Application of mesenchymal stem cells in medicine for treating acute graft versus host disease, medicine and preparation method

Abstract

The invention relates to the technical field of disease treatment drugs, and discloses application of mesenchymal stem cells in drugs for treating acute graft versus host disease, the drugs and a preparation method of the drugs, and the mesenchymal stem cells are derived from KRT8 + subgroups; the medicine comprises mesenchymal stem cells from a high-expression KRT8 < + > subgroup and pharmaceutically acceptable auxiliary materials, the culture method of the mesenchymal stem cells comprises the following steps: separating KRT8 < + > subgroups from human tissues, placing the KRT8 < + > subgroups in a 4-6% carbon dioxide incubator at 35-39 DEG C, and culturing the KRT8 < + > subgroups in DMEM / F12 added with 8-12% of fetal calf serum; then, in a 4-6% CO2 culture box at the temperature of 35-39 DEG C, culturing in RPMI-1640 added with 8-12% of FBS and 0.03-0.07 mM 2-Mer; and when the cell density reaches 70-90%, dissociating the cells by using trypsin. According to the invention, the anti-leukemia (GVL) activity of the graft can be maintained while aGVHD is prevented.

Description

Technical Field

[0001] The present invention relates to the technical field of disease treatment drugs, and in particular to use of mesenchymal stem cells in drugs for treating acute graft-versus-host disease, drugs and preparation methods. Background Art

[0002] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an important means to cure malignant blood diseases. Acute graft-versus-host disease (aGVHD) is one of the important complications that affect the success of hematopoietic stem cell transplantation and patient survival. Depending on the type of transplantation, the incidence of II-IV aGVHD is 30%-70%. The first-line drug for aGVHD is glucocorticoids, but the incidence of hormone-resistant aGVHD is 40-60%, and the mortality rate can be as high as 70-80%. Therefore, effective prevention and control of aGVHD is a key link in determining the success or failure of transplantation.

[0003] At present, the prevention and treatment of GVHD mainly use hormones and immunosuppressants (ATG, cyclosporine, cyclin D, etc.). Long-term medication may lead to side effects such as severe infection, recurrence of malignant blood diseases and organ dysfunction, which is an important factor in transplant-related death. Therefore, it is of great significance to explore new methods for the prevention and treatment of aGVHD with high efficiency and low toxicity. The basic and clinical research of the patent applicant team has repeatedly confirmed that mesenchymal stem cells (MSCs) combined with hematopoietic stem cell transplantation can help reconstruct hematopoiesis and immune function while reducing the occurrence of aGVHD; for hormone-resistant aGVHD, mesenchymal stem cell infusion can effectively control aGVHD and prolong the patient's overall survival.

[0004] The clinical study reported by the patent applicant in JCO found that sequential infusion of umbilical cord-derived mesenchymal stem cells (HUC-MSCs) after haploid hematopoietic stem cell transplantation can reduce the cumulative incidence of aGVHD without increasing the risk of leukemia relapse and infection; it was first confirmed that HUC-MSCs infusion can play an immunomodulatory role by upregulating the proportion of Th1, Treg and memory B cells in patients and reducing the proportion of Th2 and NK cells. It can be seen that MSCs are undoubtedly a promising cell therapy for aGVHD, which is both effective and has low toxicity and side effects, but the disadvantage is that the clinical effect is not very stable, and different generations, different doses, and different infusion methods have a greater impact on the efficacy, which limits the further promotion and application of MSCs in clinical practice; therefore, the development of a method to prevent aGVHD while maintaining graft-versus-leukemia (GVL) activity is still a long-term goal, and innovative therapeutic drugs are urgently needed to effectively reduce aGVHD while maintaining GVL activity. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide the use of mesenchymal stem cells in the treatment of acute graft-versus-host disease, a drug and a preparation method, which can prevent aGVHD while maintaining graft-versus-leukemia (GVL) activity.

[0006] The present invention solves the above technical problems by the following technical means:

[0007] In the first aspect, the present invention discloses a use of mesenchymal stem cells in a drug for treating acute graft-versus-host disease, wherein the mesenchymal stem cells are from the KRT8 subpopulation. Functional experiments confirmed that HUC-MSCs with enhanced KRT8 expression significantly reduced cell senescence by upregulating EZH2-mediated H3K27me3; its upregulation led to increased secretion of hepatocyte growth factor (HGF); this in turn inhibited the proliferation and activation of T cells and significantly reduced liver organoid damage in vitro. In addition, overexpression of KRT8 significantly improved aGVHD in vivo. Importantly, the enhancement of KRT8 expression did not impair GVL activity against A20 lymphoma cells.

[0008] In combination with the first aspect, the mesenchymal stem cells are one or more combinations of human adipose tissue, amniotic membrane, bone marrow, deciduous teeth, dermis, endometrium, human embryonic stem cell derivatives, oral mucosa or umbilical cord.

[0009] In combination with the first aspect, the mesenchymal stem cells are derived from one or a combination of bone marrow, umbilical cord, dermis or adipose tissue.

[0010] In combination with the first aspect, the mesenchymal stem cells are derived from umbilical cord. Mesenchymal stem cells derived from umbilical cord are HUC-MSCs. Functional validation experiments have shown that HUC-MSCs exhibit stronger immunosuppressive ability compared with MSCs from other sources. Clinical studies have further supported the superior efficacy of HUC-MSCs in treating aGVHD.

[0011] In combination with the first aspect, the mesenchymal stem cells are derived from cells that highly express KRT8 + Functional validation experiments showed that KRT8 low expression (KRT8 － ) subpopulation, HUC-MSCs expressed high levels of KRT8 (KRT8 + ) subset exhibited greater stemness and lower immunogenicity.

[0012] In a second aspect, the present invention further discloses a drug for treating acute graft-versus-host disease, characterized in that the drug comprises the above-mentioned mesenchymal stem cells and pharmaceutically acceptable excipients.

[0013] In combination with the second aspect, the drug is an infusion solution.

[0014] In a third aspect, the present invention further discloses a method for culturing mesenchymal stem cells, comprising the steps of:

[0015] Isolation of KRT8 from human tissues + The subpopulations were cultured in DMEM / F12 supplemented with 8-12% fetal bovine serum in a 4-6% CO2 incubator at 35-39°C; then cultured in RPMI-1640 supplemented with 8-12% FBS and 0.03-0.07mM 2-Mer in a 4-6% CO2 incubator at 35-39°C; when the cell density reached 70-90%, they were dissociated using trypsin.

[0016] Beneficial effects of the present invention:

[0017] 1. The HUC-MSCs with enhanced KRT8 expression in the present invention significantly reduced cell senescence by upregulating EZH2-mediated H3K27me3; upregulation led to increased secretion of hepatocyte growth factor (HGF), which in turn inhibited the proliferation and activation of T cells and significantly reduced in vitro liver organoid damage.

[0018] 2. Compared with MSCs from other sources, HUC-MSCs in the present invention show stronger immunosuppressive ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 KRT8 in Example 1 + and KRT8－ Characteristic diagram of HUC-MSCs subpopulations;

[0020] Figure 2 This is a diagram showing the verification results of Example 2, which shows that overexpression of KRT8 in HUC-MSCs enhances its therapeutic efficacy in treating GVHD while retaining the activity of GVL;

[0021] Figure 3 This is the result of RNA sequencing in Example 3, which found that KRT8 overexpression regulates lipid metabolism and H3K27me3;

[0022] Figure 4 This is a diagram showing the verification results of EZH2-mediated H3K27me3 regulating HUC-MSCs aging in Example 4;

[0023] Figure 5 This is a diagram showing the verification result that the immunomodulatory effect of KRT8 overexpression on HUC-MSCs depends on HGF in Example 5;

[0024] Figure 6 This is a diagram showing the validation results of KRT8 overexpression enhancing HUC-MSCs migration and reducing liver organoid damage in Example 6;

[0025] Figure 7 KRT8 in Example 1 + and KRT8 － Gene ontology enrichment analysis plots of subpopulations;

[0026] Figure 8 KRT8 in Example 1 + and KRT8 － Expression analysis of key genes in subgroups;

[0027] Fig. 9 This is a diagram for verifying the transfection efficiency in Example 2;

[0028] Fig.10 is a volcano plot of differentially expressed genes between HUC-MSC-KRT8 and HUC-MSC-GFP in Example 3;

[0029] Fig.11 This is a verification figure showing that KRT8 overexpression in Example 6 does not affect the migration of HUC-MSCs to the skin, spleen, lung and intestine. DETAILED DESCRIPTION

[0030] The present invention will be described in detail below with reference to specific embodiments and accompanying drawings:

[0031] The mice used in the examples were BALB / c (H-2Kd) and C57BL / 6J (H-2Kb) mice purchased from VITALRIVERexperiment+t Animal Company (Beijing, China). All mice were bred in the pathogen room of Xinqiao Hospital of Army Medical University (Chongqing, China). All mouse experiments were performed in accordance with the protocols approved by the Animal Care and Use Committee of Xinqiao Hospital of Army Medical University.

[0032] Example First, human umbilical cord mesenchymal stem cells (HUC-MSCs) were cultured as follows:

[0033] Human umbilical cord mesenchymal stem cells (HUC-MSCs) were purchased from iCell (Shanghai, China) and cultured in DMEM / F12 (GIBCO, USA) supplemented with 8-12% (10% in this example) fetal bovine serum (FBS; GIBCO, USA) in a 4-6% (5% in this example) CO2 incubator at 35-39°C (37°C in this example). A20-luc cells were purchased from Zhongqiao XinZhou Biotechnology Co., Ltd (Shanghai, China) and cultured in RPMI-1640 (GIBCO, USA) supplemented with 8-12% (10% in this example) FBS and 0.03-0.07 mM 2-Mer (0.05 mM 2-Mer in this example) in a 4-6% (5% in this example) CO2 incubator at 35-39°C (37°C in this example). When the cell density reached 70-90% (80% in this example), they were dissociated using trypsin and seeded into T25 cell culture flasks for other experiments. HUC-MSCs were identified by detecting cell surface phenotype and their multipotent potential to differentiate along the adipogenic, osteogenic, and chondrogenic lineages and maintained as described previously.

[0034] The statistical method of this example is as follows: The data of this example are shown as mean ± SEM. The weight, diarrhea, skin lesion score, GVHD and survival rate of different groups were compared using the rank sum test or log-rank test. The comparison of three means was analyzed using one-way ANOVA multiple comparison, while the comparison of two means was analyzed using an unpaired two-tailed Student's t-test (Prism, Version 6.0; GraphPad software), and P less than 0.05 was considered significant. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0035] Embodiment 1,

[0036] This example shows the cross-tissue MSC landscape at the single-cell level to identify KRT8 with superior therapeutic function +HUC-MSCs subpopulations.

[0037] In the applicant's previous study, the applicant systematically analyzed the heterogeneity of MSCs derived from four different tissues at the single-cell level: bone marrow, umbilical cord, dermis, and adipose tissue. The results highlighted significant tissue-specific characteristics in these MSCs. Functional validation experiments showed that HUC-MSCs exhibited stronger immunosuppressive capacity compared with MSCs from other sources. Clinical studies further support the superior efficacy of HUC-MSCs in the treatment of aGVHD. However, effective methods for identifying and characterizing subpopulations within HUC-MSCs that explain their functional advantages have not yet been developed.

[0038] To address this issue, the applicants performed quality control and data integration to create a comprehensive MSCs atlas, which included 256,709 single-cell transcriptomes from nine different tissue sources, including adipose tissue, amnion, bone marrow, deciduous teeth, dermis, endometrium, human embryonic stem cell-derived lines, oral mucosa, and umbilical cord. This analysis reconfirmed the strong tissue-specific characteristics exhibited by MSCs, with little similarity between cells from different tissues, as demonstrated by unsupervised clustering using the Leiden algorithm in the Scanpy toolkit (v1.10.2) ( Figure 1 AB). Further differential expression analysis identified KRT8 as a specific marker that was mainly present in umbilical cord and amniotic tissue MSCs. Notably, the expression level of KRT8 varied between HUC-MSCs subpopulations ( Figure 1 CD).

[0039] Further unsupervised cluster analysis of HUC-MSCs identified two subpopulations with high KRT8 expression (KRT8 + 1 and KRT8 + 2) and a subpopulation with low KRT8 expression (KRT8 － )( Figure 1 EF). Gene Ontology (GO) analysis highlighted KRT8 + and KRT8 － Significant functional heterogeneity among subgroups ( Figure 7 AC). Notably, the differentially expressed genes between these subgroups showed clear functional enrichment, KRT8 + Genes in the subset were related to cell cycle, cell cycle checkpoint, and hemostasis-related pathways ( Figure 7 AC). In contrast, KRT8 － Subpopulations were enriched in lipid metabolism pathways ( Figure 7 AC).

[0040] Examination of the top 10 differentially expressed genes revealed that KRT8 + The subpopulation showed increased levels of SERPINE1, CAV1, and LGALS1 (galectin-1). + The subgroup showed significantly decreased expression of APOE, MMP2, and complement system genes such as C1S and C1R ( Figure 1 G) Previous studies have shown that SERPINE1 is associated with maintaining cancer cell stemness, while CAV1 and LGALS1 are known to enhance the immunosuppressive function of MSCs. In addition, APOE promotes cell senescence by destabilizing heterochromatin by degrading nuclear lamina and heterochromatin-associated proteins, while MMP2 is associated with the development of age-related diseases and cell senescence. In addition, the analysis showed that KRT8 + The subpopulation showed significantly reduced expression of inflammation-related genes (such as IL1R), low levels of major histocompatibility complex class I (MHC-I) molecules (HLA-A, HLA-B, HLA-C), and little effect on the MHC-I molecule HLA-F and the major histocompatibility complex class II (MHC-II) molecule HLA-DMA ( Figure 1 G and Figure 8 ), indicating that KRT8 + Subpopulations are less immunogenic.

[0041] Next, the applicant used pySCENIC to predict KRT8 + and KRT8 － The results showed that key transcription factors, such as SOX5 and YY1, play important roles in maintaining stemness and are expressed in KRT8 + The subpopulation was significantly activated ( Figure 1 H). In addition, we observed enhanced activity of cell cycle regulators, including E2F1, E2F7, E2F8, and BRCA1 ( Figure 1 H), indicating that KRT8 + Cells have enhanced proliferation potential and the ability to regulate cell cycle progression and stemness. + The subpopulation showed increased activity of transcription factors associated with inflammation and aging, such as HIF1A, HMGA1, STAT5b, STAT6, RFX2, and IRF3 ( Figure 1 H), highlighting the role of KRT8 in potentially regulating stemness and inflammatory pathways. + The stemness potential of HUC MSCs was analyzed by the applicants, and it was confirmed that KRT8 － Compared with cells, this subpopulation showed greater stemness ( Figure 1I). The trajectory analysis performed by the applicant using DDRTree showed that the － Compared with HUC MSCs, KRT8 + Genes associated with MSC self-renewal, such as CAV1 and SERPINE1, were gradually upregulated in HUC MSCs, while genes associated with aging and inflammation, including MMP2, IL1R1, and HLA-C, were downregulated. Figure 1 J). Collectively, these findings suggest that KRT8 + HUC MSCs showed increased stemness with reduced senescence and inflammation. These features may enhance their potential for advanced therapeutic applications in aGVHD.

[0042] from Figure 1 As can be seen in: (A) Uniform manifold approximation and projection (UMAP) plot based on scRNA-seq data of MSCs from multiple tissue sources. Each tissue type is represented by a different color. (B) Unsupervised clustering of MSCs subpopulations based on transcriptional profiles, with each cluster represented by a unique color. (C) UMAP representation of KRT8 expression in all MSCs populations, illustrating relative expression levels. (D) Violin plot showing the expression distribution of KRT8 in MSCs from different tissues. (E) Unsupervised clustering analysis of HUC-MSCs subpopulations, with clusters labeled according to KRT8 expression levels. (F) Violin plot showing the relative expression levels of KRT8 in HUC-MSCs subpopulations. (G) Dot plot showing KRT8 + Expression of the top differentially expressed genes in the subpopulations. The dot size reflects the percentage of cells expressing each gene, and the color scale indicates the relative expression level. (H) Heat map showing KRT8-enriched + and KRT8 － Representative transcription factor activities of HUC-MSCs subsets highlighting transcriptional regulators involved in stemness, cell cycle regulation, senescence, and inflammation. (I) Using monoclonal antibodies against KRT8 + and KRT8 － Pseudo-time trajectory analysis of HUC-MSCs subpopulations. (J) Pseudo-time trajectory (black line) and pseudo-time course (color gradient) are shown. Key markers associated with stem cell self-renewal, senescence, and inflammatory response are visualized along the pseudo-time trajectory, indicating functional changes along the differentiation axis.

[0043] Embodiment 2,

[0044] This example demonstrates that overexpression of KRT8 in HUC-MSCs enhances its therapeutic efficacy in treating GVHD while retaining the activity of GVL

[0045] To further investigate how KRT8 affects the function of HUC-MSCs, the applicants conducted experiments using lentivirus carrying the EGFP (HUC-MSCs-GFP) and KRT8 (HUC-MSCs-KRT8) genes. As evidenced by the stable green fluorescence signal in more than 90% of the cells ( Fig. 9 A). Real-time quantitative PCR (RT-qPCR) confirmed that KRT8 mRNA levels increased tenfold in modified MSCs ( Fig. 9 B), Western blot analysis showed that KRT8 protein expression was significantly enhanced ( Fig. 9 C). Flow cytometry confirmed that HUC-MSCs-KRT8 cells remained positive for CD90, CD105, and CD73, and negative for CD34, CD11b, CD19, CD45, and HLA-DR ( Fig. 9 D). These results indicate that KRT8 overexpression did not alter the basic characteristics of HUC-MSCs.

[0046] The effect of KRT8 on the immunomodulatory function of HUC-MSCs in allogeneic hematopoietic stem cell transplantation was examined. First, the applicants tested whether injection of KRT8-overexpressing HUC-MSCs could ameliorate aGVHD and preserve GVL activity. Therefore, irradiated BALB / c recipients were transplanted with splenocytes (2.5 × 10 6 ) and T cell-depleted bone marrow (TCD-BM) cells (5 × 10 6 ). Recipients given TCD-BM cells alone served as a GVHD-free control. Recipients received HUC-MSCs-KRT8 or HUC-MSCs GFP control treatment on days 0, 9, and 15 after allogeneic HSCT. Consistent with previous reports, administration of HUC-MSCs-GFP significantly improved recipient survival ( Figure 2 A). More importantly, HUC-MSCs-KRT8 treatment further improved overall survival and reduced damage to GVHD target tissues (liver, small intestine, and colon) compared with HUC-MSCs-GFP ( Figure 2 AC).

[0047] To evaluate the effect of HUC-MSCs-KRT8 treatment on GVL activity, BALB / c recipients were inoculated with luciferase-transfected A20 cells (A20-Luc, 5 × 10 6 / mouse, ip). A20-Luc tumor cells grew rapidly in recipients implanted with TCD-BM, and all mice died due to uncontrolled tumor growth ( Figure 2DF). The tumor was cleared quickly in the group without HUC-MSCs treatment. However, all recipients died of severe aGVHD within 25 days after allogeneic hematopoietic stem cell transplantation ( Figure 2 DF). A20-Luc tumor cells transiently grew in HUC-MSCs-GFP-treated recipients transplanted with splenocytes and TCD-BM cells, but 80% of the recipients died of aGVHD within 30 days after allogeneic hematopoietic stem cell transplantation. A20-Luc tumor cells also transiently grew in HUC-MSCs-KRT8-treated recipients and were eliminated on day 24, and 50% of the mice survived for more than 30 days without tumor recurrence ( Figure 2 DF). Taken together, these results indicate that treatment with HUC-MSCs-KRT8 effectively controlled GVHD while preserving GVL activity mediated by alloreactive T cells.

[0048] from Figure 2 As can be seen, infusion of human umbilical cord mesenchymal stem cells overexpressing KRT8 (HUC-MSCs-KRT8) can alleviate aGVHD while preserving aGVHD. (AC) Recipient BALB / C mice were lethally irradiated and intravenously injected with 5×10 6 bone marrow (BM) cells and 2.5 × 10 6 splenocytes, with control HUC-MSCs (HUC-MSCs-GFP) or KRT8-overexpressing HUC-MSCs. (A) Survival curves. n = 13-15, from three replicate experiments. (B) Representative images of hematoxylin and eosin staining of the liver, small intestine, and colon at day 21 after transplantation (original magnification ×200 for liver, ×200 for small intestine and colon; n = 5-8 per group). (C) Histopathological scoring of the liver, small intestine, and colon. (D-F) TCD-BM cells (5 × 10 6 ) were transplanted into lethally irradiated WT BALB / c recipients with or without splenocytes (1.25 × 10 6 Then, on day 0, mice were intraperitoneally injected with A20-Luc cells (5 × 10 per mouse). 6) challenged recipients. (D) After HCT, mice were monitored for tumor growth using in vivo BLI. Representative BLI images from each mouse at each time point are shown. (EF) Photon / second curves and tumor-free survival are shown. Data are expressed as mean ± SEM. n = 10 per group from 2 replicate experiments. P values ​​were calculated using one-way ANOVA (C), nonlinear regression (curve fitting) for photon / second comparison (E), and log-rank test for survival comparison (A and F) *p<0.05, **p<0.01, ***p<0.001, ***p<0.000

[0049] Embodiment 3,

[0050] This example demonstrates that KRT8 overexpression increases the expression of H3K27me3 in HUC-MSCs

[0051] To understand the impact of KRT8 overexpression on the genome, the applicants performed bulk RNA sequencing on HUC-MSCs-KRT8 and HUC-MSCs-GFP cells. The analysis revealed 212 differentially expressed genes, of which 167 were upregulated in HUC-MSCs-KRT8 and 45 were downregulated ( Figure 3 A). This is consistent with previous results ( Fig. 9 D), MS6 markers NT5E (CD73), THY1 (CD90), and endothelin (CD105) showed no significant changes ( Fig.10 A). Upregulated genes are related to DNA transcription and repair (e.g. NFKB2, PPP1R15A, PER1, DDB2), cell cycle regulation (PLK2), and p53-related pathways (e.g. CDIP1, PIDD1). Downregulated genes are related to cell senescence and inflammatory response (e.g. IL6ST, HMGA2, TNFAIP6, XIST, PTPN13, DYNC2H1) ( Figure 3 B).

[0052] In the analysis of gene expression and related pathways, Gene Ontology (GO) enrichment highlighted that the upregulated genes were mainly related to processes such as DNA template-based transcription, protein and DNA binding, and chromatin organization ( Fig.10 B). KEGG enrichment analysis showed that the differentially expressed genes were mainly related to pathways related to cell survival, apoptosis and cell senescence. Key pathways such as p53, MAPK, Ras and PI3K / AKT signaling pathways were clearly involved ( Figure 3 C). Importantly, overexpression of KRT8 resulted in significant changes in histones, with gene set enrichment analysis (GSEA) revealing an increase in pathways associated with histone H3 lysine 27-trimethylation (H3K27me3) ( Figure 3 DE and Fig.10Specifically, a pathway associated with the PRC2 complex, which involves trimethylation of EZH2s at nucleosomes associated with the cyclin-dependent kinase inhibitor 2A (CDKN2A) promoter, was significantly elevated in HUC-MSCs-KRT8 compared with HUC-MSCs-GFP ( Figure 3 F). CDKN2A encodes the key proteins p16INK4a and p14ARF, which are essential for cell cycle regulation and cell senescence. In addition, it was found that KRT8 overexpression downregulated several KEGG pathways related to lipid metabolism, including fatty acid biosynthesis, fatty acid metabolism, and PPAR signaling pathways ( Figure 3 GI). Disturbances in lipid metabolism are considered a hallmark of cellular senescence, suggesting that the effects of KRT8 overexpression extend beyond structural integrity and include metabolic and epigenetic changes.

[0053] Further studies have shown that ZF-C2H2 transcription factors can regulate cellular senescence by regulating the expression of genes involved in cell cycle control, apoptosis, and stress response. These factors may affect the expression of key regulatory factors such as p53 and p21, as well as components of the senescence-associated secretory phenotype (S6SP). Our results showed that the ZF-C2H2 transcription factor family was significantly enriched in HUC-MSCs-KRT8 ( Figure 3 J). Collectively, these results suggest that KRT8 overexpression in HUC-MSCs profoundly affects their genomic and epigenetic signatures. Upregulation of genes associated with DNA repair, cell cycle progression, and apoptosis, together with alterations in lipid metabolism and H3K27me3 modification, suggest that KRT8 overexpression may enhance the resilience of HUC-MSCs and reshape their dynamics associated with cellular senescence.

[0054] Figure 3 (A) Bulk RNA sequencing was performed on passage 4 HUC-MSCs-KRT8 and HUC-MSCs-GFP, respectively, and 212 differentially expressed genes were identified. (B) Heat map of the top 100 differentially expressed genes. (C) Histogram of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of cellular processes and environmental information processing. (D-I) Gene set enrichment analysis (GSEA) plots of PRC2 recruited DNA methyltransferase pathway (D), PRC2 trimethylates histone H3 on lysine pathway (E), PRC2 EZH2 trimethylates nucleotides associated with CDKN2A promoter pathway (F), fatty acid biosynthesis pathway (G), fatty acid ester metabolism pathway (H), PPAR signaling pathway (I). (J) Histogram of transcription factor families in HUC-MSCs-KRT8 and HUC-MSCs-GFP. n = 3, combined from three independent experiments.

[0055] Embodiment 4,

[0056] This example demonstrates that EZH2-mediated H3K27me3 regulates the senescence of HUC-MSCs

[0057] To further confirm the bulk RNA-Seq findings at the protein level, Applicants treated HUC-MSCs-KRT8 with an EZH2 inhibitor (XNW5004), known to reduce overall H3K27me3 levels. We then performed Western blot analysis to assess the expression of H3K27me3 protein. Consistent with the RNA-Seq results, KRT8 overexpression increased the levels of H3K27me3 in HUC-MSCs compared to the control group, while the EZH2 inhibitor significantly reduced the expression of H3K27me3 ( Figure 4 A). In addition, the applicants tested cellular senescence and senescence-associated secretory phenotype (SASP) by evaluating proteins p16INK4A, p21, and p53. KRT8 overexpression reduced their expression ( Figure 4 However, EZH2 inhibition reversed this effect, as indicated by increased p16INK4A levels ( Figure 4 C). SA-β-gal assay further demonstrated that KRT8 overexpression inhibited the senescence of HUC-MSCs, an effect that was blocked by EZH2 inhibitor ( Figure 4 D).

[0058] Inhibition of T cell proliferation is generally considered a characteristic of MSCs, independent of their tissue of origin. To explore the role of H3K27me3 and reduced SASP in the inhibition of T cell activation, we tagged Thy1.2 + T cells were co-cultured with HUC-MSCs-KRT8 or HUC-MSCs-GFP. Consistent with previous findings, HUC-MSCs-KRT8 and HUC-MSCs-GFP suppressed CD3 / CD28-activated Thy1.2 on days 3 and 4 after in vitro co-culture. + T cell proliferation ( Figure 4 EF). It is worth noting that the T cell proliferation rate in the HUC-MSCs-KRT8 group was significantly reduced compared with that in the HUC-MSCs-GFP group ( Figure 4 EF). Effect of HUC-MSCs-KRT8 on mice ( Figure 4 GH) and human T cells ( Figure 4 This suppressive activity of HUC-MSCs could be partially reversed by the addition of EZH2 inhibitor, indicating that KRT8 overexpression enhanced the immunosuppressive function of HUC-MSCs in an H3K27me3-dependent manner.

[0059] Figure 4In the present study, KRT8 overexpression promoted HUC-MSCs senescence and enhanced the immunosuppressive function of HUC-MSCs through H3K27me3 regulation. (A) Western blot and (B) quantitative analysis of H3K27me3 in HUC-MSCs-GFP and HUC-MSCs-KRT8 with or without EZH2 inhibitor. (C) Western blot and (D) quantitative analysis of the expression of p16INK4A, p21, and p53 in HUC-MSCs-GFP and HUC-MSCs-KRT8 with or without EZH2 inhibitor. (EF) Representative flow cytometry patterns and the percentage of CFSE-negative cells in unstimulated T cells, activated T cells, T cells activated with HUC-MSCs-GFP, and T lymphocytes activated with HUC-MSCs-KRT8. (GH) Representative flow cytometry patterns and percentages of CFSE-negative cells in unstimulated T cells, activated T cells, T cells activated with HUC-MSCs-GFP, and activated T cells co-cultured with HUC-MSCs-KRT8 in the presence or absence of EZH2 inhibitors. (IJ) Representative flow cytometry patterns and percentages of CFSE-negative cells in unstimulated human T cells, activated human cells, human T lymphocytes activated with HUC-MSCs-GFP, and activated human T cells co-cultured with HUC-MSCs-KRT8 in the presence or absence of EZH2 inhibitors. Data represent mean ± SEM; all tests were repeated twice. P values ​​were calculated using one-way ANOVA multiple comparison test (EG) *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001.

[0060] Embodiment 5,

[0061] This example demonstrates that EZH2 controls the secretion of hepatocyte growth factor (HGF) through KRT8 overexpression in HUC-MSCs

[0062] In order to investigate the molecular mechanism of H3K27me3-mediated stem maintenance and SASP inhibition, the applicant used RT-qPCR to detect the expression levels of six molecules related to the immunomodulatory function of HUC-MSCs. The results showed that compared with HUC-MSCs-GFP, overexpression of KRT8 in HUC-MSCs significantly increased the expression levels of HGF, CXCL5, TLR46, PGE2, and TLR6 ( Figure 5 A), while reducing the levels of IL-6, TLR5, CCL-2 and TLR3 ( Figure 5 B). Further analysis showed that the HGF concentration in the HUC-MSCs-KRT8 culture medium increased significantly, and the HGF concentration decreased after EZH2 inhibition ( Figure 5C). These findings suggest that KRT8 overexpression enhances HGF secretion via H3K27me3 regulation.

[0063] The applicant then examined whether the increase in HGF secretion contributes to the inhibition of T cell proliferation. HUC-MSCs-KRT8 or HUC-MSCs-GFP was co-cultured with T cells activated by anti-CD3 / CD28 beads, and anti-HGF antibody was added to the culture medium, indicating that HUC-MSCs-KRT8 had a stronger inhibitory effect on mouse T cells compared with HUC-MSCs-GFP ( Figure 5 DE). HGF blocking antibody fully confirmed this inhibitory effect ( Figure 5 DE). Similar effects were observed when tested on human peripheral blood mononuclear cells ( Figure 5 FG), indicating that the enhanced immunosuppression of HUC-MSCs-KRT8 is HGF-dependent. Overall, overexpression of KRT8 in HUC-MSCs enhances the secretion of HGF through the regulation of H3K27me3, greatly enhancing the immunosuppressive capacity of these cells.

[0064] Figure 5 : The immunomodulatory effect of KRT8 overexpression on HUC-MSCs is HGF-dependent. (AB) Relative mRNA expression of immunomodulatory marker genes in HUC-MSCs-GFP and HUC-MSCs-KRT8. (C) Concentration of HGF in the culture medium of HUC-MSCs-GFP, HUC-MSCs-KRT8, and HUC-MSCs+EZH2i. (DE) Representative flow cytometry patterns and the percentage of CFSE-negative cells in unstimulated T cells, activated T cells, T cells activated with HUC-MSCs-GFP, and activated T cells co-cultured with HUC-MSCs-KRT8 in the presence or absence of HGF blocking antibody. (FG) Representative flow cytometry patterns and the percentage of CFSE-negative cells in unstimulated human T cells activated human T cells, human T lymphocytes activated with HUC-MSCs-GFP, and activated human T cells co-cultured with HUC-MSCs-KRT8 in the presence or absence of HGF blocking antibody. Data represent mean ± SEM; all tests were repeated twice. P values ​​were calculated using multiple comparison test (A) and one-way ANOVA multiple comparison test (Bd) *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001.

[0065] Embodiment 6,

[0066] This example demonstrates that HGF produced by KRT8-overexpressing HUC-MSCs alleviates liver injury

[0067] The therapeutic efficacy of MSCs depends largely on their ability to reach GVHD target organs. To evaluate the migration potential of HUC-MSCs in vitro, HUC-MSCs-KRT8 and HUC-MSCs-GFP cells were analyzed using the migration Transwell assay. Figure 6 As shown in AB, KRT8 overexpression significantly increased the migration of HUC-MSCs. In addition, to investigate the fate and migration pattern of HUC-MSCs in vivo, PKH26-labeled HUC-MSCs-KRT8 or HUC-MSCs-GFP were injected into allogeneic hematopoietic progenitor cell transplant recipients on days 0, 5, and 9 after allogeneic hematopoietic stem cell transplantation. Spleen, liver, lung, and intestinal tissues were collected on day 16, and PKH26-labeled HUC-MSCs-KRT8 or HUC-MSCs -GFP were analyzed by fluorescence microscopy. Our observations confirmed that KRT8 overexpression significantly enhanced the migration of HUC-MSCs to the liver in vivo, with no differences in other organs ( Figure 6 CD and Fig.11 ).

[0068] To evaluate whether KRT8-promoted HGF secretion could attenuate liver injury induced by alloreactive T cells, we established a co-culture system of T cells and liver organoids that enabled real-time monitoring of interactions and potential cytotoxicity. We observed that alloreactive T cells significantly reduced the survival rate of liver organoids, as indicated by an increase in caspase fluorescence intensity ( Figure 6 EF). However, at 24, 48, and 72 hours after the start of co-culture, the survival rate of liver organoids pulsed with HGF was more than twice that of liver organoids without HGF ( Figure 6 EF), indicating that HGF treatment provided significant protection to liver organoids from the cytotoxic effects of HLA-mismatched T cells. These findings indicate that KRT8 overexpression promotes the migration of HUC-MSCs to the liver and suggest that elevated HGF levels effectively mitigate liver damage caused by alloreactive T cells, thereby maintaining the structural integrity and functionality of liver organoids.

[0069] Figure 6:KRT8 overexpression enhances HUC-MSCs migration and alleviates liver organoid injury. (AB) Representative patterns and summary bar graphs of HUC-MSCs-GFP and HUC-MSCs-KRT8 in vitro transwell assays. n=7. (CD) Representative patterns and summary bar graphs of HUC-MSCs-GFP and HUC-MSCs-KRT8 in vivo PKH26 staining assays. n=4. (E) Representative micrographs of human T cells and liver organoids co-cultured with HGF at 0, 24, 48, and 72 hours. Scale bar, 500um. n=2-10. (F) Summary bar graph of caspase intensity at 24, 48, and 72 hours. Mean ± SEM, from 2 independent experiments. Unpaired two-tailed Student's t-test was used to compare the means *p<0.05, **p<0.01.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention. The techniques, shapes, and structural parts not described in detail in the present invention are all known technologies.

Claims

1. Use of mesenchymal stem cells in a drug for treating acute graft-versus-host disease, characterized in that: The mesenchymal stem cells are from the KRT8 subpopulation.

2. The use according to claim 1, characterized in that: The mesenchymal stem cells are one or a combination of human adipose tissue, amniotic membrane, bone marrow, deciduous teeth, dermis, endometrium, human embryonic stem cell derivatives, oral mucosa or umbilical cord.

3. The use according to claim 1 or 2, characterized in that: The mesenchymal stem cells are derived from one or a combination of bone marrow, umbilical cord, dermis or adipose tissue.

4. The use according to claim 1 or 2, characterized in that: The mesenchymal stem cells are derived from umbilical cord.

5. The use according to claim 4, characterized in that: The mesenchymal stem cells are derived from cells that highly express KRT8 + Subgroup.

6. A drug for treating acute graft-versus-host disease, characterized in that: The medicine comprises the mesenchymal stem cells according to claim 1, and pharmaceutically acceptable excipients.

7. The drug according to claim 6, characterized in that: The medicine is an infusion solution.

8. A method for culturing mesenchymal stem cells as claimed in claim 5, characterized in that the steps include: Isolation of KRT8 from human tissues + The subpopulation was cultured in DMEM / F12 supplemented with 8-12% fetal bovine serum in a 4-6% CO2 incubator at 35-39°C; then cultured in RPMI-1640 supplemented with 8-12% FBS and 0.03-0.07mM 2-Mer in a 4-6% CO2 incubator at 35-39°C; when the cell density reached 70-90%, it was dissociated using trypsin.

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