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Immunophenotypic characterization and tenogenic differentiation of mesenchymal stromal cells isolated from equine umbilical cord blood

Mohanty, Niharika, Gulati, Baldev R., Kumar, Rajesh, Gera, Sandeep, Kumar, Pawan, Somasundaram, Rajesh K., Kumar, Sandeep
In vitro cellular & developmental biology 2014 v.50 no.6 pp. 538-548
blood, collagen, culture media, fibroblasts, flow cytometry, immunocytochemistry, plastics, racehorses, reverse transcriptase polymerase chain reaction, stem cells, stromal cells, tendons, umbilical cord
Mesenchymal stem cells (MSCs) isolated from umbilical cord blood (UCB) in equines have not been well characterized with respect to the expression of pluripotency and mesenchymal markers and for tenogenic differentiation potential in vitro. The plastic adherent fibroblast-like cells isolated from 13 out of 20 UCB samples could proliferate till passage 20. The cells expressed pluripotency markers (OCT4, NANOG, and SOX2) and MSC surface markers (CD90, CD73, and CD105) by RT-PCR, but did not express CD34, CD45, and CD14. On immunocytochemistry, the isolated cells showed expression of CD90 and CD73 proteins, but tested negative for CD34 and CD45. In flow cytometry, CD29, CD44, CD73, and CD90 were expressed by 96.36 ± 1.28%, 93.40 ± 0.70%, 73.23 ± 1.29% and 46.75 ± 3.95% cells, respectively. The UCB-MSCs could be differentiated to tenocytes by culturing in growth medium supplemented with 50 ng/ml of BMP-12 by day 10. The differentiated cells showed the expression of mohawk homeobox (Mkx), collagen type I alpha 1 (Col1α1), scleraxis (Scx), tenomodulin (Tnmd) and decorin (Dcn) by RT-PCR. In addition, flow cytometry detected tenomodulin and decorin protein in 95.65 ± 2.15% and 96.30 ± 1.00% of differentiated cells in comparison to 11.30 ± 0.10% and 19.45 ± 0.55% cells, respectively in undifferentiated control cells. The findings support the observation that these cells may be suitable for therapeutic applications, including ruptured tendons in racehorses.