Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan.
PLoS One. 2010 Feb 12;5(2):e9187. doi: 10.1371/journal.pone.0009187.
Rho kinases (ROCKs) mediate cell contraction, local adhesion, and cell motility, which are considered to be important in cell differentiation. We postulated that ROCKs are involved in controlling embryonic stem (ES) cell renewal and differentiation.
METHODOLOGY/PRINCIPAL FINDINGS: CCE, a murine ES cell, was treated with Y-27632 for 48 to 96 hours and colony formation was evaluated. Y-27632 blocked CCE colony formation and induced CCE to grow as individual cells, regardless of the initial seeding cell density either at 10(4)/cm(2) ("high" seeding density) or 2x10(3)/cm(2) ("low" density). However, at high seeding density, Y-27632-treated cells exhibited reduction of alkaline phosphatase (AP) staining and Oct3/4 expression. They expressed SOX-1, nestin, and MAP2c, but not betaIII-tubulin or NG-2. They did not express endoderm or mesoderm lineage markers. After removal of Y-27632, the cells failed to form colonies or regain undifferentiated state. Silencing of ROCK-1 or ROCK-2 with selective small interference RNA induced CCE morphological changes similar to Y-27632. Silencing of ROCK-1 or ROCK-2 individually was sufficient to cause reduction of AP and Oct3/4, and expression of SOX-1, nestin, and MAP2c; and combined silencing of both ROCKs did not augment the effects exerted by individual ROCK siRNA. Y-27632-treated CCE cells seeded at 2x10(3) or 6.6x10(3) cells/cm(2) did not lose renewal factors or express differentiation markers. Furthermore, they were able to form AP-positive colonies after removal of Y-27632 and reseeding. Similar to ROCK inhibition by Y-27632, silencing of ROCK-1 or ROCK-2 in cells seeded at 2x10(3)/cm(2) did not change renewal factors.
CONCLUSIONS/SIGNIFICANCE: We conclude that ROCKs promote ES cell colony formation, maintain them at undifferentiated state, and prevent them from neural differentiation at high seeding density. ROCK inhibition represents a new strategy for preparing large numbers of neural progenitor cells.
Rho 激酶（ROCK）介导细胞收缩、局部黏附以及细胞运动，这些过程被认为在细胞分化中起着重要作用。我们假设 ROCK 参与控制胚胎干细胞（ES）细胞的更新和分化。
方法/主要发现：用 Y-27632 处理 CCE（一种鼠 ES 细胞系）48 到 96 小时后，评估集落形成情况。Y-27632 抑制 CCE 集落形成，并诱导 CCE 以单个细胞的形式生长，而与初始接种细胞密度无关，初始接种细胞密度分别为 10(4)/cm(2)（“高”接种密度）或 2x10(3)/cm(2)（“低”密度）。然而，在高接种密度下，Y-27632 处理的细胞碱性磷酸酶（AP）染色和 Oct3/4 表达减少。它们表达 SOX-1、巢蛋白和 MAP2c，但不表达βIII-微管蛋白或 NG-2。它们不表达内胚层或中胚层谱系标志物。去除 Y-27632 后，细胞无法形成集落或恢复未分化状态。用选择性小干扰 RNA 沉默 ROCK-1 或 ROCK-2 可诱导 CCE 形态发生变化，类似于 Y-27632 的作用。单独沉默 ROCK-1 或 ROCK-2 足以导致 AP 和 Oct3/4 减少，以及 SOX-1、巢蛋白和 MAP2c 的表达；而同时沉默两种 ROCK 并不能增强单独使用 ROCK siRNA 的作用。以 2x10(3)或 6.6x10(3)细胞/cm(2)接种的 Y-27632 处理的 CCE 细胞不会失去更新因子或表达分化标志物。此外，在去除 Y-27632 并重新接种后，它们能够形成 AP 阳性集落。与 Y-27632 抑制 ROCK 相似，以 2x10(3)/cm(2)接种的细胞中沉默 ROCK-1 或 ROCK-2 不会改变更新因子。
结论/意义：我们得出结论，ROCK 促进 ES 细胞集落形成，使它们保持未分化状态，并防止它们在高接种密度下向神经分化。ROCK 抑制为制备大量神经祖细胞提供了一种新策略。
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