Laboratories of Cell Function Regulation, Korea University, Seoul 136-701, Korea.
Stem Cells. 2010 May;28(5):874-84. doi: 10.1002/stem.412.
The self-renewal of embryonic stem cells involves a balance between processes governed by crosstalk between intrinsic and extrinsic factors. We hypothesized that protein serine/threonine phosphatase 2A (PP2A) may play a central role in the signaling pathways that regulate human embryonic stem cell (hESC) self-renewal. Biochemical analyses revealed that PP2A activity gradually increases over the course of hESC differentiation; PP2A/C and PP2A/A levels also increased. The overexpression of PP2A/C or the addition of PP2A activator C2-ceramide promoted hESC differentiation. Accordingly, the addition of PP2A inactivator okadaic acid (OA) maintained hESC self-renewal in the absence of basic fibroblast growth factor (bFGF). The hESCs maintained with OA expressed pluripotency markers and exhibited substantial telomerase activity with normal karyotypes. The hESCs were able to differentiate into derivatives of the three germ layers, both in vitro and in vivo. Furthermore, the addition of OA and bFGF enabled the maintenance of hESC self-renewal without feeder cells, even in chemically defined xeno-free media. These findings shed a light on the role of PP2A in hESC differentiation and provide a novel strategy for maintaining the self-renewal capability of hESC in bFGF-free, feeder cell-free, and xeno-free media through the optimal suppression of PP2A activity using OA.
胚胎干细胞的自我更新涉及到内在和外在因素之间相互作用的信号通路的平衡。我们假设蛋白丝氨酸/苏氨酸磷酸酶 2A（PP2A）可能在调节人类胚胎干细胞（hESC）自我更新的信号通路中发挥核心作用。生化分析表明，PP2A 活性在 hESC 分化过程中逐渐增加；PP2A/C 和 PP2A/A 水平也增加。PP2A/C 的过表达或添加 PP2A 激活剂 C2-神经酰胺促进 hESC 分化。因此，添加 PP2A 失活剂 okadaic acid（OA）可在没有碱性成纤维细胞生长因子（bFGF）的情况下维持 hESC 的自我更新。用 OA 维持的 hESC 表达多能性标记物，并表现出具有正常核型的大量端粒酶活性。hESC 能够在体外和体内分化为三个胚层的衍生物。此外，添加 OA 和 bFGF 可在无饲养细胞的情况下，甚至在化学定义的无动物源、无饲养细胞的培养基中维持 hESC 的自我更新。这些发现揭示了 PP2A 在 hESC 分化中的作用，并提供了一种新的策略，通过使用 OA 对 PP2A 活性进行最佳抑制，在无 bFGF、无饲养细胞和无动物源的培养基中维持 hESC 的自我更新能力。
