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 (hESCs). Currently, there are few well-defined protein epitopes that definitively mark hESCs. Here we combine immunotranscriptional profiling of hESC lines with membrane-polysome translation state array analysis (TSAA) to determine the full set of genes encoding potential hESC surface marker proteins. Three independently isolated hESC lines (HES2, H9, and MEL1) grown under feeder and feeder-free conditions were sorted into subpopulations by fluorescence-activated cell sorting based on coimmunoreactivity to the hESC surface markers GCTM-2 and CD9. Colony-forming assays confirmed that cells displaying high coimmunoreactivity to GCTM-2 and CD9 constitute an enriched subpopulation 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 hESCs identified 1,492 mRNAs encoding actively translated plasma membrane and secreted proteins. Combining these data sets, 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 hESCs. This comprehensive listing of both hESCs 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.
Kolle
Gabriel
G
Institute for Molecular Bioscience, The University of Queensland, Queensland, Australia.
Ho
Mirabelle
M
Zhou
Qi
Q
Chy
Hun S
HS
Krishnan
Keerthana
K
Cloonan
Nicole
N
Bertoncello
Ivan
I
Laslett
Andrew L
AL
Grimmond
Sean M
SM
D000951:Antigens, Neoplasm / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D000954:Antigens, Surface / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D015820:Cadherins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D000071858:Epithelial Cell Adhesion Molecule; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D010641:Phenotype; D040081:Protein Array Analysis / Q000379:methods; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism
D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D015415:Biomarkers; C512197:CDH3 protein, human; D015820:Cadherins; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D008565:Membrane Proteins; D012333:RNA, Messenger