Large-Scale Extraction and Characterization of CD271+Multipotential Stromal Cells From Trabecular Bone in Health and Osteoarthritis Implications for Bone Regeneration Strategies Based on Uncultured or Minimally Cultured Multipotential Stromal Cells

Objective. To test the hypothesis that CD45(low)CD271+ bone marrow multipotential stromal cells (MSCs) are abundant in the trabecular bone niche and to explore their functional fitness in health and osteoarthritis (OA). Methods. Following enzymatic extraction, MSC release was evaluated using colony-forming unit-fibroblast (CFU-F) and colony-forming unit-osteoblast assays, flow cytometry, and confocal microscopy. CD45(low)CD271+ cells isolated by fluorescence-activated cell sorting were enumerated and expanded under standard and clonal conditions. Their proliferative and osteogenic potencies were assessed in relation to donor age and compared with those of aspirated CD45(low)CD271+ cells. In vitro and in vivo MSC aging was measured using quantitative polymerase chain reaction-based telomere length analysis, and standard differentiation assays were utilized to demonstrate multipotentiality. Results. Cellular isolates from trabecular bone cavities contained similar to 65-fold more CD45(low)CD271+ cells compared with aspirates (P < 0.0001) (median 1.89% [n = 39] and 0.029% [n = 46], respectively), concordant with increased CFU-F release. Aspirated and enzymatically released CD45(low)CD271+ cells had identical MSC phenotypes (similar to 100% CD73+CD105+CD13+, similar to 50-60% CD146+CD106+CD166+) and contained large proportions of highly clonogenic multipotential cells. In vitro osteogenic potency of freshly isolated CD45(low)CD271+ cells was comparable with, and often above, that of early-passage MSCs (8-14%). Their frequency and in vivo telomere status in OA bone were similar to those in bone from age-matched controls. Conclusion. Our findings show that CD45(low)CD271+ MSCs are abundant in the trabecular bone cavity and indistinguishable from aspirated CD45lowCD271+ MSCs. In OA they display aging-related loss of proliferation but no gross osteogenic abnormality. These findings offer new opportunities for direct study of MSCs in musculoskeletal diseases without the requirement for culture expansion. They are also relevant for direct therapeutic exploitation of prospectively isolated, minimally cultured MSCs in trauma and OA.