The roles of subtype specific bone marrow stromal cell secretomes

Understanding of bone marrow stromal cell (BMSC) functions is complicated by heterogeneity. Current cell-surface markers are insufficiently selective for functionality, which has profound implications for development of BMSC based therapies. In this thesis I investigated secreted factors of immortalised BMSC clones and their effects on BMSC functions.
I compared the effects of secreted factors from two phenotypically distinct clones on BMSC phenotype. I used Y201, a multipotent and migratory BMSC, and a nullipotent non-migratory clone, Y202. Y201 conditioned media (Y201CM) treatment of Y202 cells resulted in larger colonies in a CFU-F assay. Subsequent tracking of Y202 cells in culture indicated that they migrated further in Y201CM. Increased migration of Y202 cells was driven by heat-labile secreted factors, and was replicated by culturing Y202 on BMSC derived extracellular matrix (ECM). This ECM migration was reduced with a focal adhesion kinase inhibitor.
Proteomics of conditioned media revealed that Y201 secreted proteins significantly enriched for ECM. Periostin and aggrecan were identified as candidate ECM components that could contribute to Y201 phenotype due to high abundance and large fold-changes versus Y202. Extracellular vesicle (EV) protein and miRNAs were also compared between BMSC subtypes, with upregulated Y201 EV proteins enriched for pathways and terms associated with ECM. Nanostring analysis revealed significant upregulation of 10 versus 2 miRNAs in EVs from Y201 and Y202 respectively. Predicted targets of Y201 upregulated miRNAs also indicated ECM involvement.
Immunofluorescence staining of ECM proteins identified by proteomics identified possible BMSC locations in human and murine tissue, with Y201 upregulated ECM components predominantly in endosteal regions. Rare CD271+ bone-lining stromal cells were found on top of aggrecan and periostin in trabecular regions of human bone.
I conclude that BMSC subtype heterogeneity is mirrored in secreted factors and that the secretome and ECM components provide important markers of functionality and localization.