Comparison of the osteogenic potential of equine mesenchymal stem cells from bone marrow, adipose tissue, umbilical cord blood, and umbilical cord tissue.

OBJECTIVE: To determine the optimal osteogenic source of equine mesenchymal stem cells (eMSCs) and optimize collection of and expansion conditions for those cells. ANIMALS: 10 adult Quarter Horses and 8 newborn Thoroughbred foals. PROCEDURES: eMSCs were isolated from bone marrow (BM), adipose tissue, and umbilical cord blood and tissue, and the osteogenic potential of each type was assessed. Effects of anatomic site, aspiration volume, and serum type on eMSC yield from BM were investigated. RESULTS: BM-eMSCs had the highest overall expression of the osteogenic genes Cbfa1, Osx, and Omd and staining for ALP activity and calcium deposition. There was no significant difference in BM-eMSC yield from the tuber coxae or sternum, but yield was significantly greater from the first 60-mL aspirate than from subsequent aspirates. The BM-eMSC expansion rate was significantly higher when cells were cultured in fetal bovine serum instead of autologous serum (AS). CONCLUSIONS AND CLINICAL RELEVANCE: eMSCs from BM possessed the highest in vitro osteogenic potential; eMSCs from adipose tissue also had robust osteogenic potential. The tuber coxae and the sternum were viable sources of BM-eMSCs in yearlings, and 60 mL of BM aspirate was sufficient for culture and expansion. Expanding BM-eMSCs in AS to avoid potential immunologic reactions decreased the total yield because BM-eMSCs grew significantly slower in AS than in fetal bovine serum. Additional studies are needed to determine optimal ex vivo eMSC culture and expansion conditions, including the timing and use of growth factor­supplemented AS.

Osteogenic comparison of expanded and uncultured adipose stromal cells.

BACKGROUND AIMS: Adipose stromal cells (ASC) are a promising alternative to progenitor cells from other tissue compartments because of their multipotential and capacity to retrieve significantly more progenitor cells. Initial cell samples are heterogeneous, containing a collection of cells that may contribute to tissue repair, but the sample becomes more homogeneous with each passage. Therefore, we hypothesized that the osteogenic potential of culture-expanded ASC would differ from uncultured ASC. METHODS: Adipose tissue was collected from a yearling colt, and ASC were isolated and expanded using standard protocols or prepared by a commercial vendor using proprietary technology (proprietary stromal vascular fraction, SVFp). Cells were seeded on collagen sponges and maintained in osteogenic culture conditions for up to 21 days to assess osteogenic potential. The ability of each population to stimulate neovascularization and bone healing was determined upon implanting cell-loaded sponges into a rodent calvarial bone defect. Neovascularization was measured 3 weeks post-implantation, while bone formation was monitored over 12 weeks using in vivo microcomputed tomography (microCT). RESULTS: SVFp exhibited increased intracellular alkaline phosphatase activity compared with cultured ASC but proliferated minimally. Histologic analysis of explanted tissues demonstrated greater vascularization in defects treated with cultured ASC compared with SVFp. We detected increases in bone volume for defects treated with cultured cells while observing similar values for bone mineral density, regardless of cell type. CONCLUSIONS: These results suggest that expanded ASC are advantageous for neovascularization and bone healing in this model compared with SVFp, and provide additional evidence of the utility of ASC in bone repair.

Oxygen tension modulates neurite outgrowth in PC12 cells through a mechanism involving HIF and VEGF.

Cell-based approaches are a promising therapeutic strategy for treating injuries to the nervous system, but the optimal means to promote neurite extension and direct cellular behavior are unclear. Previous studies have examined the behavior of neural-like cells in ambient air (21% oxygen tension), yet these conditions are not representative of the physiological oxygen microenvironment of neural tissues. We hypothesized that neuronal differentiation of a model neural cell line (PC12) could be controlled by modulating local oxygen tension. Compared to ambient conditions, PC12 cells cultured in reduced oxygen exhibited significant increases in neurite extension and total neurite length, with 4% oxygen yielding the highest levels of both indicators. We confirmed neurite extension was mediated through oxygen-responsive mechanisms using small molecules that promote or inhibit HIF-1alpha stabilization. The hypoxic target gene Vegf was implicated as a neurotrophic factor, as neurite formation at 21% oxygen was mimicked with exogenous VEGF, and a VEGF-neutralizing antibody attenuated neurite formation under reduced oxygen conditions. These findings demonstrate that behavior of neural-like cells is driven by the oxygen microenvironment via VEGF function, and suggest promising approaches for future applications in neural repair.