Raw data related to the article "Impact of aging on CD146+ mesenchymal stromal cells-mediated regulation of bone marrow CD34+ hematopoietic stem/progenitor cell mobilization"

ABSTRACT: Background: The study of age-associated changes in the bone marrow (BM), a key organ for hematopoiesis and immune regulation, is crucial to understanding inflammatory processes linked to cardiovascular diseases. Notably, aging is associated with impaired mobilization of BM-derived cardioprotective CD34+ hematopoietic stem/progenitor cells (HSPCs), resulting in a lower frequency in the circulation and poorer cardiovascular outcomes. HSPCs pharmacological mobilizers (G-CSF, AMD3100) and sympathetic signaling stimulator (norepinephrine, NE) function mainly by acting on CD146+ Bone marrow stromal cells (BMSCs), key cellular players in this process. In this study, we developed a human in vitro BM simplified experimental model to assess the impact of aging on CD146+ BMSCs in this context. Methods: CD146+ BMSCs were isolated from 29 human subjects, stratified into middle-aged (middle-aged, ≤66 years; N = 13) and older adults (older adult, ≥75 years; N = 16). For functional assays, bone marrow HSPCs from a single donor (female, 64 years) were used. The in vitro BM simplified experimental model was developed by co-culturing CD146+ BMSC and CD34+ HSPCs in a transwell system. After treatment with mobilizing agents, CD146+ BMSCs' viability, HSPC migration, transcriptome, metabolism, and paracrine activity were assessed. Results: AMD3100 enhanced HSPC migration in the model using MA-derived CD146+ BMSCs but not in OA-derived cells. RNA sequencing identified 9 age-associated genes, with validated downregulation of NRK and upregulation of PDK4, AQP1, and LMO2 in OA CD146+ BMSC. Moreover, a differential gene expression response to mobilizing treatments was observed between groups. Cell-conditioned media from OA CD146+ BMSCs showed stronger chemoattractant effects on peripheral blood mononuclear cells and presented increased VCAM-1 levels. No age-related effects on oxidative respiration were observed. Conclusion: In this study, a BM in vitro co-culture system was developed to study CD146+ BMSC-dependent CD34+ mobilization in a subject-specific manner, and the complexity of the impact of aging on CD146+ BMSC and their response to mobilizing agents was highlighted. Keywords: In vitro models; aging; bone marrow; hematopoietic stem/progenitor cells; migration; stromal cells; vasculature.摘要： 背景：骨髓（bone marrow, BM）是造血与免疫调控的核心器官，对其衰老相关变化的研究，对于解析与心血管疾病关联的炎症进程至关重要。值得注意的是，衰老会导致骨髓来源的具有心脏保护作用的CD34+造血干/祖细胞（hematopoietic stem/progenitor cells, HSPCs）动员功能受损，使其循环频率降低，并伴随更差的心血管预后。造血干/祖细胞的药理学动员剂（如粒细胞集落刺激因子G-CSF、AMD3100）以及交感信号刺激剂（如去甲肾上腺素norepinephrine, NE）主要通过作用于CD146+骨髓基质细胞（bone marrow stromal cells, BMSCs）发挥功能，而这类细胞是该过程中的关键细胞组分。本研究构建了简化的人骨髓体外实验模型，以评估衰老在此场景下对CD146+骨髓基质细胞的影响。 方法：研究人员从29名人类受试者体内分离得到CD146+骨髓基质细胞，并将其分为中年组（≤66岁，n=13）与老年组（≥75岁，n=16）。功能实验所用的骨髓造血干/祖细胞均来自1名64岁女性供体。本研究通过Transwell小室共培养CD146+骨髓基质细胞与CD34+造血干/祖细胞，构建简化的骨髓体外实验模型。在使用动员剂处理后，研究人员对CD146+骨髓基质细胞的活性、造血干/祖细胞迁移能力、转录组、代谢水平以及旁分泌活性进行了检测。 结果：研究发现，AMD3100可在中年供体来源的CD146+骨髓基质细胞模型中增强造血干/祖细胞的迁移能力，但在老年供体来源的细胞模型中无此效果。RNA测序共鉴定出9个衰老相关基因，经验证，老年组CD146+骨髓基质细胞中NRK基因表达下调，而PDK4、AQP1与LMO2基因表达上调。此外，两组细胞对动员剂处理的基因表达响应存在显著差异。老年组CD146+骨髓基质细胞的条件培养基对外周血单个核细胞的趋化活性更强，且VCAM-1蛋白水平升高。未观察到衰老对氧化呼吸功能产生显著影响。 结论：本研究构建了骨髓体外共培养系统，可实现供体特异性的CD146+骨髓基质细胞依赖型CD34+造血干/祖细胞动员研究，并揭示了衰老对CD146+骨髓基质细胞及其对动员剂响应的复杂影响。 关键词：体外模型；衰老；骨髓；造血干/祖细胞；迁移；基质细胞；脉管系统。