Identification of human embryonic stem cell surface markers by combined membrane-polysome translation state array analysis and immunotranscriptional profiling

Surface marker expression forms the basis for characterization and isolation of human embryonic stem cells (hESC). Currently, there are few well defined protein epitopes that definitively mark hESCs. Here we combine immunotranscriptional profiling of hESC cell lines with membrane-polysome translation state array analysis (TSAA) to determine the full set of genes encoding potential hESC cell surface marker proteins. Three independently isolated hESC lines (HES2, H9 and MEL1) grown under feeder and feeder-free conditions were sorted into subpopulations by FACS based on co- immunoreactivity to the hESC surface markers GCTM-2 and CD9. Colony forming assays confirmed that cells displaying high co-immunoreactivity to GCTM-2 and CD9 constitute an enriched sub-population displaying multiple stem cell properties. Following microarray profiling, 820 genes were identified that were common to the GCTM-2(high)/CD9(high) stem-cell like subpopulation. Membrane-polysome TSAA analysis of hESC identified 1,492 mRNAs encoding actively translated plasma membrane and secreted proteins. Combining these datasets, 88 genes encode proteins that mark the pluripotent subpopulation, of which only four had been previously reported. Cell surface immunoreactivity was confirmed for two of these markers: TACSTD1/EpCAM and CDH3/P-Cadherin, with antibodies for EpCAM able to enrich for pluripotent hESC. This comprehensive listing of both hESC and spontaneous differentiation associated transcripts and survey of translated membrane-bound and secreted proteins provides a valuable resource for future study into the role of the extracellular environment in both the maintenance of pluripotency and directed differentiation.