Xu Qing, Wang Rui, Sui Ke, Xu Yuxi, Zhou Ya, He Yuxuan, Hu Ziyi, Wang Qi, Xie Xiaodong, Wang Xiaoqi, Yang Shijie, Zeng Lingyu, Zhong Jiang F, Wang Zheng, Song Qingxiao, Zhang Xi
School of Life Sciences, Chongqing University, Chongqing, 405200, China.
Medical Center of Hematology, Xinqiao Hospital of Army Medical University, Chongqing, 400037, China.
Stem Cell Res Ther. 2025 May 1;16(1):222. doi: 10.1186/s13287-025-04321-6.
Acute graft-versus-host disease (aGVHD) remains a major life-threatening complication of allogeneic haematopoietic cell transplantation (allo-HSCT), often limiting the therapeutic efficacy of allo-HSCT. Recent studies have suggested that mesenchymal stem cells (MSCs) may be beneficial for the treatment of aGVHD. However, the therapeutic potential of MSCs is often negatively impacted by their heterogeneity.
To investigate MSCs heterogeneity, we conducted single-cell transcriptomic analysis of human umbilical cord-derived MSCs (HUC-MSCs) and identified key feature genes that distinguish MSCs subpopulations. The function of the newly discovered biomarker CRISPLD2 was also explored. We engineered human umbilical cord-derived MSCs (HUC-MSCs) to overexpress the CRISPLD2 gene using lentiviral vectors. The downstream regulatory effects of CRISPLD2 overexpression were assessed through bulk RNA sequencing. Additionally, we evaluated its impact on cellular senescence using Western blotting and β-galactosidase (SA-β-gal) staining. The immunoregulatory capability of HUC-MSCs was tested through coculture experiments with T cells and liver organoids in vitro. Mitochondrial function was analysed via flow cytometry and electron microscopy. The in vivo therapeutic effects of HUC-MSCs on aGVHD were evaluated using an aGVHD murine model. The graft-versus-leukaemia (GVL) effect was measured via the inoculation of luciferase-positive A20 cells, and tumour growth was monitored via bioluminescence imaging.
Our findings indicated that the CRISPLD2 gene is heterogeneously expressed in HUC-MSCs subsets characterized by stemness and immunosuppressive properties. Transcriptomic analysis revealed that CRISPLD2 overexpression suppressed calcium ion binding and G protein-coupled receptor signalling. In vitro studies demonstrated a marked increase in IL-10 secretion, which enhanced T-cell suppression in CRISPLD2-modified HUC-MSCs. The in vivo results demonstrated that transfusion of CRISPLD2-overexpressing HUC-MSCs ameliorated aGVHD while maintaining GVL activity. Mechanistically, CRISPLD2 overexpression overcomes the mitochondrial damage mediated by extracellular ATP and LPS in HUC-MSCs by inhibiting P2Y11 receptor signalling, thereby preserving their stemness and IL-10-mediated immunosuppressive functions.
Our study revealed that CRISPLD2 is a novel marker for identifying HUC-MSCs subpopulation with enhanced immunosuppressive functions. CRISPLD2 overexpression enhances the immunosuppressive function of HUC-MSCs by inhibiting P2Y11 receptor signalling. Targeting CRISPLD2 is a promising strategy to improve the therapeutic efficacy of HUC-MSCs in aGVHD while maintaining GVL activity.
急性移植物抗宿主病（aGVHD）仍然是异基因造血细胞移植（allo-HSCT）的一种主要的危及生命的并发症，常常限制了allo-HSCT的治疗效果。最近的研究表明间充质干细胞（MSCs）可能对aGVHD的治疗有益。然而，MSCs的治疗潜力常常受到其异质性的负面影响。
为了研究MSCs的异质性，我们对人脐带源MSCs（HUC-MSCs）进行了单细胞转录组分析，并鉴定了区分MSCs亚群的关键特征基因。还探索了新发现的生物标志物CRISPLD2的功能。我们使用慢病毒载体对人脐带源MSCs（HUC-MSCs）进行基因工程改造，使其过表达CRISPLD2基因。通过大量RNA测序评估CRISPLD2过表达的下游调节作用。此外，我们使用蛋白质免疫印迹法和β-半乳糖苷酶（SA-β-gal）染色评估其对细胞衰老的影响。通过与T细胞和肝类器官进行体外共培养实验来测试HUC-MSCs的免疫调节能力。通过流式细胞术和电子显微镜分析线粒体功能。使用aGVHD小鼠模型评估HUC-MSCs对aGVHD的体内治疗效果。通过接种荧光素酶阳性A20细胞来测量移植物抗白血病（GVL）效应，并通过生物发光成像监测肿瘤生长。
我们的研究结果表明，CRISPLD2基因在以干性和免疫抑制特性为特征的HUC-MSCs亚群中呈异质性表达。转录组分析显示，CRISPLD2过表达抑制钙离子结合和G蛋白偶联受体信号传导。体外研究表明，IL-10分泌显著增加，这增强了CRISPLD2修饰的HUC-MSCs对T细胞的抑制作用。体内结果表明，输注过表达CRISPLD2的HUC-MSCs可改善aGVHD，同时维持GVL活性。从机制上讲，CRISPLD2过表达通过抑制P2Y11受体信号传导克服了细胞外ATP和LPS介导的HUC-MSCs线粒体损伤，从而保留了它们的干性和IL-10介导的免疫抑制功能。
我们的研究表明，CRISPLD2是一种用于鉴定具有增强免疫抑制功能的HUC-MSCs亚群的新型标志物。CRISPLD2过表达通过抑制P2Y11受体信号传导增强了HUC-MSCs的免疫抑制功能。靶向CRISPLD2是一种有前景的策略，可提高HUC-MSCs在aGVHD中的治疗效果，同时维持GVL活性。
