OBJECTIVE To produce a clonal equine myoblast cell line that retains the ability to divide for multiple passages and differentiate into multinucleated myotubes during specific conditions. SAMPLE Cultured primary equine skeletal muscle-derived cells from a healthy Thoroughbred. PROCEDURES Cell cultures were transfected by electroporation with a plasmid (pNIT) that expresses the temperature-sensitive simian vacuolating virus 40 large T antigen (TAg), which can be controlled by a doxycycline-responsive promoter. Cells that stably integrated the TAg were selected and expanded to passage 25. For each passage, differentiation and fusion properties of the cells were determined and immunocytochemical analyses were performed to evaluate expression of TAg and other muscle-specific proteins. Optimum conditions that led to cell differentiation into myotubes were also determined. RESULTS Compared with nontransfected control cells, myogenic, desmin-positive cells expressed the TAg when incubated at 33°C and could be maintained in culture for numerous passages. Reduced expression of TAg was identified in cells incubated at 37°C or when incubated with doxycycline at 33°C. Expression of TAg was not detected when cells were incubated with doxycycline at 37°C, and when serum was withdrawn from the culture medium, those clones differentiated into a pure population of multinucleated myotubes. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that production of an immortalized clonal equine skeletal muscle cell line was possible. A clonal equine skeletal muscle cell line will be a valuable in vitro tool for use in equine physiology and disease research.

OBJECTIVE To assess efficiency of gravity filtration to enhance recovery of equine bone marrow elements including stem and progenitor cells. ANIMALS 12 healthy adult horses. PROCEDURES Bone marrow aspirates were collected from the fifth sternebral body and filtered by gravitational flow to obtain bone marrow elements. Raw and harvested bone marrow and marrow effluent were evaluated for WBC and platelet counts, automated and cytomorphologic cell differential counts, mesenchymal stem cell CFUs, cell viability, and differentiation capacity. Isolated cells were analyzed for CD90 and major histocompatibility complex (MHC) class I and II antigens. RESULTS Mean cell viability of harvested bone marrow was 95.9%. Total WBCs and platelets were efficiently captured on the filter (> 95%), and mean recovery in harvested bone marrow was 30%. Cytologic cell differential counts indicated that the percentage of neutrophils was significantly less and the progenitor cell population was significantly higher and concentrated 1.56-fold in harvested bone marrow, compared with results for raw bone marrow. Flow cytometry and cell culture were used to characterize harvested bone marrow cells as positive for expression of CD90 and negative for MHCI and MHCII, which indicated stem cells with a multipotent phenotype that differentiated into chondrocytes, osteocytes, adipocytes, and tenocytes. CONCLUSIONS AND CLINICAL RELEVANCE Gravitational filtration of bone marrow efficiently yielded platelets and cells and produced a progenitor-enriched, leukocyte-reduced product, compared with raw bone marrow.

OBJECTIVE To compare the characteristics and multipotential and in vivo cartilage formation capabilities of porcine adipose-derived stem cells (pASCs) with those of porcine skin-derived stem cell–like cells (pSSCs). ANIMALS Three 6-month-old female pigs and four 6-week-old female athymic mice. PROCEDURES Adipose and skin tissue specimens were obtained from each pig following slaughter and digested to obtain pASCs and pSSCs. For each cell type, flow cytometry and reverse transcription PCR assays were performed to characterize the expression of cell surface and mesenchymal stem cell markers, and in vitro cell cultures were performed to determine the adipogenic, osteogenic, and chondrogenic capabilities. Each cell type was then implanted into athymic mice to determine the extent of in vivo cartilage formation after 6 weeks. RESULTS The cell surface and mesenchymal stem cell marker expression patterns, multipotential capability, and extent of in vivo cartilage formation did not differ significantly between pASCs and pSSCs. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that pSSCs may be a viable alternative to pASCs as a source of progenitor cells for tissue engineering in regenerative medicine.