Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Lower Kent Ridge Road, Singapore, 119074, Singapore.
Stem Cell Rev Rep. 2010 Mar;6(1):86-95. doi: 10.1007/s12015-009-9107-8. Epub 2009 Dec 15.
The Rho associated coiled coil protein kinase (ROCK) dependent signaling pathway plays an important role in numerous physiological functions such as cell proliferation, adhesion, migration and inflammation. Human embryonic stem cells (hESCs) undergo differentiation and poor survival after single cell dissociation in culture thus limiting their expansion for cell based therapies. We evaluated the role of the selective ROCK inhibitor Y-27632 on hESC colonies and disassociated single hESCs from two different hESC lines. Karyotypically normal hESCs (HES3) and variant hESCs (BG01V) were treated with Y-27632 at 5, 10 and 20 muM concentrations for 72 h and its effects on hESC self renewal, colony morphology, cell cycle and pluripotency were evaluated. Increased cell proliferation of both HES3 and BG01V were observed for all three concentrations compared to untreated controls following passaging of cell clusters or dissociated single cells and some of these increases were statistically significant. Cell cycle assay demonstrated normal cell cycle progression with no peaks evident of apoptosis. No morphological differentiation was evident following treatment with the highest concentration of Y-27632 (20 muM) and the stemness related genes continued to be highly expressed in both HES3 and BG01V cells compared to untreated controls. The results confirmed that Y-27632 is a useful agent that aids in the expansion of undifferentiated hESC numbers for downstream applications in regenerative medicine.
Rho 相关卷曲螺旋蛋白激酶（ROCK）依赖性信号通路在许多生理功能中发挥重要作用，如细胞增殖、黏附、迁移和炎症。人类胚胎干细胞（hESC）在培养过程中经单细胞解离后会发生分化和存活率降低，从而限制了其用于基于细胞的治疗方法的扩展。我们评估了选择性 ROCK 抑制剂 Y-27632 对 hESC 集落和两种不同 hESC 系分离的单个 hESC 的作用。用 Y-27632 处理正常核型 hESC（HES3）和变异 hESC（BG01V），浓度分别为 5、10 和 20 μM，持续 72 小时，评估其对 hESC 自我更新、集落形态、细胞周期和多能性的影响。与未处理的对照组相比，细胞集落或分离的单个细胞传代后，所有三种浓度均观察到 HES3 和 BG01V 的细胞增殖增加，其中一些增加具有统计学意义。细胞周期分析表明细胞周期进程正常，无凋亡峰。用最高浓度的 Y-27632（20 μM）处理后，未观察到明显的形态分化，与未处理的对照组相比，HES3 和 BG01V 细胞中的干细胞相关基因持续高表达。结果证实，Y-27632 是一种有用的试剂，可帮助未分化 hESC 数量的扩增，用于再生医学中的下游应用。
