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, which are critical for skeletal formation and function. They can differentiate into multiple lineages, possess anti-inflammatory properties, and provide specialised niches for regulating hematopoietic stem/progenitor cell and blood vessel formation and function. hBM MSCs are heterogeneous and can contain both multipotent stem and partially differentiated mesenchymal progenitor cells. Their functional heterogeneity represents a major challenge in developing isolation, potency and release assays which predict their functionality prior to transplantation. 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 then assessed the lineage (i.e. adipogenic {A}, osteogenic {O} and/or chondrogenic {C}) potential of individual hBM MSC-derived CFU-F clones and determined whether 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. Of 133 CFU-F clones isolated at P0, 95% had osteogenic potential (AOC, OC, AO, O), 64% tri-lineage AOC potential, 31% were bi-potent (AO, AC, OC), 4% were unipotent (O or C) and 1% nullipotent. The vascular supportive potency of the CFU-F clones varied from very poor to strong and showed donor variability. CFU-F with the highest vascular supportive ability all shared osteogenic potential in common, principally occurring in the tri-lineage AOC and osteo-chondrogenic (OC) bi-lineage groups and quantitatively having a higher capacity for osteogenesis. A correlation existed between CFU-F osteogenic and vascular supportive capacity potential for all 133 clones (Pearson's correlation coefficient (r) = 0.3855; p <0.0001). However, when comparing 3 donor bone marrows, the strength of this association was highest and strongly positive for donor 2 (r = 0.6822, p <0.0001), followed by donor 3 (r = 0.4126) and poor for donor 1 (r = 0.1578). The strength of the association between adipogenic and their vascular tubule supportive potential was low (r = 0.1523). CFU-F gene fingerprints to predict differences in potentiality were assessed using RNAseq. 100 viable single cells from each of 16 CFU-F clones (that supported vascular networks in the upper or lower quartile ranges) were flow sorted and cDNA libraries generated. Genes with average expression levels across all clones in the upper quartile range, compared with those in the lower quartile range, were enriched for specific signalling pathways. The samples were also divided into different groups based on their tri-lineage, osteogenic, chondrogenic and adipogenic potential, and EdgeR used to explore the degree of differential expression dependent on each attribute. Osteogenic potential showed the most dynamic changes in gene expression, with 161 genes (FDR <0.05) or 332 genes (FDR <0.1) differentially expressed. The more highly expressed transcripts in clones with a higher osteogenic potential included those involved in angiogenesis. In conclusion, we demonstrate a correlation between increased osteogenic potential and increased vascular supportive activity of MSC derived CFU-F that is donor dependent. These studies will inform on future selection of the most suitable recipients to benefit from autologous hBM MSC cell therapies, which require osteo/angiogenic cell coupling.