Does osteogenic potential of clonal human bone marrow mesenchymal stem/stromal cells correlate with their vascular supportive ability?

Human bone marrow-derived mesenchymal stem/stromal cells (hBM MSCs) have multiple functions, critical for skeletal formation and function. Their functional heterogeneity, however, represents a major challenge for their isolation and in developing and potency and release assays to predict their functionality prior to transplantation. Additionally, potency, biomarker profiles and defining mechanisms of action in a particular clinical setting are increasing requirements of Regulatory Agencies for release of hBM MSCs as Advanced Therapy Medicinal Products (ATMPs) for cellular therapies. Since the healing of bone defects in larger bone grafts depends on the coupling of new blood vessel formation with osteogenesis, we hypothesised that a correlation between the osteogenic and vascular supportive potential of individual hBM MSC-derived CFU-F (colony forming unit-fibroblastoid) clones might exist. We tested this by assessing the lineage (i.e. adipogenic {A}, osteogenic {O} and/or chondrogenic {C}) potential of individual hBM MSC-derived CFU-F clones and determining if their osteogenic {O} potential correlated with their vascular supportive profile in vitro using lineage differentiation assays, endothelial-hBM MSC vascular co-culture assays and transcriptomic (RNAseq) analyses. Our results demonstrate that the majority of CFU-F (95%) possessed tri-lineage, bi-lineage or uni-lineage osteogenic capacity, with 64% of the CFU-F exhibiting tri-lineage AOC potential. We found a correlation between the osteogenic and vascular tubule supportive activity of CFU-F clones, with the strength of this association being donor dependent. RNAseq of individual clones defined gene fingerprints relevant to this correlation. 16 samples from two donors (8 from each donor), possessing high or low osteogenic potential.

人骨髓源性间充质/基质干细胞（human bone marrow-derived mesenchymal/stromal cells, hBM MSCs）具备多种功能，对骨骼的形成与生理功能至关重要。然而，其功能异质性仍是一大挑战：不仅给细胞分离纯化工作带来阻碍，也为开发移植前预测细胞功能的效能检定与放行检测方案增添了难度。此外，监管机构对作为细胞治疗用先进治疗药物产品（Advanced Therapy Medicinal Products, ATMPs）放行的hBM MSCs，提出了愈发严格的要求，包括明确其效能、生物标志物特征以及特定临床场景下的作用机制。
鉴于大型骨移植物中的骨缺损修复，有赖于新生血管形成与成骨过程的协同耦合，我们提出假说：单个hBM MSCs来源的成纤维细胞集落形成单位（colony forming unit-fibroblastoid, CFU-F）克隆的成骨潜能与血管支持潜能之间可能存在相关性。为验证该假说，我们首先评估了单个hBM MSCs来源的CFU-F克隆的谱系分化潜能（即成脂{A}、成骨{O}及/或软骨分化{C}潜能），并借助体外谱系分化检测、内皮细胞-hBM MSCs血管共培养检测以及转录组学（RNA测序，RNAseq）分析，探究其成骨{O}潜能是否与血管支持特征相关。
研究结果显示，95%的CFU-F克隆具备三系、双系或单系成骨能力，其中64%的CFU-F克隆展现出三系AOC分化潜能。我们发现，CFU-F克隆的成骨活性与血管管状结构支持活性之间存在相关性，且该关联的强度具有供者依赖性。对单个克隆的RNA测序分析，明确了与该相关性相关的基因特征谱。本研究共纳入来自2名供者的16份样本（每名供者8份），这些样本的成骨潜能存在高低差异。