Department of Biomedical Science, College of Life Science, CHA University, Pochon, 487-010, Korea.
Arch Pharm Res. 2010 Aug;33(8):1253-60. doi: 10.1007/s12272-010-0816-y. Epub 2010 Aug 28.
The optimization of in vitro culture conditions for embryonic stem cells (ESCs) is a matter of critical importance; a prompt supply of a sufficient population of cells that retain their pluripotency capabilities must be secured in order to make possible future cell therapies. Despite a number of reports asserting that a variety of cytokines, signaling ligands, and small molecules can help in maintaining the pluripotency of ESCs, mammalian feeder cells continue to be broadly accepted as the method of choice for ESC cultures. This appears to be because mammalian feeder cells seem to produce some as-yet-unidentified factor that makes them very effective as feeder cells. In this study, we investigated wild-type p53 inducible phosphatase (Wip1), the knockdown of which increases Wnt inhibitory factor-1 expression, in its feeder functions toward mouse embryonic stem cells, lowering the effect of Wnt, one of key signaling in maintaining stemness of ESCs. For this purpose, Wip1 was stably expressed in mouse embryonic fibroblast cell line (STO) using retro-viral gene delivery system and then the function as a feeder cell was monitored either with or without leukemia inhibitory factor (LIF) in culture medium. We demonstrated that mouse embryonic stem cells grown with Wip1 expressing STO showed higher alkaline phosphatase activity and sustained Oct-4 expression level even under LIF deprivation condition compared to both control and Wip1 phosphatase activity dead mutant expressing STO. These results imply that Wip1 phosphatase activity in feeder cells is important to retain pluripotency of mouse embryonic stem cells under LIF deprivation conditions. These results indicate that genetically engineered feeder cells such as Wip1 expressing cell lines, are alternative strategy for the optimization of maintenance and expansion of mouse embryonic stem cells.
优化胚胎干细胞（ESC）的体外培养条件至关重要；必须迅速提供足够数量的保持其多能性的细胞，以便将来进行细胞治疗。尽管有许多报道称，各种细胞因子、信号配体和小分子有助于维持 ESC 的多能性，但哺乳动物饲养细胞仍然被广泛认为是 ESC 培养的首选方法。这似乎是因为哺乳动物饲养细胞似乎产生了一些尚未确定的因子，使它们作为饲养细胞非常有效。在这项研究中，我们研究了野生型 p53 诱导磷酸酶（Wip1），其敲低会增加 Wnt 抑制因子-1 的表达，从而降低 Wnt 的作用，Wnt 是维持 ESC 干性的关键信号之一。为此，我们使用逆转录病毒基因传递系统将 Wip1 稳定表达在小鼠胚胎成纤维细胞系（STO）中，然后在培养基中有无白血病抑制因子（LIF）的情况下监测其作为饲养细胞的功能。我们证明，与对照和表达 Wip1 磷酸酶活性缺失突变体的 STO 相比，在 LIF 缺乏条件下生长的表达 Wip1 的 STO 饲养的小鼠胚胎干细胞表现出更高的碱性磷酸酶活性和持续的 Oct-4 表达水平。这些结果表明，饲养细胞中的 Wip1 磷酸酶活性对于在 LIF 缺乏条件下维持小鼠胚胎干细胞的多能性很重要。这些结果表明，表达 Wip1 的工程化饲养细胞等遗传工程化饲养细胞是优化维持和扩增小鼠胚胎干细胞的替代策略。
