Two‐dimensional gel electrophoresis database of murine R1 embryonic stem cells

Abstract

Embryonic stem (ES) cell lines represent a population of undifferentiated pluripotent cells capable of multilineage differentiation in vitro. Although very useful for studying developmental processes, human ES cell lines have also been suggested as a potential and unlimited source for cellular transplantation and the treatment of human disease. The proteomic basis of embryonic stemness (pluripotentiality and multilineage differentiation) and the transitions that lead to specific cell lineages however, remain to be defined. As an important first step in defining these processes, we have performed a proteomic analysis of undifferentiated mouse R1 ES cell lines using pH 3-10, 4-7 and 6-11 two-dimensional electrophoresis gels, matrix-assisted laser desorption/ionization and tandem mass spectrometry. Of the 700 gel spots analyzed, 241 distinct protein species were identified corresponding to 218 unique proteins, with a significant proportion functionally related to protein expression.

Proteome analysis of the culture environment supporting undifferentiated mouse embryonic stem and germ cell growth

“…Among these proteins, lamin A has already been identified in the CM of MEFs [37,38] and therefore should be a CM contaminant. More interestingly, among the proteins identified here, Hsp-70 protein 8, the proteasome 26S subunit protein and the inorganic pyrophosphatase have already been identified in hESC proteome studies [39,40], and vinculin, the 26S proteasome subunit and the stress-70 protein have been identified in mESC proteome studies [41,42].…”

“…Others culture cells without feeders but with their secretomes by taking the risk of contaminating their extracts with proteins resulting from differentiated cells [39]. Some teams adopt the plating approach, which consists in reducing the contamination of MEFs, based on the hypothesis that MEFs adhere more rapidly on gelatin-coated dishes than ESCs on MEFs [41]. All these factors may induce stress, differentiation or modify metabolism, add gelatin carryover or MEF contaminants thus modifying these cells' proteomes.…”

“…Different attempts have been performed to establish a protein pluripotency signature, mainly at the transcriptional level [43][44][45][46], but also at the translational level [40]. Although mouse ESC and EGC proteomes are increasingly studied [41,42,[47][48][49][50], the proteome of the culture environment supporting their growth has only been poorly investigated. Reports on this subject had either not taken these factors in consideration [40] or had used culture conditions that limit non-ESCs contaminant proteins, regardless of the stress it can induce in cells.…”

“…Reports on this subject had either not taken these factors in consideration [40] or had used culture conditions that limit non-ESCs contaminant proteins, regardless of the stress it can induce in cells. Indeed, some groups culture cells for 16 h without serum to simplify the proteome [38], others culture cells without MEFs during several spreadings [41,42], or harvest cells by scratching or trypsinating them. Others culture cells without feeders but with their secretomes by taking the risk of contaminating their extracts with proteins resulting from differentiated cells [39].…”

Nuclear proteome analysis of undifferentiated mouse embryonic stem and germ cells

“…These proteins are interesting because like the well-known Oct-3/4 pluripotency marker, they are expressed in germ cell lines during development [56]. Among the identified proteins, 17 proteins have already been identified in previous mESC and hESC proteome studies: NUBP1, SRM, CFL1, IDH3, PCBP1, ARBP, hnRNPL, UMPS, TDH, DJ1, LDH2, RPA2, PPA1, GSTA4, STMN1, PRMT7, and UPP1 [57][58][59]. Concerning their cellular location, most of the identified proteins have a nuclear location (60% of the identified proteins) indicating a good nuclear enrichment.…”