Nakagawa Toshiyuki, Lee Sang Yang, Reddi A Hari
University of California-Davis, Sacramento, CA, USA.
Arthritis Rheum. 2009 Dec;60(12):3686-92. doi: 10.1002/art.27229.
Human embryonic stem cells (ESCs) provide an unlimited supply of pluripotent cells for articular cartilage tissue engineering and regenerative medicine applications. Articular cartilage is an avascular tissue with precise polarity and organization comprising 3 distinct functional zones: surface, middle, and deep. To date, attempts at differentiating human ESCs into articular chondrocytes have been unsuccessful. The majority of studies have focused on chondrogenic (but not specifically articular cartilage) differentiation. Furthermore, previous investigations of induction of chondrogenesis by human ESCs required embryoid body formation; however, embryoid body formation often results in heterogeneous differentiation. The present study was undertaken to determine the in vitro chondrogenic potential of bone morphogenetic protein 7 (BMP-7) and transforming growth factor beta1 (TGFbeta1)-induced human ESC differentiation toward the articular cartilage phenotype.
Dissociated single human ESCs were cultured and passaged on a gelatin-coated flask. The human ESCs were cultured as an aggregate in a pellet culture system for 14 days in basal chondrogenic medium (CM), CM with TGFbeta1, CM with BMP-7, or CM with both TGFbeta1 and BMP-7.
The size and wet weight of the cartilage pellets and glycosaminoglycan levels increased, with the smallest, intermediate, and greatest increases, respectively, observed with CM plus TGFbeta1 treatment, CM plus BMP-7 treatment, and CM plus TGFbeta1 and BMP-7 treatment (compared with CM treatment alone). The largest size and highest weight of the pellet was in the group in which TGFbeta1 and BMP-7 were added to the medium. However, expression of the genes for cartilage-specific aggrecan and type II collagen II, as assessed by determination of messenger RNA levels, was highest in the BMP-7-treated group. Superficial zone protein (SZP)/lubricin, a marker of the superficial zone articular chondrocyte, was not detectable under identical culture conditions.
These results demonstrate an efficient and reproducible model system of human ESC-induced chondrogenesis, using a novel direct plating method in which intervening embryoid body formation does not occur. Further work is needed for optimization of conditions to obtain the articular cartilage phenotype that includes the superficial zone marker as demonstrated by SZP/lubricin synthesis.
人类胚胎干细胞（ESCs）为关节软骨组织工程和再生医学应用提供了无限的多能细胞来源。关节软骨是一种无血管组织，具有精确的极性和组织结构，由三个不同的功能区组成：表层、中层和深层。迄今为止，将人类胚胎干细胞分化为关节软骨细胞的尝试均未成功。大多数研究集中在软骨形成（但不是特异性关节软骨）分化上。此外，先前关于人类胚胎干细胞诱导软骨形成的研究需要形成胚状体；然而，胚状体的形成往往导致异质性分化。本研究旨在确定骨形态发生蛋白7（BMP - 7）和转化生长因子β1（TGFβ1）诱导人类胚胎干细胞向关节软骨表型分化的体外软骨形成潜能。
将解离的单个人类胚胎干细胞在明胶包被的培养瓶中培养并传代。人类胚胎干细胞在沉淀培养系统中聚集成团培养14天，培养于基础软骨形成培养基（CM）、添加TGFβ1的CM、添加BMP - 7的CM或同时添加TGFβ1和BMP - 7的CM中。
软骨沉淀的大小、湿重和糖胺聚糖水平均增加，分别在CM加TGFβ1处理组、CM加BMP - 7处理组和CM加TGFβ1和BMP - 7处理组（与单独CM处理相比）观察到最小、中等和最大程度的增加。添加TGFβ1和BMP - 7的培养基组沉淀的大小最大、重量最高。然而，通过测定信使核糖核酸水平评估，软骨特异性聚集蛋白聚糖和II型胶原蛋白基因的表达在BMP - 7处理组中最高。在相同培养条件下未检测到表层区蛋白（SZP）/润滑素，这是表层区关节软骨细胞的标志物。
这些结果证明了一种高效且可重复的人类胚胎干细胞诱导软骨形成的模型系统，采用了一种新的直接接种方法，其中不发生中间胚状体的形成。需要进一步优化条件以获得包括由SZP/润滑素合成所证明的表层区标志物的关节软骨表型。
