Influence Of Rho-kinase Inhibitor On The Derivation And Biological Characteristics Of Mouse Embryonic Stem Cells

The member of small G-proteins family Rho is substantially involved in intracellular signaling. The Rho regulates both cytoskeletal reorganization and gene expression. As with other Rho GTPases, Rho acts as a molecular switch, cycling between an active GTP-bound state and an inactive GDP-bound state. The best-characterized downstream effector of Rho is Rho-kinase (ROCK), which mediates various cellular functions, including migration, division, apoptosis, adhesion, Phagocytosis. There are two isoforms of ROCK: ROCK I and ROCK II. The genes expressing human ROCK I and ROCK II are located on chromosome 18 and chromosome 2, respectively. ROCK I and ROCK II are highly homologous, sharing 65% homology in amino acid sequence and 92% homology in their kinase domains. Although there are two isoforms, it has not exposed differentiation between these in the resent study.The pyridine derivative Y-27632 ([(+)-(R)-trans-4-(1-aminoethyl)-N-4-pyridyl) cyclohexanecarboxamide dihydrochloride]) is commonly used as a Rho kinase inhibitor. Y-27632 is a cell permeable, ATP competitive inhibitor, that inhibits the activities of ROCK-I and ROCK-II in vitro. Y-27632 is therefore a reasonably selective ROCK-I and ROCK-II inhibitor, and provides a useful pharmacological tool to study the role of Rho kinase in (patho)physiological events, both in vitro and in vivo.In this study, we use mouse embryonic stem cells as experimental materials to investigate biological effects of Y-27632 in mouse embryonic stem cells, obtaining some foundational research datas.Y-27632 could change the morphology of ES colonies. Y-27632 can make tight cell-cell connection into loose and flat connection. In passage, Y-27632 could improve the efficiency. In the Y-27632 under the conditions of long-standing, ES cells remain stability of chromosome number, high alkaline phosphatase activity, and differentiate into teratomas in vivo, cardiomyocytes in vitro. Suggest Y-27632 could not affect self-renewal and pluripotency of embryonic stem cells, moreover, it could be applied to the relative manipulations of embryonic stem cells.In the generation of chimeric mice, we get perfect cell shape via Y-27632 treatment for short term. The cells change as spherical, soft, and enable needle to select, collect easily, reducing the microinjection time in vitro. Moreover, Y-27632 has not affected production efficiency of chimeric mice.For derivation of ES cells, the joint use of accutase and Y-27632 could improve the production of sub-colonies. Therefore we establish a simple and efficient approach to get mouse ES cells. And the obtaining ES cell lines by this means conform biological characteristics and stabilize passage.