Exploiting knowledge of mesenchymal stromal cells in vivo for bone disease therapy development

This thesis aims to demonstrate how understanding of the nature of MSCs in vivo can be used to guide bone disease therapy development. It first address the unpredictable MSC content of bone marrow (BM) aspirates used for therapy; then examines MSC enrichment using clinical grade immunomagnetic cell selection. Induction of osteogenesis was subsequently explored; by examining an induced membrane (IM) used for bone regeneration. Finally the potential of a janus kinase (JAK) inhibitor to block osteogenesis was assessed, by studying its effect on MSCs in vitro. Flow cytometry was used to enumerate cells expressing a CD45-/low CD271+ phenotype in BM aspirate and this was compared to colony forming unit fibroblast (CFU-F) content. MSCs were enriched from BM, enzymatically treated femoral heads (FH) and intramedullary canal aspirates and enumerated. The composition morphology, MSC content and differentiation potential of IM was compared to periosteum. The potential effects of JAK inhibition on MSC colony formation, expansion and differentiation potential were examined. The concentration of cells expressing a CD45-/low CD271+ phenotype strongly correlated with CFU-F concentration (R=0.812, p<0.001). Immunomagnetic cell selection resulted in an increase in the proportion of MSCs in BM, FH and intramedullary canal aspirates by 204, 14.1 and 291-fold respectively. The regenerative potential of periosteum and IM were comparable. JAK inhibition did not affect MSC growth, osteogenesis or chondrogenesis but caused an increase in adipogenesis at concentrations ≥100nM compared to controls (1.38 fold, p=0.041). Flow cytometry may be used to rapidly and accurately predict the MSC content of BM. Clinical grade immunomagnetic selection can substantially increase the purity of MSCs from bone cavities. The similarity, in terms of regenerative potential, of periosteum and IM gives insight into its use for bone regeneration. JAK inhibition did not affect in vitro osteogenesis but has potential to affect in vivo osteogenesis through stimulation of adipogenesis.