table 2signal pathways that may be active in hescsnameproteinmrnatgf βstat1 + + + + + + pai - 1 / serpine1 + + + - smad2 / 3 + + + + jun + + + smad4 / dpc4 + + + endoglin + - wntctbp2 + + + + + + + pp2a catalytic alpha / ppp2ca + + + + + + + ebp50 / slc9a3r1 + + + + + + beta - catenin / ctnnb1 + + + + cyclin d3 / ccnd3 + + + + gsk - 3 beta + + + + jun + + + casein kinase ii alpha / csnk2a1 + + + + jak - statstat1 + + + + + + crk + + + + stat3 / 2 + + + + + stat6 + + + + + ptp1b + + + + + jak1 + + - glucocorticoid r / nr3c1 + + - thrombin receptor / par1 / f2r + + + shps - 1 / ptpns1 + + + + mcm5 + + + + + smad2 / 3 + + + + tyk2 + + + + jun + + + bcl - x / bcl2l1 + + + + smad4 / dpc4 + + + stat5a + + gpcrb2 bradykinin receptor / bdkrb2 + + + + - neurotensin receptor 3 / sort1 + + + - endopeptidase 3. 4. 24. 16 / nln + + + + ip3r - 3 + + + + shc + + + + gap junctioncdk1 / cdc2 + + + + + + grb2 + + + + + + mek1 / map2k1 + + + + + + pka c + + - pka ri alpha + + - pkc alpha + + - c - raf / raf1 + + + + zo - 1 / tjp1 + + + + + connexin - 43 / gja1 + + + + igfpkc iota + + + + + + mek1 / map2k1 + + + + + + rsk / rps6ka1 + + + + + grb2 + + + + + + mek2 / map2k2 + + + + + + pi3kinase / pik3r1 + + + + + pan erk / mapk1 + + + + + crk + + + + eif - 4e + + + + + shcc + + + - pai - 1 / serpine1 + + + - c - raf + + + + shc + + + + + pkc beta / prkcb1 + + + + nck + + + + pkb alpha / akt + + + gsk - 3 beta + + + + ercc - 1 + + + + fatty acid synthase / fasn + + + + + jun + + + raft1 / frap + + + + ptp1d / shp2 / ptpn11 + + + + scamp1 + + + + bcl - x / bcl2l1 + + + + p70s6k / rps6kb1 + - pi3 - kinase p170 / pik3c2a + + ptp1b / ptpn1 + + + dok1 / p62dok + + + pi3 - kinase p110 alpha / pik3ca + - erbbepha4 / sek + + + + - shcc / shc3 + + + - c - erb - b2 / erbb2 + + + + c - raf / raf1 + + + + shc / shc1 + + + + + gdnfi kappa b epsilon / nfkbie + + + + + + grb2 + + + + + + mek2 + + + + + + nck + + + + + c - raf + + + + ras - gap / rasa1 + + + + shc + + + + + gdnfr - alpha / gfra1 + + - jun + + + ikk beta + + + + pan - jnk / sapk1 / mapk10 + + + nbs1 / artn + + dok1 / p62dok + + + tight junctionpten + + + + + + pp2a catalytic alpha + + + + + + + pkc iota + + + + + + sec8 / sec8l1 + + + + + beta - catenin / ctnnb1 + + + + cdc42 + + + + + af6 / mllt4 + + + + + pkc alpha + + - yes + + + + rho / arha + + + + + zo - 1 / tjp1 + + + + + cask + + + symplekin / sympk + + - ras / nras + + + + casein kinase ii alpha / csnk2a1 + + + + vap33 / vapa + + + alpha - catenin / ctnna1 + + + mapkpan erk + + + + + + mek1 + + + + + + rsk + + + + + + erk2 + + + + + + mek2 / map2k2 + + + + + + mst3 / stk25 + + + + + + erk1 + + + + + cdc42 + + + + + c - raf + + + + p38 alpha / sapk2a + + - g3bp + + + + + tfii - i / gtf2ird1 + + + + mst1 / stk4 + + + + mkp2 / dusp4 + + + + ras + + + + phospho - p38mapk ( t180 / y182 ) + + + pan - jnk / sapk1 + + + inhibitor2 / ppp1r2 + + + + abp - 280 + + + + + + 14 - 3 - 3 epsilon / ywhae + + + + mapkapk - 5 + + + tao1 + * pbk + + + mkk3b / map2k3 + + protein expression level: > 10, 000: + + + +; 5, 000 – 10, 000: + + +; 1, 000 – 5, 000: + +; 100 – 1, 000: + mrna gene expression level: > 5, 000: + + + +; 1, 000 – 5, 000: + + +; 100 – 1, 000: + +; 30 – 100: + *: not included in the gene expression arraythis independent confirmation of known networks led us to examine other pathways that showed a similar correlation but have not been identified as key regulators of either self - renewal or differentiation, or suggest unappreciated characteristics of hescs. four signaling pathways ( igf, erbb2, gpcr, and gdnf ) and the tight junction complex were highlighted by this analysis ( table 2 ), and expression of key proteins in these pathways was confirmed. a detailed study demonstrating the importance of the igf and erbb2 pathways in hesc self - renewal has been performed and enabled the development of a defined medium for hesc maintenance ( tcs and ajr, submitted ). tight junctions are apical cell - cell junctions found in epithelia that establish a barrier to the extracellular environment and a border for apical - basolateral polarity. while hescs grow in colonies that are highly reminiscent of epithelia, and have been shown to be coupled by gap junctions [ 40 ], the formation of tight junction complexes has not been described. hescs expressed the zo1 and occludin tight junction proteins along cell borders as expected in polarized epithelia. the distribution of zo1 expression changed dramatically as hescs proliferated in culture. when tight junction complexes were disrup ted by disagg reagation to single cells, only a subset of cells showed zo1 staining 4 days after plating ( fig. 5 ). continued proliferation to a confluent monolayer on day 7 was accompanied by widespread expression of zo1, suggesting the formation of a general tight junction barrier. these cultures were undifferentiated and retained uniform expression of oct4 protein ( not shown ). erbb2 and 3 are members of the epidermal growth factor ( egf ) - receptor family, which regulate epithelial proliferation via egf - family ligands. erbb2 and 3 transcripts are expressed by hescs [ 8 ], are known to function as a heterodimer [ 41 ], and transmit a strong proliferative signal for hescs by heregulin 1β ( an egf - family ligand ) ( tcs and ajr, submitted ). immunoflu orescence revealed general cell surface expression of erbb2 on hescs. conversely, erbb3 was highly localized to a concentrated area, and observed in cells that also expressed zo1. epithelial cells are known to localize erbb receptors to the basolateral side of tight junctions, which serves to functionally separate receptors from ligands [ 42, 43 ]. this is a basic epithelial wound healing mechanism, whereby disrup tion of the tight junction barrier by injury immediately exposes receptors to extracelluar ligands [ 43 ]. these staining patterns are also suggestive of basolateral sorting of erbb3 in hescs. the pathways and complexes identified by these analyses lay a framework for future functional analyses of signaling networks in hescs. figure 5tight junction proteins and erbb2 / 3 expression in hescs. bg01 hescs were disagg regated to single cells using accutase [ 52 ] and cultured in defined conditions. ( a ) zo1 expression four and ( b ) seven days after plating, indicating progressive tight junction formation. ( c ) occludin expression 5 days after plating. ( d ) general cell surface expression of erbb2, in the same field of view as ( a ). ( e ) localized expression of erbb3, in the same field of view as ( b ). ( f ) higher magnification of erbb3 localization in zo1 expressing bg01 cells, 5 days after plating. nuclei were stained with dapi. attempts to harness the potential of hescs for models of human embryogenesis and cell therapy applications will be greatly enhanced by a detailed understanding of their molecular characteristics. this includes definition of the transcripts, splice variants, and protein isoforms expressed by these cells. post - translational modifications such as phosphorylation and glyco sylation, and the receptors and signaling pathways active in the pluripotent state, or during early differentiation, also need to be determined. this should also be complemented by an understanding of epigenetic characteristics of pluripotency, including methylation, imprinting and chromatin conformation. such a comprehensive definition of the molecular state of hescs will enable more ac curate prediction and testing of the conditions used for growth and differentiation of hescs, by precise genetic modification or application of specific growth factor cocktails and reagents. for example, a scalable, fully defined and gmp - certified culture system will need to be developed for the eventual development of hesc - based cellular therapies.