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

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Abstract

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.

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Acknowledgments

The authors wish to thank Remount Veterinary Services and Equine Breeding Stud, Hisar, Haryana, India for providing access for sampling. We thank NRCE, Hisar for infrastructure support. Financial support to Niharika from Indian Council of Agricultural Research (ICAR) and to Baldev R Gulati from Department of Biotechnology, Government of India is duly acknowledged. The assistance in flow cytometry by BD-FACS academy, New Delhi is duly acknowledged.

Conflict of interest

None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.

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Authors and Affiliations

  1. Department of Veterinary Physiology and Biochemistry, College of Veterinary Sciences, LLR University of Veterinary & Animal Sciences, Hisar, 25004, Haryana, India

    Niharika Mohanty, Sandeep Gera & Sandeep Kumar

  2. National Research Centre on Equines, Sirsa Road, Hisar, 125001, Haryana, India

    Baldev R. Gulati & Rajesh Kumar

  3. Department of Veterinary Anatomy, College of Veterinary Sciences, LLR University of Veterinary & Animal Sciences, Hisar, 125004, Haryana, India

    Pawan Kumar

  4. Equine Breeding Stud, Hisar, 125001, Haryana, India

    Rajesh K. Somasundaram

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  1. Niharika Mohanty
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  2. Baldev R. Gulati
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Correspondence to Baldev R. Gulati.

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Mohanty, N., Gulati, B.R., Kumar, R. et al. Immunophenotypic characterization and tenogenic differentiation of mesenchymal stromal cells isolated from equine umbilical cord blood. In Vitro Cell.Dev.Biol.-Animal 50, 538–548 (2014). https://doi.org/10.1007/s11626-013-9729-7

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