HESCs have the unique ability to self-renew and give rise to ectodermal, mesodermal, and endodermal lineages 
. This capacity to differentiate into cells of all three germ layers provides an excellent system to study human development and model disease states. Additionally, as hESCs are a continuously self-replicating population of cells, they have the potential to be a stable source of numerous cell types for regenerative medicine. Unfortunately, hESCs are sensitive to even the most routine manipulations, such as passaging and cryopreservation, illustrating the need for technical advancements to realize their full potential 
Cell dissociation induced apopotosis has been attributed to the inherent sensitivity of hESCs and has duly received much attention. A significant breakthrough for ameliorating this problem was the identification of Y-27632, a selective inhibitor of the p160-Rho-associated coiled kinase (ROCK) 
, as a factor that enhanced hESC survival upon single cell dissociation 
. Subsequently, Y-27632 has been used in various applications in stem cell research where extensive cell death occurs. The post-thaw survival rate was enhanced by the addition of Y-27632 to hESCs grown in feeder-dependent and independent conditions and feeder-independent human induced pluripotent stem cells (hiPSCs) 
. Improved recovery from cryopreservation was also reported from the addition of Y-27632 to other stem cell types including non-human primate embryonic stem cells 
and bone marrow-derived mesenchymal stem cells 
. Inhibition of ROCK also improved the survival upon dissociation of hESC-derived cardiomyocyte and non-cardiomyocyte cells 
. The recovery upon dissociation as well as the transplantation of neural precursor cells derived from mouse embryonic stem cells was positively impacted by the addition of Y-27632 
. During differentiation, Y-27632 was applied to cells to improve survival upon differentiation of hESCs to retinal cells 
. In reprogramming, Y-27632 has been used after viral transduction at culture media exchange to aid in the establishment of hiPSCs 
. Y-27632 has also been applied to improve survival of additional cell types such as endothelial cells 
and retinal ganglion cells 
. All together, this body of research has demonstrated the utility and safety of Y-27632 for a variety of applications and cell types.
The utilization of cell surface markers, including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and SSEA-1 to characterize hESCs is widely accepted 
. The ability to use these and other surface markers in combination with FACS to consistently isolate hESCs and their differentiated progeny would facilitate many applications. These applications include the removal of contaminating secondary cell types such as feeders, hESC-derived fibroblasts and spontaneously differentiating cells, as well as the identification and isolation of pure subpopulations, genetically modified cells, and single-cell clones. Thus far, the reported use of FACS to isolate and consistently recover hESCs has been limited and variable, necessitating the generation of robust and standardized sorting methods. HESCs have been sorted by the use of light scatter gating 
and by the expression of GFP in genetically altered hESCs lines 
. A combination of sorting using a fluorescent reporter and SSEA-3 labeling of hESCs with the successful recovery of cells has also been reported 
. Interestingly, the only study to date that used more than one endogenously expressed cell surface marker (SSEA-4 and TRA-1-81) to sort hESCs resulted in an inability to recover viable cultures post-sort 
. All the previously mentioned studies that successfully sort hESCs report low recovery. Although a low recovery is to be expected since sorting of hESCs requires that the cells be dissociated to a single cell state, it also highlights the necessity for an improvement in sorting conditions for hESCs.
Since previous studies have shown that the addition of Y-27632 improves the survival of hESCs that have been dissociated to single cells (for review see 
), we examined whether Y-27632 would improve the recovery of hESCs after sorting. Specifically, we describe that Y-27632 improved the recovery upon sorting of hESCs using three cell surface markers, SSEA-3 and TRA-1-81 for pluripotency and SSEA-1 to exclude spontaneously differentiating cells in the culture. Cell sorting could be performed on cells grown in feeder-dependent and feeder-independent growth conditions. After long-term culturing, sorted cells expressed markers for pluripotency, differentiated in vitro
and in vivo
to all three germ layers, and maintained a stable karyotype.