromal cells is negative compared with that of stem cells.

[0025] In the application of the present invention, the expression levels of mRNAs encoding SOX2, DPPA4, and MYCN in the mesenchymal stromal cells are negative compared to stem cells.

[0026] In the application of this invention, the mesenchymal matrix cells home to the neovascularization formed by endothelial cells at the site of acute stroke injury, and the homing time window coincides with the time window of angiogenesis and / or injury signals.

[0027] In the application of this invention, the homing time window is 6 to 72 hours after the damage occurs.

[0028] In the application of this invention, the mesenchymal matrix cells home to the neovascularization formed by endothelial cells at the site of acute stroke injury, and the time window for promoting the formation of neovascular network and / or maintaining the structure of neovascular network is 24 to 36 hours after the injury.

[0029] In the application of this invention, the mesenchymal stromal cells home to the site of acute stroke injury in an effective number, promoting at least one of the following events:

[0030] a. Reduce serum inflammatory factor levels;

[0031] b. Increase the level of nerve growth factor;

[0032] c. Reduces apoptosis of nerve cells surrounding the infarct;

[0033] d. Increase the number of CD31-positive microvessels in the peri-infarct area.

[0034] In the application of this invention, the effective number of mesenchymal stromal cells is 1×10⁻⁶. 5 / kg~5×10 7 / kg.

[0035] In the application of this invention, the mesenchymal stromal cells are isolated from one or more of adipose tissue, umbilical cord, placenta, or bone marrow, and undergo at least two passages in cell culture.

[0036] The present invention also provides a drug for treating acute stroke, comprising the above-mentioned cell drug;

[0037] The therapeutic drug contains an effective therapeutic number of mesenchymal stromal cells;

[0038] The effective treatment quantity of the mesenchymal stromal cells is 1×10⁻⁶. 5 / kg~5×10 7 / kg.

[0039] The present invention also provides the application of the above-mentioned cell drugs in promoting the functional maturation of immature blood vessels, wherein the application is for non-disease diagnosis or treatment purposes.

[0040] In the application of this invention, the cellular drug is administered during the angiogenesis window period when promoting the functional maturation of immature blood vessels.

[0041] The cell-based drugs of this invention can be used to treat vascular injury, and can also be used for non-disease diagnosis or treatment purposes, such as research on the mechanism of action of mesenchymal stromal cells, development of related drugs, and efficacy evaluation.

[0042] The present invention has found that applying the cell-based drug of the present invention during the angiogenesis phase following tissue injury or ischemic stroke can achieve effective therapeutic results.

[0043] The cell-based drugs of the present invention also include pharmaceutically acceptable excipients.

[0044] Those skilled in the art can select excipients based on common technical knowledge to assist in the delivery of active ingredients and the exertion of their efficacy.

[0045] In the cell drug of the present invention, the excipient is one or more of a carrier, excipient or diluent.

[0046] The cell drug of the present invention can be used alone or in combination with other functional components to promote the functional maturation of immature blood vessels.

[0047] The microenvironment influences cell phenotype and cell function. Any difference in conditions during the preparation process can lead to differences in the cultured cells. Differences in all aspects of the preparation process, such as different donors, different parts of the cell source, different original sources, different culture media, and different culture conditions, will result in different gene expression, cell phenotype, and cell function in the obtained cells. Therefore, the discovery of effective cell drugs is not always obvious.

[0048] The beneficial effects of this invention are at least as follows:

[0049] The cell drug of the present invention has significantly reduced impurity cell components, which fully ensures the safety and effectiveness of disease treatment. It can promote the functional maturation of immature blood vessels and provides a new method for the diagnosis and treatment of related diseases. Attached Figure Description

[0050] Figure 1 shows the results of single-cell transcriptome analysis of mesenchymal stromal cells and stem cells. In Figure 1, A represents the differences in single-cell transcriptome profiles among mesenchymal stromal cells, adult stem cells, and pluripotent stem cells. In Figure 2, B represents the differential signaling pathways based on the enrichment of highly expressed genes in mesenchymal stromal cells, adult stem cells, and pluripotent stem cells. The length of the horizontal bar represents the number of highly expressed genes in the signaling pathway, and the color of the horizontal bar represents the significance of the enrichment. The color gradient from blue to red indicates increased significance. The FDR method was used to adjust the p-value. In Figure 3, C represents the pseudo-temporal developmental trajectory of mesenchymal stromal cells, adult stem cells, and pluripotent stem cells based on the expression profiles of genes related to self-renewal and differentiation. The dashed arrows indicate the developmental trajectory from pluripotent stem cells to mesenchymal stromal cells. In Figure 4, D represents the time series of pluripotent stem cells, adult stem cells, and mesenchymal stromal cells along the developmental trajectory.

[0051] Figure 2 shows the expression of three key genes, SOX2, DPPA4, and MYCN, which are crucial for maintaining cell self-renewal and differentiation functions, in mesenchymal stromal cells and stem cells. In Figure 2, A represents the expression levels (red intensity) and positive cell ratio (circle size) of SOX2, DPPA4, and MYCN in different mesenchymal stromal cells and stem cells, calculated based on single-cell gene expression profiles. In Figure 2, B shows the trajectory analysis results of the expression intensity of SOX2, DPPA4, and MYCN genes in the development of pluripotent stem cells and adult stem cells into mesenchymal stromal cell lineages. Red represents the intensity of gene expression in cells, with deeper red indicating higher intensity.

[0052] Figure 3 shows the expression of SOX2, DAPP4 and MYCN in 42 AD-MSCs samples. In the figure, A is the proportion of positive cells in the AD-MSCs samples that simultaneously express the above three genes; B is the distribution of the percentage of positive cells; C is the proportion of negative cells in the AD-MSCs samples that negatively express all three genes; and D is the distribution of the percentage of negative cells.

[0053] Figure 4 shows the effect of intravenous infusion of AD-MSCs into a rat model at different time points after MCAO surgery on the area of ​​cerebral infarction.

[0054] Figure 5 shows the homing and co-localization immunofluorescence results of AD-MSCs administered intravenously at different time points. In Figure 5, A shows the homing of AD-MSCs administered intravenously at different time points after stroke at the site of brain injury, as observed by immunofluorescence. The arrows in the figure indicate AD-MSCs carrying red fluorescence. B shows the co-localization results of AD-MSCs (red) and endothelial cells (green). The lower figure is an enlarged view of the dotted line area in the upper figure.

[0055] Figure 6 shows an image of blood vessel formation in Example 5.

[0056] Figure 7 shows the statistical results of key angiogenesis indicators in Example 5.

[0057] Figure 8 shows a comparison of vascular structure imaging at 4h and 24h time points in Example 5.

[0058] Figure 9 shows the statistical analysis of the ratios of key vascular indicators in Example 5.

[0059] In each figure (if any), * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and NS represents no significant difference. Detailed Implementation

[0060] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0062] Example 1

[0063] (1) Obtaining adipose-derived mesenchymal stromal cells

[0064] Under sterile conditions, adipose tissue was collected from the donor and washed twice with DPBS to remove blood cells. 100 mL of adipose tissue was transferred to 10 50 mL centrifuge tubes, and 30 mL of DPBS was added to each centrifuge tube. The tubes were shaken thoroughly to wash the fat. The tubes were centrifuged at 400 g for 5 min, and the upper layer of adipose tissue was transferred to a new centrifuge tube. The washing steps were repeated until the adipose tissue was completely cleaned.

[0065] Approximately 30 mL of adipose tissue was obtained and divided into 5 portions of 6 mL each. Twice the volume of 1 mg / mL type I collagenase was added to each portion of adipose tissue, and the mixture was transferred to a preheated constant temperature air shaker at 37°C and digested at 120 rpm for 1 hour to obtain a suspension of adipose tissue and adipose mesenchymal matrix cells.

[0066] The digested tissue was centrifuged at 500g for 8 minutes at room temperature. After centrifugation, it separated into an upper lipid layer, a middle adipose tissue layer, a lower digestive fluid layer, and a bottom cell pellet. The upper lipid layer, middle adipose tissue layer, and lower digestive fluid layer were discarded. The bottom cell pellet was resuspended in DPBS and filtered through a 100μm sieve into a 50mL centrifuge tube. The tube was centrifuged at 500g for 5 minutes, and the supernatant was removed to obtain a cell pellet containing P0 generation adipose-derived mesenchymal matrix cells. An equal volume of complete culture medium was added to the centrifuge tube, and the mixture was stirred to allow the digested cells to be fully freed in the complete culture medium. The cell suspension was collected after filtering through a 40μm cell sieve. The centrifugation and washing steps were repeated twice. The harvested cells were the P0 generation adipose-derived mesenchymal matrix cells.

[0067] (2) Culture of adipose-derived mesenchymal stromal cells

[0068] Adipose-derived mesenchymal stromal cells were cultured and proliferated using a medium (DMEM / F-12 + 5% Helios UltraGRO-Advanced, DMEM / F-12 purchased from Thermo Fisher Scientific, and Helios UltraGRO-Advanced purchased from Helios BioScience). The specific steps are as follows:

[0069] The cell pellet was resuspended in a culture medium of equal volume to that of the adipose tissue. 1.5 mL of the cell suspension was seeded into a T75 cell culture flask pre-filled with 8.5 mL of culture medium. The T75 cell culture flask was labeled and transferred to a cell culture incubator and cultured at 37°C and 5% CO2. After 24 hours, the adipose-derived mesenchymal matrix cells had basically adhered to the culture vessel. The supernatant was removed and 10 mL of culture medium was added. The medium was changed every three days thereafter. When the adipose-derived mesenchymal matrix cells reached 70% confluence and exhibited short, spindle-shaped, fibrous structures, they were passaged.

[0070] The passage procedure is as follows: Discard the supernatant from the T75 cell culture flask, wash once with 10 mL of DPBS and remove the washing solution, then add 1.5 mL of digestion solution Tryple. TM - Express (1×) digest for 1-2 min until most cells become rounded and detach. Gently tap the culture flask and add 4.5 mL of DPBS to stop digestion. Collect the digested liquid in a 50 mL centrifuge tube, wash once with 10 mL of DPBS, centrifuge at 400 g for 5 min, remove the supernatant, collect the white cell pellet from multiple centrifuge tubes into one centrifuge tube, resuspend the cells in culture medium, and adjust the volume to 30 mL to obtain expanded P0 generation mesenchymal matrix cells. Take samples for cell counting, centrifuge at 400 g for 5 min, remove the supernatant, resuspend the cells in culture medium, mix well by pipetting, adjust the volume, and seed in cell culture flasks to achieve a cell density of 5000-6000 cells / cm³. 2 Label the cell batch, passage number, and culture time on the cell culture flasks, place them in a cell culture incubator, and when the cell confluence reaches about 90%, harvest the P1 generation mesenchymal matrix cells and passage them again in proportion; harvest the P2 generation mesenchymal matrix cells and freeze them in liquid nitrogen as seed cells.

[0071] When using, rapidly thaw frozen P2 generation mesenchymal stromal cells in a 37°C water bath, and then administer at a dose of 9 × 10⁻⁶ cells / day. 3 / cm 2 The cells were seeded into new T75 cell culture flasks, 15 mL of culture medium was added, and the cells were cultured and passaged at 37°C in a 5% CO2 incubator. P5 generation mesenchymal stromal cells were harvested, resuspended in physiological saline, and washed three times by centrifugation. After resuspending again, the cells were filtered through a 40 μm cell sieve to form a single-cell suspension, and the cell density was adjusted to 2 × 10⁶ cells / mL. 6 / mL to form a mesenchymal matrix cell preparation sample for subsequent operations.

[0072] Example 2

[0073] Stem cells possess self-renewal and differentiation capabilities, but cultured stromal cells exhibit almost no self-renewal or differentiation in vivo, although they can be induced to differentiate in vitro. To elucidate the properties of the cellular drug of the present invention, this embodiment analyzes and compares the single-cell transcriptome characteristics of mesenchymal stromal cells and stem cells.

[0074] (1) Sources of single-cell RNA sequencing data

[0075] Single-cell RNA sequencing data were obtained for mesenchymal stromal cells (MSCs), adult stem cells (ASCs), and pluripotent stem cells (PSCs). MSCs included adipose-derived MSCs (AD-MSCs), umbilical cord-derived MSCs (UC-MSCs), placental-derived MSCs (PM-MSCs), and bone marrow-derived MSCs (BM-MSCs); adult stem cells included hematopoietic stem cells (HSCs) and neural stem cells (NSCs); and pluripotent stem cells included embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) (Table 1).

[0076] in, a The method for obtaining single-cell RNA sequencing data of AD-MSCs is as follows:

[0077] The AD-MSCs samples prepared in Example 1 were used to prepare single-cell libraries (each sample came from a different donor, and each sample was prepared as a single-cell library), and single-cell RNA sequencing (scRNA-seq) was performed using the BD Rhapsody platform.

[0078] Cells were randomly dispersed into single-cell sorting plates, and capture beads modified with oligonucleotide barcodes were added, pairing one bead with one cell. Cell lysis buffer was added to ligate mRNA to probes on the surface of the capture beads. The capture beads were then recovered into centrifuge tubes, and complementary DNA was synthesized by reverse transcription. Each complementary DNA molecule was labeled with a unique molecular identifier (UMI) and a cell barcode at its 5' end. Subsequently, second-strand complementary DNA was generated and ligated using universal amplification adapters. Random PCR was initiated to enrich the 3' ends of transcripts, yielding a single-cell library. The single-cell library was sequenced using the Illumina NextSeq platform, with each 100 bp segment being end-paired.

[0079] scRNA-seq data from the BD platform were analyzed using BD Rhapsody cwlrefrunner (v3.1.2) to construct a gene × cell expression matrix. The gene × cell expression matrix was processed using the Seurat (v4.3.0) package in R, including quality control, standardization, data normalization, sample integration, dimensionality reduction, and clustering: genes expressed in more than three cells were retained; cells with fewer than 200 genes or mitochondrial content exceeding 25% were defined as low-quality data and excluded. For the remaining cells, the gene expression count data for each cell was standardized using the NormalizedData function, normalized using a scaling factor of 10000, and the top 2000 hypervariable genes were extracted using the FindVariableFeatures function. All cell sample data were merged to eliminate batch effects, and the data was scaled using the ScaleData function for UMI regression. Principal component analysis (PCA) and t-SNE were performed using the RunPCA and RunUMAP functions, respectively, for dimensionality reduction, and 30 principal components were selected for further analysis. The clustering results were visualized using UMAP projection. Use the Wilcoxon test in the FindAllMarkers function to identify differentially expressed genes.

[0080] Table 1. Single-cell RNA sequencing data of mesenchymal stromal cells, adult stem cells, and pluripotent stem cells.

[0081] The gene expression differences among mesenchymal stromal cells, adult stem cells, and pluripotent stem cells were analyzed (Figure 1, A). Functional enrichment analysis showed that genes highly expressed in pluripotent stem cells (ESCs and iPSCs) are mainly involved in self-renewal, differentiation, and proliferation; genes highly expressed in adult stem cells (HSCs and NSCs) are mainly involved in differentiation and metabolic processes; and genes highly expressed in MSCs are mainly involved in cell metabolism and interactions (Figure 1, B).

[0082] To determine the developmental states and interrelationships of different cell types, this embodiment performed a pseudo-temporal developmental trajectory analysis based on the expression levels of genes involved in self-renewal and differentiation. The results showed that the expression of genes involved in self-renewal and differentiation occurred in the early, middle, and late stages of the time trajectory, corresponding to pluripotent stem cells, adult stem cells, and mesenchymal stromal cells (C and D in Figure 1), respectively. Therefore, mesenchymal stromal cells represent a cell type in the later stages of differentiation, indicating that self-renewal and differentiation are key characteristics distinguishing all stem cells from mesenchymal stromal cells.

[0083] SOX2, DPPA4, and MYCN are key genes for maintaining stem cell self-renewal and differentiation. SOX2 is a transcription factor that plays an important role in maintaining the self-renewal function of undifferentiated ESCs; DPPA4 mainly helps stem cells maintain their self-renewal capacity and pluripotent state; and MYCN can promote self-renewal in some stem cell types. This study examined the expression of SOX2, DPPA4, and MYCN in mesenchymal stromal cells (MSCs) to determine whether MSCs could be distinguished from stem cells based on their self-renewal and differentiation capabilities. The results showed that SOX2, DPPA4, and MYCN were almost not expressed in MSCs (Figure 2A). The expression of these three genes gradually decreased along the trajectory from pluripotent stem cells to MSCs, and at the end of the trajectory, their expression was absent in MSCs (Figure 2B). Therefore, the expression of SOX2, DPPA4, and MYCN helps distinguish MSCs from stem cells.

[0084] The proportions of mesenchymal stromal cells and stem cells expressing SOX2, DPPA4, and MYCN were further calculated. The results showed that at least one of these three genes was expressed in only 0.156% of cells. a AD-MSCs and BM-MSCs were expressed in 1.201% of cells; conversely, at least one of these three genes was expressed in 99.636% of ESCs, 98.400% of iPSCs, 75.989% of HSCs and 66.947% of NSCs (Table 2).

[0085] Table 2. Proportion of mesenchymal stromal cells and stem cells expressing SOX2, DPPA4, and MYCN.

[0086] This embodiment further analyzes the proportion of cells expressing SOX2, DAPP4, and MYCN in 42 AD-MSCs samples prepared according to the method of Example 1. The proportion of AD-MSCs samples expressing all three genes simultaneously ranged from 0% to 0.341%, with an average of 0.156% (Figure 3A). The proportion of positive cells followed a normal distribution, with a 95% confidence interval (CI) of 0.136% to 0.177% (Figure 3B). The proportion of AD-MSCs samples expressing all three genes negatively ranged from 99.669% to 100%, with an average of 99.844% (Figure 3C). The proportion of negative cells also followed a normal distribution, with a 95% confidence interval of 99.834% to 99.865% (Figure 3D).

[0087] The above analysis indicates that mesenchymal stromal cells do not possess the self-renewal and differentiation characteristics of stem cells.

[0088] Example 3

[0089] Since mesenchymal stromal cells (MSCs) are stromal cells, not stem cells, their clinical application must follow the mechanisms of action of stromal cells. This example uses AD-MSCs to explore the in vivo mechanisms of action of mesenchymal stromal cells.

[0090] Following tissue damage, cells undergo apoptosis, resulting in a significant reduction in the number of mesenchymal stromal cells (MSCs) in the damaged tissue, necessitating exogenous replenishment. Homing is crucial for the functional mechanism of MSCs, and this study investigated the optimal time window for sufficient homing of exogenously replenished MSCs.

[0091] A rat model of ischemic stroke due to middle cerebral artery occlusion (MCAO) was established using SD rats. At 6 h, 24 h, 36 h, 48 h, and 72 h post-MCAO surgery, rats were treated with AD-MSCs preparations prepared in Example 1 via intravenous infusion. Each time window included a sham-operated group, a model control group, and a cell therapy group, with 10 rats in each group. The sham-operated group and the model control group were administered solvent + cyclosporine A (10 mg / kg) at the corresponding time points, while the cell therapy group was administered AD-MSCs (1 × 10⁻⁶ mg / kg) at the corresponding time points. 7 / kg)+cyclosporine A, cyclosporine A was injected intraperitoneally every day before and after modeling; the endpoint of the experiment was 28 days after the drug was administered.

[0092] TCC staining was performed uniformly 28 days after MCAO surgery to measure the infarct area after intravenous infusion of AD-MSCs at different time points post-stroke. The formula for calculating the infarct area (%) is as follows:

[0093] Infarct area (%) = (area of ​​healthy hemisphere - area of ​​normal brain on the infarcted side) / (area of ​​healthy hemisphere × 2) × 100%.

[0094] The results (Table 3) showed that, compared with the corresponding sham-operated group, the rats in the model control group had significant cerebral infarction (P < 0.001). Compared with the model control group at the corresponding time points, the cell therapy groups at 6h, 24h, 36h, 48h, and 72h after modeling reduced the infarct area to varying degrees, with improvement rates of 1.1% (P > 0.05), 20.7% (P < 0.01), 15.3% (P < 0.01), 5.8% (P > 0.05), and 4.5% (P > 0.05), respectively.

[0095] Table 3 Effects of AD-MSCs on cerebral infarction area (%) in rats with permanent cerebral ischemia (n=10, mean±SD)

[0096] Note: Compared with the sham surgery group. ### P<0.001; compared with the model group, **P < 0.01. Improvement rate % = (Model group - Drug-treated group) / Model group × 100%.

[0097] Compared with the model control group, the improvement rate of cerebral infarction area at 6 hours after drug administration was not significant, with no statistically significant difference. The improvement rates of cerebral infarction area at other time points (24h, 36h, 48h and 72h) were significant and statistically different (P<0.05), with the improvement rates of cerebral infarction area at 24h and 36h after drug administration being significantly higher than those in other time windows (Figure 4).

[0098] Based on the above results, angiogenesis begins approximately 24 hours after stroke, and exogenous mesenchymal stromal cells (MSCs) only function effectively after hometing to immature vessels. The effective time window for administering AD-MSCs to treat acute stroke is 24–72 hours post-stroke. Therefore, to ensure therapeutic efficacy, 24–36 hours post-stroke is selected as the time window for AD-MSCs treatment of acute stroke. This selection reflects the scientific reasoning regarding the mechanism of action of AD-MSCs and is feasible in the clinical setting of acute stroke patients.

[0099] Example 4

[0100] AD-MSCs were labeled with PKH26 (red fluorescence) to detect their homing in injured tissue. When AD-MSCs were infused 6 hours post-stroke, no AD-MSCs were detected at the injury site; however, when infused 24 or 36 hours post-stroke, a large number of AD-MSCs were detected at the injury site (Figure 5A). Importantly, AD-MSCs co-localized with endothelial cells (exhibiting CD31 green fluorescence) (Figure 5B). Mesenchymal stromal cells (MSCs) are primarily located around the capillary walls formed by endothelial cells. Under acute stroke conditions, the microvascular structure in injured tissue collapses, disrupting the original niche of MSCs. Ischemia or hypoxia stimulates the production of VEGF and other factors in injured tissue, promoting angiogenesis, accompanied by endothelial cell budding, proliferation, and the formation of immature luminal structures at the edge of the injury area. These immature vessels composed solely of endothelial cells require the stabilization of MSCs to form functionally mature vessels and restore blood flow. Therefore, immature blood vessels composed of endothelial cells provide a "nest" for MSCs, and the interaction between MSCs and endothelial cells forms the basis for MSC homing.

[0101] Example 5

[0102] Using an in vitro two-dimensional angiogenesis assay, we explored the role of mesenchymal stromal cells in promoting angiogenesis in vitro by inducing human umbilical vein endothelial cells to form a vascular network on the Matrigel surface.

[0103] Three batches of AD-MSCs formulation samples were prepared. After cell culture, the culture supernatant was collected and used to prepare conditioned medium as the experimental group (the three batches of formulation samples were prepared from three different donors using the same method as in Example 1). The complete MSCs culture medium served as the control group. Both groups of samples were mixed with a suspension containing human umbilical vein endothelial cells in equal proportions and cultured in microwells pre-coated with Matrigel gel for 6 hours. The samples were then observed and imaged under a microscope. As shown in Figure 6, microscopic observation revealed that human umbilical vein endothelial cells induced with conditioned medium on the Matrigel surface for 6 hours by three batches (S1-S3, three replicates per batch) could form a network-like vascular structure. Visually, the experimental group showed a greater number and integrity of vascular rings than the control group.

[0104] ImageJ software was used for full-frame image analysis to obtain data on four indicators: the number of branch points (Nb master junction), the number of branch trunks (Nb master segments), the total length of branch trunks (Tot.master segments length), and the number of vascular rings (Nb meshes). The results of three batches of tests were combined to form the experimental group data. The experimental data are expressed as mean ± standard deviation (±s). If the variances of the experimental group and the control group are homogeneous, a one-sample t-test was used; if the variances are unequal, a nonparametric Mann-Whitney test was used to assess whether there are significant differences in the above indicators between the experimental group and the control group.

[0105] As shown in Figure 7, after 6 hours of induction with AD-MSCs conditioned medium on the surface of human umbilical vein endothelial cells (HVVEs) on Matrigel, a clear vascular network structure was observed under a microscope. Key angiogenesis indicators in the experimental group, such as the number of branch points, the number of main branches, the total length of main branches, and the number of vascular rings, were significantly higher than those in the control group (P < 0.05 or P < 0.001). Induction of HVVEs on Matrigel with AD-MSCs conditioned medium for 6 hours promoted the formation of more vascular structures, indicating that mesenchymal stromal cells (MSCs) have an in vitro angiogenesis-promoting effect.

[0106] By adding mesenchymal stromal cells after constructing primary vascular structures in vitro, we explored the role of mesenchymal stromal cells in maintaining primary angiogenesis and reducing the rate of disintegration in vitro.

[0107] Human umbilical vein endothelial cells (HUVECs) were induced for 4 hours using a culture medium containing human serum substitutes, allowing them to form a rich vascular network structure on Matrigel gel. After removing the induction medium, the supernatant from three batches of AD-MSCs preparation samples was used to prepare conditioned medium as the experimental group (the three batches of preparation samples were prepared from three different donors using the same method as in Example 1), and the complete MSCs culture medium served as the control group. Both were mixed in equal proportions with basal culture medium containing HUVECs and added to the gel wells of the induced vascular network HUVECs. All were incubated at 37°C and 5% CO2 for 20 hours. The changes in vascular structure were observed and imaged under a microscope at 4 hours (replaced with zero-value conditioned medium), 6 hours, 9 hours, 12 hours, and 24 hours. Microscopic observation at the 24-hour time point (Figure 8) showed that the vascular network integrity in the experimental group was superior to that in the control group.

[0108] ImageJ software was used to quantitatively analyze full-frame images of the incubation start (4h) and end (24h) of conditioned medium. Four indicators were obtained: number of branch points (Nb masterjunction), number of branch trunks (Nb master segments), total length of branch trunks (Tot. master segments length), and number of vascular rings (Nb meshes). To eliminate the baseline differences between wells at the starting point, the ratio (%) of each indicator at the end of 24h to each indicator at the starting point of 4h was used as the evaluation index for vascular structure maintenance. The results of three batches of tests were combined to form the experimental group data. The experimental data are expressed as mean ± standard deviation (±s). The variances between the experimental group and the control group were homogeneous, and the variances were not homogeneous, so the nonparametric Mann-Whitney test was used to analyze the differences between the experimental group and the control group in each indicator.

[0109] As shown in Figure 9, after the experimental group was replaced with AD-MSCs conditioned medium for 20 hours, the number of branch points, the number of branch trunks, the total length of branch trunks, and the ratio of the number of vascular rings were significantly higher than those in the control group (P<0.05 or P<0.01), and the vascular structure disintegration was slowed down.

[0110] Example 6

[0111] A rat model of ischemic stroke due to middle cerebral artery occlusion (MCAO) was established using SD rats. Treatment was administered via intravenous infusion of AD-MSCs preparations (prepared in Example 1). Five groups were established: sham-operated group, model control group, low-dose AD-MSCs treatment group, medium-dose AD-MSCs treatment group, and high-dose AD-MSCs treatment group, with 10 rats in each group. The sham-operated group and model control group received solvent + cyclosporine A 24 hours after model establishment; the AD-MSCs treatment group received the corresponding dose of AD-MSCs (5 × 10⁻⁶) 24 hours after model establishment. 6 cells / kg, 1×10 7 cells / kg, 2×10 7 (cells / kg) + cyclosporine A; the endpoint of the experiment was 28 days after drug administration.

[0112] As shown in Table 4, the serum levels of pro-inflammatory factors IL1β, IL6, and IFNγ in the model control group rats were significantly increased (P<0.05–0.001). Compared with the model control group, the levels of IL1β, IL6, and IFNγ in the model control group rats were significantly increased (P<0.05–0.001). 7 cells / kg, 2×10 7 AD-MSCs at a concentration of cells / kg reduced the levels of pro-inflammatory factors IL1β, IL6, and IFNγ in rat serum to varying degrees 24 hours after administration (P<0.01–0.001).

[0113] Table 4. Effects of AD-MSCs on serum inflammatory factors in rats with permanent cerebral ischemia (n=10, mean±SD)

[0114] Note: Compared with the sham surgery group. # P<0.05, ### P<0.001; compared with the model control group, ** P<0.01, *** P<0.001.

[0115] As shown in Table 5, the levels of SDF1, HGF (hepatocyte growth factor), and NGF (nerve growth factor) in the brain tissue of rats in the model control group were significantly decreased (P<0.001). Compared with the model control group, the dosage was 5×10 6 cells / kg, 1×10 7 cells / kg, 2×10 7 AD-MSCs at a concentration of cells / kg could increase the levels of HGF and NGF in rat brain tissue to varying degrees (P<0.05–0.001).

[0116] Table 5. Effects of AD-MSCs on neurotrophic factors in brain tissue of rats with permanent cerebral ischemia (n=10, mean±SD)

[0117] Note: Compared with the sham surgery group. ### P<0.001; compared with the model control group, * P<0.05, ** P<0.01, *** P<0.001.

[0118] CD31 positivity is observed in vascular endothelial cells and is used to identify microvessels. As shown in Table 6, compared with the sham-operated group, the number of CD31-positive microvessels in the peri-infarct area of ​​rats in the model control group was increased (P<0.001). Compared with the model control group, the dose was 1×10 7 cells / kg, 2×10 7 AD-MSCs at a concentration of cells / kg significantly increased the number of CD31-positive microvessels in the peri-infarct area of ​​rats (P<0.05).

[0119] Table 6. Effects of AD-MSCs on microvascular formation in the peri-infarct area of ​​rats with permanent cerebral ischemia (n=10, mean±SD)

[0120] Note: Compared with the sham surgery group. ### P<0.001; compared with the model control group, * P<0.05.

[0121] As shown in Table 7, compared with the sham-operated group, the number of apoptotic astrocytes and neurons in the peri-infarct area of ​​rats in the model control group was significantly increased (P<0.05–0.001); compared with the model control group, the dose was 1×10 7 cells / kg, 2×10 7 AD-MSCs at a concentration of cells / kg significantly reduced neuronal apoptosis in the peri-infarct area of ​​rats (P<0.05) and showed a trend of reducing astrocyte apoptosis, but there was no statistical difference (P>0.05).

[0122] Table 7. Effects of AD-MSCs on apoptosis of neurons in the peri-infarct area of ​​rats with permanent cerebral ischemia (n=10, mean±SD)

[0123] Note: Compared with the sham surgery group. # P<0.05, ### P<0.001; compared with the model control group, * P<0.05.

[0124] This embodiment established a rat model of permanent cerebral ischemia (MCAO) and administered different doses of AD-MSCs intravenously 24 hours after modeling to study the treatment mechanism. Serum inflammatory factor detection results 24 hours after drug administration showed that a dose of 1×10⁻⁶ was most effective. 7 cells / kg and 2×10 7 AD-MSCs at a concentration of cells / kg significantly reduced the levels of pro-inflammatory factors IL1β, IL6, and IFNγ in rat serum; brain tissue neurotrophic factor assays showed that a dose of 1×10⁻⁶ cells / kg was effective. 7 cells / kg and 2×10 7 AD-MSCs at a dose of 1×10⁶ cells / kg significantly increased the levels of HGF and NGF in rat brain tissue; brain tissue neuronal apoptosis detection results showed that a dose of 1×10⁶ cells / kg significantly increased the levels of HGF and NGF in rat brain tissue. 7 cells / kg and 2×10 7 AD-MSCs at a concentration of 1 × 10⁻⁶ cells / kg significantly reduced neuronal apoptosis in the peri-infarct region of rat cerebral infarction and showed a trend towards reducing astrocyte apoptosis; immunohistochemical results of CD31 in the peri-infarct region of rat cerebral infarction indicated that a dose of 1 × 10⁻⁶ cells / kg was effective. 7 cells / kg and 2×10 7 AD-MSCs at a concentration of cells / kg significantly increased the number of CD31-positive microvessels in the peri-infarct area of ​​rats.

[0125] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. Industrial applicability

[0126] The cellular drug provided by this invention contains over 90% intermediate mesenchymal matrix cells. Following the characteristics and mechanism of action of matrix cells, the cellular drug of this invention homes to the neovascularization formed by endothelial cells at the site of injury within the effective time window, promoting / maintaining angiogenesis and remodeling the microenvironment of the injured site. This fully ensures the safety and effectiveness of disease treatment and provides a new method for the diagnosis and treatment of related diseases.

Claims

1. A cell-based drug, characterized in that, The product includes mesenchymal stromal cells and pharmaceutically acceptable excipients, wherein the expression levels of mRNAs encoding self-renewal and differentiation in the mesenchymal stromal cells are negative compared to those in stem cells; The mesenchymal matrix cells account for more than 90% of the content of the cell drug.

2. The cell-based drug according to claim 1, characterized in that, Compared with stem cells, the expression levels of mRNAs encoding SOX2, DPPA4, and MYCN in the mesenchymal stromal cells were negative.

3. The cell-based drug according to claim 1 or 2, characterized in that, The mesenchymal stromal cells home to the newly formed blood vessels from endothelial cells, promoting the following events: a. Promotes the formation of new blood vessels, and / or b. Maintain the structure of the newly formed vascular network.

4. The cell-based drug according to any one of claims 1-3, characterized in that, The mesenchymal stromal cells promote an increase in the number of branch points, the number of main branches, the total length of the main branches, and the number of vascular rings in new blood vessels.

5. The cell-based drug according to any one of claims 1-4, characterized in that, The mesenchymal stromal cells maintain the stability of the number of branch points, the number of main branches, the total length of the main branches, and the number of vascular rings in the neovascular network.

6. The cell-based drug according to any one of claims 1-5, characterized in that, The mesenchymal stromal cells home to the injury site in an effective number, promoting at least one of the following events: a. Reduce serum inflammatory factor levels; b. Increase the level of nerve growth factor; c. Reduces apoptosis of nerve cells surrounding the infarct; d. Increase the number of CD31-positive microvessels in the peri-infarct area.

7. The cell-based drug according to any one of claims 1-6, characterized in that, The mesenchymal stromal cells are isolated from one or more of adipose tissue, umbilical cord, placenta, or bone marrow, and have undergone at least two passages in cell culture.

8. The application of mesenchymal stromal cells in the preparation of drugs for the treatment of acute stroke, characterized in that, Compared to stem cells, the expression levels of mRNAs encoding self-renewal and differentiation in the mesenchymal stromal cells were negative.

9. The application according to claim 8, characterized in that, Compared with stem cells, the expression levels of mRNAs encoding SOX2, DPPA4, and MYCN in the mesenchymal stromal cells were negative.

10. The application according to claim 8 or 9, characterized in that, The mesenchymal matrix cells home to the neovascularization formed by endothelial cells at the site of acute stroke injury, and the homing time window coincides with the time window of angiogenesis and / or injury signals.

11. The application according to claim 10, characterized in that, The homing time window is 6–72 hours after the injury occurs.

12. The application according to claim 8 or 9, characterized in that, The mesenchymal matrix cells home to the neovascularization formed by endothelial cells at the site of acute stroke injury, and the time window for promoting the formation of neovascular networks and / or maintaining the structure of neovascular networks is 24 to 36 hours after the injury.

13. The application according to any one of claims 8-12, characterized in that, The mesenchymal stromal cells home to the site of acute stroke injury in an effective number, promoting at least one of the following events: a. Reduce serum inflammatory factor levels; b. Increase the level of nerve growth factor; c. Reduces apoptosis of nerve cells surrounding the infarct; d. Increase the number of CD31-positive microvessels in the peri-infarct area.

14. The application according to claim 13, characterized in that, The effective number of mesenchymal stromal cells is 1×10⁻⁶. 5 / kg~5×10 7 / kg.

15. The application according to any one of claims 8-14, characterized in that, The mesenchymal stromal cells are isolated from one or more of adipose tissue, umbilical cord, placenta, or bone marrow, and have undergone at least two passages in cell culture.

16. A drug for treating acute stroke, characterized in that, Includes the cell-based drug according to any one of claims 1-7; The therapeutic drug contains an effective therapeutic number of mesenchymal stromal cells; The effective treatment quantity of the mesenchymal stromal cells is 1×10⁻⁶. 5 / kg~5×10 7 / kg.

17. The use of the cell drug according to any one of claims 1-7 in promoting the functional maturation of immature blood vessels, wherein the use is not for disease diagnosis or treatment purposes.

18. The application according to claim 17, characterized in that, The cellular drug is administered during the angiogenesis window to promote the functional maturation of immature blood vessels.

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