Mohanty Niharika, Gulati Baldev R, Kumar Rajesh, Gera Sandeep, Kumar Pawan, Somasundaram Rajesh K, Kumar Sandeep
Department of Veterinary Physiology and Biochemistry, College of Veterinary Sciences, LLR University of Veterinary & Animal Sciences, Hisar, 25004, Haryana, India.
In Vitro Cell Dev Biol Anim. 2014 Jun;50(6):538-48. doi: 10.1007/s11626-013-9729-7. Epub 2014 Jan 11.
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, respect vely in undifferentiated control cells. The findings support the observation that these cells may be suitable for therapeutic applications, including ruptured tendons in racehorses.
从马脐带血（UCB）中分离出的间充质干细胞（MSC）在多能性和间充质标志物表达以及体外成腱分化潜能方面尚未得到充分表征。从20份UCB样本中的13份分离出的贴壁成纤维细胞样细胞可增殖至第20代。通过逆转录聚合酶链反应（RT-PCR），这些细胞表达多能性标志物（OCT4、NANOG和SOX2）和MSC表面标志物（CD90、CD73和CD105），但不表达CD34、CD45和CD14。免疫细胞化学检测显示，分离出的细胞表达CD90和CD73蛋白，但CD34和CD45检测为阴性。在流式细胞术中，分别有96.36±1.28%、93.40±0.70%、73.23±1.29%和46.75±3.95%的细胞表达CD29、CD44、CD73和CD90。通过在补充有50 ng/ml BMP-12的生长培养基中培养10天，UCB-MSCs可分化为肌腱细胞。通过RT-PCR，分化后的细胞显示出莫霍克同源框（Mkx）、I型胶原α1（Col1α1）、硬骨素（Scx）、肌腱调节蛋白（Tnmd）和核心蛋白聚糖（Dcn）的表达。此外，流式细胞术检测到，与未分化的对照细胞中分别为11.30±0.10%和19.45±0.55%的细胞相比，95.65±2.15%和96.30±1.00%的分化细胞中存在肌腱调节蛋白和核心蛋白聚糖。这些发现支持了以下观察结果：这些细胞可能适用于治疗应用，包括赛马的肌腱断裂。
