Conclusions
The protocol for isolation of ovine foetal bone marrow derived MSCs was simple to perform, and the cultural method of obtaining pure spindle morphology cells was established. Criteria proposed for defining MSCs by this study includes the cell adherence to culture plates, specific surface protein profiles and differentiation to osteogenic lineage. The MSCs and osteogenic differentiated cells in this ovine animal model may serve as a large source for stem cell applications in regenerative medical therapies.
Methods
Isolation and culture of sheep foetal bone marrow cells were done and uniform clonally derived MSC population was collected. The cells were characterized using cytochemical, immunophenotyping, biochemical and molecular analyses. The cells with defined characteristics were differentiated into osteogenic lineages and analysis for differentiated cell types was done. The cells were analyzed for cell surface marker expression and the gene expression in undifferentiated and differentiated osteoblast was checked by reverse transcriptase PCR (RT PCR) analysis and confirmed by sequencing using genetic analyzer.
Results
Ovine foetal samples were processed to obtain mononuclear (MNC) cells which on culture showed spindle morphology, a characteristic oval body with the flattened ends. MSC population CD45⁻/CD14⁻ was cultured by limiting dilution to arrive at uniform spindle morphology cells and colony forming units. The cells were shown to be positive for surface markers such as CD44, CD54, integrinβ1, and intracellular collagen type I/III and fibronectin. The osteogenically induced MSCs were analyzed for alkaline phosphatase (ALP) activity and mineral deposition. The undifferentiated MSCs expressed RAB3B, candidate marker for stemness in MSCs. The osteogenically induced and uninduced MSCs expressed collagen type I and MMP13 gene in osteogenic induced cells. Interpretation & conclusions: The protocol for isolation of ovine foetal bone marrow derived MSCs was simple to perform, and the cultural method of obtaining pure spindle morphology cells was established. Criteria proposed for defining MSCs by this study includes the cell adherence to culture plates, specific surface protein profiles and differentiation to osteogenic lineage. The MSCs and osteogenic differentiated cells in this ovine animal model may serve as a large source for stem cell applications in regenerative medical therapies.