Abstract
BackgroundRho 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 FindingsCCE, 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 104/cm2 (“high” seeding density) or 2×103/cm2 (“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 βIII-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 2×103 or 6.6×103 cells/cm2 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 2×103/cm2 did not change renewal factors.Conclusions/SignificanceWe 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.
Highlights
The mammalian Rho-associated coiled-coil forming protein kinase (ROCK or ROK) comprises ROCK-1 (ROKb) and ROCK-2 (ROKa) which contain highly conserved aminoterminal and substantially different carboxy-terminal domains [1,2]
Our results show that ROCK-1 and ROCK-2 are constitutively expressed and active in CCE murine embryonic stem (ES) cells under normal culture conditions
By using pharmacological inhibitors and genetic silencers of ROCK-1 and ROCK-2, our data provide evidence for a critical role that both ROCKs play in promoting ES cell growth as colonies and maintaining ES cell at undifferentiated state
Summary
The mammalian Rho-associated coiled-coil forming protein kinase (ROCK or ROK) comprises ROCK-1 (ROKb) and ROCK-2 (ROKa) which contain highly conserved aminoterminal and substantially different carboxy-terminal domains [1,2]. RhoA binds to the coiled coil region of ROCK and activates ROCK catalytic activity [3]. Activated ROCK mediates actinmyosin contraction, stress fiber formation and local adhesion by targeting downstream kinases and phosphatases resulting in increased myosin light chain phosphorylation [4]. Activated ROCK induces neurite retraction [5] while selective ROCK inhibitor, Y-27632, as well as ROCK dominant negative mutants promote neurite formation [6]. It was proposed that ROCKs are required for contraction of the cleavage furrow [8], and ROCK inhibition was reported to retard cytokinesis and impair cytokinetic segregation of glial filaments [9]. 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
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