High-content screening of feeder-free human embryonic stem cells to identify pro-survival small molecules

Abstract

The propensity of human embryonic stem cells to die upon enzymatic disaggregation or low-density plating is an obstacle to their isolation and routine use in drug discovery and basic research. Equally, the very low rate of establishment of implanted cells hinders cell therapy. In the present study we have developed a high-content assay for human embryonic stem cell survival and used this to screen a range of libraries of 'lead-like' small molecules and known bioactives. From this we identified 18 confirmed hits with four structural classes being represented by multiple compounds: a series of 5-(acyl/alkyl-amino)indazoles, compounds with a 4-(acylamino)pyridine core, simple N⁶,N⁶-dialkyladenines and compounds with a 5-(acylamino)indolinone core. In vitro kinase profiling indicated that the ROCK (Rho-associated kinase)/PRK2 (protein kinase C-related kinase 2) protein kinases are of pivotal importance for cell survival and identified previously unreported compound classes that inhibited this important biological activity. An evaluation using an extensive panel of protein kinases showed that six of our hit compounds exhibited better selectivity for ROCK inhibition than the routinely used commercially available ROCK inhibitor Y-27632. In this screen we also identified the K(+)-ATP channel opener pinacidil and show that it probably promotes cell survival, by 'off-target' inhibition of ROCK/PRK2. We have therefore identified novel pro-survival compounds of greater specificity, equivalent potency and reduced toxicity relative to the routinely employed ROCK inhibitor Y-27632.

Sialyl-lactotetra, a Novel Cell Surface Marker of Undifferentiated Human Pluripotent Stem Cells

“…For isolation of glycosphingolipids, the two hECS lines SA121 and SA181 were cultured under feeder-free conditions and harvested, as previously described (10, 14). The cell pellets obtained were stored at −80 °C.…”

Structural Complexity of Non-acid Glycosphingolipids in Human Embryonic Stem Cells Grown under Feeder-free Conditions

“…Expansion and Harvest of Human Embryonic Stem CellshESC were grown and passaged as described previously (11). In brief, two cell lines (SA121 and SA181) were generated from two separate leftover human in vitro fertilized embryos.…”

Human pluripotent stem cells on artificial microenvironments: a high content perspective

“…This strategy isolated the antihypertensive drug pinacidil as a promoter of hES cells survival. Independently, the same molecule was found using hES cells feeder free with conditioned medium; here the authors showed pinacidil as well as other compounds to be structurally related to classic ROCK inhibitors (Andrews et al, 2010). Similar approaches have been tried elsewhere and identified compounds that improved survival by inhibiting ROCK or protein kinase C (Damoiseaux et al, 2009; Sherman et al, 2011).…”