Isolation and characterisation of novel stromal cell populations from human bone marrow
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Mesenchymal stem cells (MSCs) hold great promise for application in the field of regenerative medicine due to their multipotent capacity and immuno-regulatory potential. Paramount to their implementation in the clinic is adherence to EU and British regulatory guidelines requiring a clear definition of the previously heterogenic MSC population. Single cell cloning studies have revealed the presence of cell subpopulations with defined differentiation potential confirming that within the bone marrow reside cell subtypes with lineage-specific differentiation capacities. Isolation of these cell subtypes may provide an opportunity to apply defined cell populations to specific clinical therapies. Isolation of an osteo/chondro progenitor based on cell surface marker expression will allow for specific application in orthobiologic therapies. The cell surface proteins tissue non-specific alkaline phosphatase (TNAP) and syndecan-2 (SDC2) are expressed on bone marrow MSCs and in tissues of mesenchymal descent and therefore are highlighted as prime candidates for assessment. Therefore, the aim of this thesis was to isolate MSCs from human bone marrow using magnetic-activated cell sorting (MACS) and fluorescent-activated cell sorting (FACS) and characterise the resultant subpopulations based on expression of TNAP and SDC2. MACS of TNAP-expressing (TNAPEnr) MSCs revealed that in addition to enriching for the colony-forming unit fibroblasts (CFU-Fs), characteristic of MSCs, TNAPEnr cells maintained a proliferative capacity, cell surface antigen expression, immuno-suppressive properties and differentiation potential comparable to Parent, unseparated controls. TNAP transcript expression increased during both osteogenic and adipogenic differentiation. While TNAP expression may be used as a tool for MSC isolation, selection using MACS was not appropriate due to a methodology-associated increase in cell death. Therefore, fluorescent activated cell sorting (FACS) was investigated as an alternative isolation technique. Bone marrow mononuclear cells were isolated by FACS based on expression of TNAP and SDC2. SDC2 is a cell surface proteoglycan, involved in angiogenesis, with functional roles in embryonic developmental processes. Four cell subpopulations TNAP-SDC2-, TNAP+SDC2+, TNAP+SDC2-, and TNAP-SDC2+ (T-S-, T+S+, T+S-, and T-S+, respectively) were isolated. Only T+ populations adhered to tissue culture plastic and formed colonies. Both T+S- and T+S+ isolations enriched for CFU-Fs, 1:70 and 1:24 respectively, and maintained cell surface antigen expression and immuno-suppressive potential compared to Parent MSCs. Both selected populations also exhibited greater proliferation potential. While T+S- and T+S+ MSCs showed equivalent osteogenic and chondrogenic differentiation capacity to Parent MSCs, T+S+ cells showed significantly reduced adipogenic potential, indicating T+S+ is an osteo/chondral specific subpopulation. SDC2 transcript expression during differentiation demonstrated no regulated expression during adipogenesis, significant down-regulation on initiation of osteogenesis, and at day 14 of chondrogenesis. To determine if T+S+ cells retain enhanced osteogenic capacity after implantation, subcutaneous bone formation assays were conducted using the Parent, T+S-, and T+S+ cells in combination with hydroxyapatite/tri-calcium phosphate scaffolds. Although the metabolic activity of the cells was maintained upon loading and their distribution through the scaffold was uniform, bone formation was not observed after 8 weeks in vivo. Minimal indications of bone formation was present in the Parent control samples, however both T+S- and T+S+ loaded samples were devoid of bone. Passage 0-2 or osteogenically-primed MSCs were used in the majority of published in vivo bone studies. The negative results described in this study may be reflective of implantation of un-primed, passage 5 MSCs with significantly higher cumulative population doublings of both T+S- and T+S+ cells. Therefore, while T+ expression identifies the CFU-F population from BM, SDC2 expression further divides progenitors into subpopulations with in vitro tri-potent and bi-potent capacities. The in vivo potential of these cell populations, however, remains to be elucidated with the development of more robust assays. This study advances the-state-of the-art by identifying MSC markers which can be utilised as a means to isolate osteochondral progenitors for applications in orthobiologic therapies. The identification of a progenitor population with specific osteo/chondral potential provides a unique tool for applications in orthobiologic therapies and therefore advances the state-of-the-art in MSC selection.
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