Isolation, characterization, and in vitro proliferation of canine bone marrow, adipose tissue, muscle, and periosteum-derived mesenchymal stem cells

Objective: To isolate and characterize mesenchymal stem cells (MSCs) from canine muscle and periosteum, and to compare the proliferative capacity of bone marrow, adipose tissue, muscle, and periosteum-derived MSCs (BMSCs, AMSCs, MMSCs, and PMSCs, respectively).

Sample Population: Seven canine cadavers.

Procedures: Characterization of MSCs was based on their plastic adherence and morphology, immunofluorescence of MSC-associated cell surface markers, and expression of pluripotency-associated transcription factors. Morphological and histochemical methods were used to evaluate differentiation of MSCs cultured in adipogenic, osteogenic, and chondrogenic media. Passage one MSCs, cultured in triplicate, were counted at 24, 48, 72, and 96 hours to determine tissue specific-MSC proliferative capacity. Mesenchymal stem cell yield/gram of tissue was calculated for confluent passage one MSCs.

Results: Successful isolation of BMSCs, AMSCs, MMSCs, and PMSCs was based on their plastic adherence and morphology, positive expression of CD44 and CD90, negative expression of CD34, CD45, and CD 146, mRNA expression of SOX2, OCT4, and NANOG, and adipogenic and osteogenic differentiation. The proliferative capacity was not significantly different between BMSCs, AMSCs, MMSCs, and PMSCs over a four day culture period. However, periosteum provided a significantly higher MSC yield/gram of tissue once confluent in passage one (mean ± SD of 19,400,000 ± 12,800,000 of PMSCs/gram of periosteum obtained in a mean ± SD of 13 ± 1.64 days).

Conclusions and Clinical Relevance: Canine muscle and periosteum are sources of MSCs. Periosteum is a superior tissue source for MSC yield, and may be useful in allogenic applications.