Fülber Joice, Maria Durvanei A, da Silva Luis Cláudio Lopes Correia, Massoco Cristina O, Agreste Fernanda, Baccarin Raquel Y Arantes
Department of Internal Medicine, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), Avenida Prof. Orlando Marques de Paiva, 87, 05508-270, São Paulo, SP, Brazil.
Laboratory of Biochemistry and Biophysics, Butantan Institute, Avenida Vital Brasil 1500, São Paulo, 05503-900, SP, Brazil.
Stem Cell Res Ther. 2016 Mar 5;7:35. doi: 10.1186/s13287-016-0294-3.
Bone marrow and adipose tissues are known sources of mesenchymal stem cells (MSCs) in horses; however, synovial tissues might be a promising alternative. The aim of this study was to evaluate phenotypic characteristics and differentiation potential of equine MSCs from synovial fluid (SF) and synovial membrane (SM) of healthy joints (SF-H and SM-H), joints with osteoarthritis (SF-OA and SM-OA) and joints with osteochondritis dissecans (SF-OCD and SM-OCD) to determine the most suitable synovial source for an allogeneic therapy cell bank.
Expression of the markers CD90, CD105, CD44, and CD34 in SF-H, SM-H, SF-OA, SM-OA, SF-OCD and SM-OCD was verified by flow cytometry, and expression of cytokeratin, vimentin, PGP 9.5, PCNA, lysozyme, nanog, and Oct4 was verified by immunocytochemistry. MSCs were cultured and evaluated for their chondrogenic, osteogenic and adipogenic differentiation potential. Final quantification of extracellular matrix and mineralized matrix was determined using AxioVision software. A tumorigenicity test was conducted in Balb-C(nu/nu) mice to verify the safety of the MSCs from these sources.
Cultured cells from SF and SM exhibited fibroblastoid morphology and the ability to adhere to plastic. The time elapsed between primary culture and the third passage was approximately 73 days for SF-H, 89 days for SF-OCD, 60 days for SF-OA, 68 days for SM-H, 57 days for SM-OCD and 54 days for SM-OA. The doubling time for SF-OCD was higher than that for other cells at the first passage (P < 0.05). MSCs from synovial tissues showed positive expression of the markers CD90, CD44, lysozyme, PGP 9.5, PCNA and vimentin and were able to differentiate into chondrogenic (21 days) and osteogenic (21 days) lineages, and, although poorly, into adipogenic lineages (14 days). The areas staining positive for extracellular matrix in the SF-H and SM-H groups were larger than those in the SF-OA and SM-OA groups (P < 0.05). The positive mineralized matrix area in the SF-H group was larger than those in all the other groups (P < 0.05). The studied cells exhibited no tumorigenic effects.
SF and SM are viable sources of equine MSCs. All sources studied provide suitable MSCs for an allogeneic therapy cell bank; nevertheless, MSCs from healthy joints may be preferable for cell banking purposes because they exhibit better chondrogenic differentiation capacity.
骨髓和脂肪组织是马间充质干细胞（MSCs）的已知来源；然而，滑膜组织可能是一个有前景的替代来源。本研究的目的是评估来自健康关节（SF-H和SM-H）、骨关节炎关节（SF-OA和SM-OA）以及剥脱性骨软骨炎关节（SF-OCD和SM-OCD）的滑液（SF）和滑膜（SM）中马MSCs的表型特征和分化潜能，以确定同种异体治疗细胞库最合适的滑膜来源。
通过流式细胞术验证SF-H、SM-H、SF-OA、SM-OA、SF-OCD和SM-OCD中标志物CD90、CD105、CD44和CD34的表达，并通过免疫细胞化学验证细胞角蛋白、波形蛋白、PGP 9.5、增殖细胞核抗原（PCNA）、溶菌酶、纳米基因（nanog）和八聚体结合转录因子4（Oct4）的表达。培养MSCs并评估其成软骨、成骨和成脂分化潜能。使用AxioVision软件对细胞外基质和矿化基质进行最终定量。在Balb-C(nu/nu)小鼠中进行致瘤性试验，以验证这些来源的MSCs的安全性。
来自SF和SM的培养细胞呈现成纤维细胞样形态并具有贴壁能力。原代培养至第三次传代的时间，SF-H约为73天，SF-OCD为89天，SF-OA为60天，SM-H为68天，SM-OCD为57天，SM-OA为54天。SF-OCD在第一代时倍增时间高于其他细胞（P < 0.０5）。滑膜组织来源的MSCs显示标志物CD90、CD44、溶菌酶、PGP 9.5、PCNA和波形蛋白呈阳性表达，并能够分化为成软骨谱系（21天）和成骨谱系（21天），虽然分化为脂肪谱系的能力较弱（14天）。SF-H和SM-H组中细胞外基质染色阳性区域大于SF-OA和SM-OA组（P < 0.05）。SF-H组中矿化基质阳性区域大于所有其他组（P < 0.05）。所研究的细胞未表现出致瘤作用。
SF和SM是马MSCs的可行来源。所有研究的来源都能为同种异体治疗细胞库提供合适的MSCs；然而，健康关节来源的MSCs可能更适合用于细胞库保存，因为它们表现出更好的成软骨分化能力。
