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

Niharika Mohanty; Baldev R. Gulati; Rajesh Kumar; Sandeep Gera; Pawan Kumar; Rajesh K. Somasundaram; Sandeep Kumar

doi:10.1007/s11626-013-9729-7

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

Niharika Mohanty, Baldev R. Gulati, Rajesh Kumar, Sandeep Gera, Pawan Kumar, Rajesh K. Somasundaram, Sandeep Kumar

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36 Scopus citations

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.

Original language	English
Pages (from-to)	538-548
Number of pages	11
Journal	In Vitro Cellular and Developmental Biology - Animal
Volume	50
Issue number	6
DOIs	https://doi.org/10.1007/s11626-013-9729-7
State	Published - Jun 2014
Externally published	Yes

Bibliographical note

Funding Information:
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.

Keywords

Horsemesenchymal stromal cell
Pluripotency
Tenogenic differentiation
Umbilical cord blood

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10.1007/s11626-013-9729-7

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title = "Immunophenotypic characterization and tenogenic differentiation of mesenchymal stromal cells isolated from equine umbilical cord blood",

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.",

keywords = "Horsemesenchymal stromal cell, Pluripotency, Tenogenic differentiation, Umbilical cord blood",

author = "Niharika Mohanty and Gulati, {Baldev R.} and Rajesh Kumar and Sandeep Gera and Pawan Kumar and Somasundaram, {Rajesh K.} and Sandeep Kumar",

note = "Funding Information: 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.",

year = "2014",

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AU - Mohanty, Niharika

AU - Gulati, Baldev R.

AU - Kumar, Rajesh

AU - Gera, Sandeep

AU - Kumar, Pawan

AU - Somasundaram, Rajesh K.

AU - Kumar, Sandeep

N1 - Funding Information: 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.

PY - 2014/6

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N2 - 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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Immunophenotypic characterization and tenogenic differentiation of mesenchymal stromal cells isolated from equine umbilical cord blood

Abstract

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Keywords

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