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This work is concerned with the characterization of mesenchymal stem cells (MSC) specifically from bone marrow samples derived from patients with osteoarthritis (OA). The multilineage potential of mesenchymal stem cells as well as their ease of exvivo expansion makes these cells an attractive therapeutic tool for applications such as autologous transplantation and tissue engineering. Bone marrow is considered a source of MSC. However, there is a general assumption that the occurrence of MSCs and their activity in bone marrow diminishes with age and disease. This prompted us to isolate and identify multipotential and self-renewing cells from patients with the degenerative disease osteoarthritis, with the view of using these cells for autologous cell therapies. It is therefore of great potential benefit to investigate the isolation and characterization of stem cell/progenitors from bone marrow samples of patients with osteoarthritis in greater detail. We employed a single cell clone culture method in order to develop clonal cell populations from three bone marrow samples and characterized them based on their proliferation and differentiation capabilities. The clonal populations were grouped into fast-growing and slow-growing clones based on their proliferation rates. The fastgrowing clones displayed 20-30% greater proliferation rate than the slow-growing clones. The study also revealed that the proliferation rates were directly proportional to their differentiation capacities. Most of the fast-growing clones were found to be tripotential for osteogenic, chondrogenic and adipogenic lineages, whereas the slow growing clones were either uni or bipotential. Flow cytometry analysis for the phenotype determination using putative MSC surface markers did not reveal any difference between the two clonal populations indicating a need for further molecular studies. Two approaches were employed to further investigate the molecular processes involved in the existence of such varying populations. In the first method gene expression studies were performed between the fast-growing (n=3) and slow-growing (n=3) clonal populations to identify potential genetic markers associated with cell 'sternness' using the Stem Cell RT2 ProfilerTM PCR Array comprising a series of 84 genes related to stem cell pathways. Ten genes were identified to be commonly and significantly over represented in the fast-growing stem cell clones when compared to slow-growing clones. This included expression of transcripts beyond MSC lineage specification such as SOX2, NOTCH1 and FOXA2 which signified that stem cell maintenance requires a coordinated regulation by multiple signalling pathways. The second study involved an extensive protein expression profiling of the fast growing (n=2) and slow growing (n=2) clonal populations using off-line Two Dimensional Liquid Chromatography (2D-LC)/Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry (MS). A total of 67 proteins were identified, of which 11 were expressed at significantly different levels between the subpopulations. Protein ontology revealed these proteins to be associated with cellular organization, cytokinesis, signal transduction, energy pathways and cell stress response. Of particular interest was the differential presentation of the proteins calmodulin, tropomyosin and caldesmon between fast- and slow-growing clones. Based on their reported roles in the regulation of cell proliferation and maintenance of cell integrity, we draw an association between their expression and the altered status in which the subpopulations exist. Based on our observations, these proteins may be prospective molecular markers to distinguish between the fast-growing and slow-growing subpopulations. In summary, this study demonstrated the existence of potential stem cells of therapeutic importance in spite of a supposedly smaller stem cell compartment in patients with osteoarthritis. Furthermore, the differentially expressed genes between the sub-populations highlight the 'sternness' of the potential clones, an observation supported by the expression of proteins which act as effective modulators in the maintenance of cell integrity and cell cycle regulation. This study provides a basis for more detailed investigations in search of selective cell surface markers

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Background: The focus of this study was to investigate how producing human bone marrow (hBM) derived mesenchymal stromal cell (MSC) extracellular vehicles (EVs) in a high purity isolation system would affect their established characterization criteria and address the validity of these methods of EV production. Additionally, we set out to functionally characterize the hBM-MSC-EVs for their identified immunomodulatory ability while also assessing the presence of novel MSC-EV marker NRP1 identified by our group to further affirm its validity as a functional MSC-EV identity marker. Methods: Each hBM-MSC-EV donor was cultured in a hollow-fiber bioreactor system in non-stimulating serum/xeno-free conditions for 25 days to produce EVs persistently under quiescent conditions to characterize the hBM-MSC-EVs in their native form. EVs were isolated by traditional PEG-based precipitation for preliminary characterization to monitor bioreactor production wherein they were characterized using multimodal tangential flow filtration coupled with fast protein liquid chromatograph (FPLC) size exclusion/high-affinity purifications to obtain the final highly purified EV sample. Additionally, functional analysis of their immunomodulatory ability, EVs and MSCs were incubated with activated peripheral blood mononuclear cells (PBMCs) as an in-vitro model to evaluate their potency. Results: The hBM-MSC-EVs produced from the bioreactor system showed consistent characterization in accordance with the MISEV2018 establish criteria. We were also able to demonstrate their functional ability by observing statistically significantly immunomodulatory ability of activated PBMCs equivalent to native MSC ability. We were also able to validate the present of NRP1 on all hBM-MSC-EV samples produced confirming its validity as a MSC-EV marker. Conclusion: The significance of the results obtained from this study confirms the production of MSC-EV using a bioreactor and high purity isolation for obtaining consistent MSC-EVs for downstream investigation. Additionally, we were able to demonstrate the significance of MSC-EVs on MSC signaling for immunomodulation by showing equivalent functional potency when tested in-vitro. These results contribute to further understanding the biological attributes of MSC-EVs and contribute to the validation of currently established characterization guidelines.