Comparison of cell isolates within anatomical regions of the ovine umbilical cord for improved isolation of putative mesenchymal stem cells.

The umbilical cord is a source of mesenchymal stem cells (MSCs) for potential MSC therapy but studies isolating MSCs from human cords have shown highly variable results leading to the establishment of the ISCT criteria for defining human MSCs. MSC therapy has achieved early successes in veterinary regenerative medicine but still the lack of standardized characterization is problematic and researchers have attempted to use the ISCT human criteria to define animal MSCs. However, the unique characteristics of animal-derived MSCs made the use of human guidelines difficult leading to the proposal of modified guidelines tailored to veterinary medicine. To address the potential variability arising from anatomical region contamination, we carefully dissected different regions of sheep Wharton's jelly prior to expansion of putative MSCs contained within. The morphology of cells derived demonstrated the benefit of pre-dissection of Wharton's jelly due to the absence of contaminating cells from other regions. Following cell expansion, we used the proposed guidelines for animal-MSCs to characterize the isolated cells. In the absence of species-specific antibodies for reliable detection of marker proteins, we utilized ddPCR to determine accurate gene expression analysis of MSC positive markers CD73, CD90 and CD105 and negative markers CD34, and CD45 utilizing sheep specific primer-probe assays. The results were indicative of MSC phenotype alone in sheep Wharton's jelly isolates, with no evidence of cellular contamination from other regions. Moreover, the putative MSCs isolated from different regions of sheep umbilical Wharton's jelly were not significantly different by ddPCR analysis of positive and negative MSC markers.