The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers.
The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers.
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DOI：
10.1371/journal.pone.0012148
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发表时间：
2010-08-13
期刊：
影响因子：
3.7
通讯作者：
Carson CT
中科院分区：
文献类型：
--
作者：
Emre N;Vidal JG;Elia J;O'Connor ED;Paramban RI;Hefferan MP;Navarro R;Goldberg DS;Varki NM;Marsala M;Carson CT
由于人胚胎干细胞（hESC）对操作的固有敏感性，荧光激活细胞分选（FACS）后hESC的恢复和存活可能较低。此外，尚未描述使用多细胞表面标记物对hESC进行FACS的充分表征和稳健的方法。p160-Rho相关卷曲激酶（ROCK）抑制剂Y-27632先前已被鉴定为在单细胞解离后增强hESC的存活，以及增强从冷冻保存中的恢复。在这里，我们研究了Y-27632在FACS后对hESC的应用，以改善饲养层依赖性和饲养层非依赖性生长条件下的存活率。使用SSEA-3、TRA-1-81和SSEA-1的标记物分选HESC。在存在或不存在Y-27632的情况下分选24小时后将细胞铺板。在饲养层依赖性和饲养层非依赖性条件下，当Y-27632在分选后应用于hESC时，细胞存活率更高。具体而言，用Y-27632处理细胞将分选后回收率提高了四倍。为了确定在应用和不应用Y-27632的情况下分选的长期效果，进一步分析了hESC。具体而言，在添加和不添加Y-27632的情况下分选的hESC保留正常形态，表达通过免疫细胞化学和流式细胞术测量的hESC特异性标志物，并保持稳定的核型。此外，hESCs在体外和体内的饲养层依赖和饲养层非依赖的生长条件下都可以分化为三个胚层。在细胞分选后将Y-27632应用于hESC改善了细胞回收，对多能性没有观察到影响，并且能够通过使用多种表面标志物的FACS一致地回收hESC。这种用于hESC细胞分选的改进方法将有助于许多应用，例如从次级细胞类型中去除hESC，鉴定和分离干细胞亚群，以及产生单细胞克隆。最后，这些结果证明了ROCK抑制在hESC研究中的额外应用。
Due to the inherent sensitivity of human embryonic stem cells (hESCs) to manipulations, the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be low. Additionally, a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK) inhibitor, Y-27632, previously has been identified as enhancing survival of hESCs upon single-cell dissociation, as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions. HESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions, cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically, treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632, hESCs were further analyzed. Specifically, hESCs sorted with and without the addition of Y-27632 retained normal morphology, expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry, and maintained a stable karyotype. In addition, the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions. The application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types, identification and isolation of stem cell subpopulations, and generation of single cell clones. Finally, these results demonstrate an additional application of ROCK inhibition to hESC research.
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影响因子：
2.7
作者：
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