Fortier L A, Nixon A J, Williams J, Cable C S
Comparative Orthopaedics Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
Am J Vet Res. 1998 Sep;59(9):1182-7.
To isolate mesenchymal stem cells from adult horses and determine specific monolayer culture conditions required to enhance biochemically and phenotypically defined chondrocytic differentiation.
2 adult horse bone marrow donors without skeletal or hematologic abnormalities.
Bone marrow was aspirated from the sternebra, and mesenchymal stem cells were isolated by centrifugation and cultured in monolayers. Subcultures were established in 24-well plates on day 13. Culture medium was harvested every 2 days, and culture of 12 of the 24 wells was terminated on day 6 and of the remaining wells on day 12. Medium proteoglycan content was determined for all samples, and proteoglycan monomeric size was determined for pooled samples from days 2-6 and 8-12. Total nucleated cell numbers were determined at culture termination on days 6 and 12. Histologic, histochemical, and collagen immunohistochemical analyses of multiwell chamber slides harvested on day 6 or 12 were performed.
Mesenchymal cells were an abundant cellular constituent of bone marrow aspirates, and separation of hematopoietic elements was achieved by centrifugation and delayed medium exchange. The remaining mesenchymal stem cells progressed from large, spindyloid, fibroblastic-appearing cells to a rounder shaped cell which formed colony plaques; isolated cells remained more spindyloid. Mesenchymal cell transformation toward a chondrocytic phenotype was verified by a shift in expression from collagen type I to type II, and an increase in quantity and molecular size of proteoglycans synthesized over time.
Mesenchymal stem cells obtained from adult horses have the capacity to undergo chondrogenic differentiation in monolayer cultures and may provide a locally recruitable or transplantable autogenous cell source for articular cartilage repair.
从成年马中分离间充质干细胞，并确定增强生化和表型定义的软骨细胞分化所需的特定单层培养条件。
2 匹无骨骼或血液学异常的成年马骨髓供体。
从胸骨抽吸骨髓，通过离心分离间充质干细胞并进行单层培养。在第 13 天建立传代培养于 24 孔板中。每 2 天收集培养基，24 孔中的 12 孔培养在第 6 天终止，其余孔在第 12 天终止。测定所有样品的培养基蛋白聚糖含量，并测定第 2 - 6 天和第 8 - 12 天合并样品的蛋白聚糖单体大小。在第 6 天和第 12 天培养终止时测定总核细胞数。对在第 6 天或第 12 天收获的多孔腔室玻片进行组织学、组织化学和胶原免疫组织化学分析。
间充质细胞是骨髓抽吸物中丰富的细胞成分，通过离心和延迟培养基更换实现造血成分的分离。剩余的间充质干细胞从大的、梭形的、成纤维细胞样细胞发展为更圆形的细胞，形成集落斑块；分离的细胞仍更呈梭形。间充质细胞向软骨细胞表型的转变通过从 I 型胶原到 II 型胶原表达的转变以及随着时间推移合成的蛋白聚糖数量和分子大小的增加得到证实。
从成年马获得的间充质干细胞有能力在单层培养中进行软骨形成分化，并可能为关节软骨修复提供局部可募集或可移植的自体细胞来源。
