2‐D DIGE identification of differentially expressed heterogeneous nuclear ribonucleoproteins and transcription factors during neural differentiation of human embryonic stem cells

Abstract

Neural stem cells (NSC) are progenitors that can give rise to all neural lineages. They are found in specific niches of fetal and adult brains and grow in vitro as non-adherent colonies, the neurospheres. These cells express the intermediate filament nestin, commonly considered an NSC marker. NSC can be derived as neurospheres from human embryonic stem cells (hESC). The mechanisms of cellular programming that hESC undergo during differentiation remain obscure. To investigate the commitment process of hESC during directed neural differentiation, we compared the nuclear proteomes of hESC and hESC-derived neurospheres. We used 2-D DIGE to conduct a quantitative comparison of hESC and NSC nuclear proteins and detected 1521 protein spots matched across three gels. Statistical analysis (ANOVA n = 3 with false discovery correction) revealed that only 2.1% of the densitometric signal was significantly changed. The ranges of average ratios varied from 1.2- to 11-fold at a statistically significant p-value <0.05. MS/MS identified 15 regulated proteins previously shown to be involved in chromatin remodeling, mRNA processing and gene expression regulation. Notably, three members of the heterogeneous nuclear ribonucleoprotein family (AUF-1, and FBP-1 and FBP-2) register a 54, 70 and 99% increased expression, highlighting them as potential markers for NSC in vitro derivation. By contrast, Cpsf-6 virtually disappears with differentiation with an 11-fold drop in NSC, highlighting this protein as a novel marker for undifferentiated ESC.

Differential Association of Chromatin Proteins Identifies BAF60a/SMARCD1 as a Regulator of Embryonic Stem Cell Differentiation

“…Unlike proteome analysis of whole-extract samples from nuclei of ESCs and differentiated cells (Barthé lé ry et al, 2009;Kurisaki et al, 2005;Lu et al, 2009), D-CAP enables one to distinguish between proteins that are expressed roughly at similar levels but display differential association to chromatin, as we observed for SMARCC1 and SMARCD1. Although their protein levels in ESCs and NPCs remained unaltered, the association of these proteins with chromatin changed, possibly suggesting a different role for these as well as other proteins in ESCs and during differentiation.…”

Vertebrate nucleoplasmin and NASP: egg histone storage proteins with multiple chaperone activities

“…NASP is widely distributed throughout eukaryotes in a wide range of tissue types, with multiple isoforms present in the same species, as supported by biochemical and phylogenetic analysis (33, 64). NASP is either directly or indirectly essential for eukaryotic DNA replication (14, 65–72), cell proliferation (70, 73), normal cell cycle progression (14, 65–74), blastocyst development (14, 65–72), cellular growth (70, 73), histone storage (1, 18, 25–27), histone transport (33, 34, 65, 74, 75), stem cell proliferation (76), neural stem cell differentiation (77), and the pluripotency of human embryonic stem cells (hESCs; ref. 76) ( Fig.…”

Quantitative Neuroproteomics: Classical and Novel Tools for Studying Neural Differentiation and Function

“…2D-DIGE has been poorly adopted for the investigation of neural differentiation processes. Recently, this strategy has been applied to the study of the commitment of human embryonic stem cells during directed neural differentiation by comparing the nuclear proteomes of hESC and hESC-derived neurospheres [66].…”