Lee H Janice, Yu Christopher, Chansakul Thanissara, Varghese Shyni, Hwang Nathaniel S, Elisseeff Jennifer H
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
Stem Cells Dev. 2008 Jun;17(3):555-63. doi: 10.1089/scd.2007.0177.
Enhancing the specific differentiation of pluripotent embryonic stem (ES) cells has been a challenge in the field of tissue engineering. Previously, hepatic cells have been shown to secrete various soluble morphogenic factors to direct mesodermal differentiation of ES cells. In this study, we hypothesized that factors secreted by hepatic cells possess chondrogenic-differentiating effects, and, therefore, the co-culture of hepatic cells would enhance chondrogenesis of ES cells. ES-derived cells(ESDCs) were co-cultured with hepatic cells (HEPA-1C1c7) in three-dimensional bilayered hydrogels. After 3 weeks culture, the histological and biochemical analysis of the HEPA-co-cultured ESDCs revealed a four-fold increase in glycosaminoglycan (GAG) compared to ESDCs cultured alone. This result was supported by real-time PCR analysis, which demonstrated an 80-fold increase in aggrecan expression in co-cultured ESDCs. Additionally, type IIB collagen expression was observed only with co-cultured ESDCs, and immunohistochemical analysis resulted in significantly more positive type II collagen staining with co-cultured ESDCs. Moreover, at day 21, gene expression of other lineages in HEPA-co-cultured ESDCs was either comparable to or lower than those of ESDCs cultured alone. These results indicated that co-culture of ESDCs with hepatic cells significantly enhanced specific chondrogenic differentiation of ESDCs.
增强多能胚胎干细胞（ES细胞）的特异性分化一直是组织工程领域的一项挑战。此前已表明，肝细胞可分泌多种可溶性形态发生因子来引导ES细胞的中胚层分化。在本研究中，我们假设肝细胞分泌的因子具有软骨生成分化作用，因此，肝细胞的共培养将增强ES细胞的软骨生成。将ES衍生细胞（ESDCs）与肝细胞（HEPA-1C1c7）在三维双层水凝胶中共培养。培养3周后，对与肝细胞共培养的ESDCs进行组织学和生化分析，结果显示与单独培养的ESDCs相比，糖胺聚糖（GAG）增加了四倍。实时PCR分析支持了这一结果，该分析表明共培养的ESDCs中聚集蛋白聚糖的表达增加了80倍。此外，仅在共培养的ESDCs中观察到IIB型胶原蛋白表达，免疫组织化学分析显示共培养的ESDCs中II型胶原蛋白染色阳性明显更多。此外，在第21天，与肝细胞共培养的ESDCs中其他谱系的基因表达与单独培养的ESDCs相当或更低。这些结果表明，ESDCs与肝细胞的共培养显著增强了ESDCs的特异性软骨生成分化。
