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

Abstract

The therapeutical interest of pluripotent cells and ethical issues related to the establishment of human embryonic stem cell (ESC) or embryonic germ cell (EGC) lines raise the understanding of the mechanism underlying pluripotency to a fundamental issue. Establishing a protein pluripotency signature for these cells can be complicated by the presence of unrelated proteins produced by the culture environment. Here, we have analyzed the environment supporting ESC and EGC growth, and established 2-D reference maps for each constituent present in this culture environment: mouse embryonic fibroblast feeder cells, culture medium (CM) and gelatin. The establishment of these reference maps is essential prior to the study of ESC and EGC specific proteomes. Indeed, these maps can be subtracted from ESC or EGC maps to allow focusing on spots specific for ESCs or EGCs. Our study led to the identification of 110 unique proteins from fibroblast feeder cells and 23 unique proteins from the CM, which represent major contaminants of ESC and EGC proteomes. For gelatin, no collagen-specific proteins were identified, most likely due to difficulties in resolution and low quantities. Furthermore, no differences were observed between naive and conditioned CM. Finally, we compared these reference maps to ESC 2-D gels and isolated 17 ESC specific spots. Among these spots, proteins that had already been identified in previous human and mouse ESC proteomes were identified but no apparent ESC-specific pluripotency marker could be identified. This work represents an essential step in furthering the knowledge of environmental factors supporting ESC and EGC growth.

Nuclear proteome analysis of undifferentiated mouse embryonic stem and germ cells

“…Then, in-gel digestion was performed with an automated protein digestion system, MassPREP station (Waters, Milford, Massachusetts, USA) as previously described [48]. The peptide extracts were analyzed by MALDI-TOF-MS and/or nano-LC-MS/MS as previously described [48].…”

“…This was performed to bring a better knowledge of these constituents and to use them to study ESC and EGC specific proteomes. Indeed, these reference maps were subtracted from ESC or EGC maps to allow focusing exclusively on spots specific for ESCs or EGCs [48]. Following the nuclear enrichment, at least five 2-D gels were generated for MEFs, CM, ESCs, and 8.5/11.5 dpc EGCs to ensure reproducibility.…”

“…2-D separation and silver staining were performed as previously described [48]. Silver-stained 2-D gels were scanned using a high-resolution scanner (GS-800 Calibrated Imaging Densitometer, Bio-Rad, ref.…”

“…CM (supplemented with FCS and LIF) protein extracts were prepared by precipitation with a TCA/acetone approach, as previously described [48].…”

“…In a previous study [48], we have demonstrated that because of the particular culture conditions required for maintaining ESCs and EGCs, a subtractive approach (subtracting the CM and MEFs contaminating spots) was necessary in order to reveal the ESCs and EGCs specific spots. The same approach previously applied to total protein extracts was applied here to study the nuclear proteomes as most of the pluripotency markers are nuclear proteins.…”