Reptin52 expression during in vitro neural differentiation of human embryonic stem cells.

Human embryonic stem cells (hESCs) give rise to all somatic cell types, including neural cells such as astrocytes, oligodendrocytes and neurons. Commitment of hESC to a neural fate can be achieved via selection and expansion of developing neural stem cells, which, grown into non-adhering colonies called neurospheres, express nestin, a neurofilament marker. Analysis of hESC and hESC-derived neural stem cell nuclear extracts revealed an increased expression of Reptin52 in neurosphere nuclei. The increase in Reptin52 was evident throughout directed neuronal differentiation as assessed by western blotting, quantitative RT-PCR and immunocytochemistry. Reptin52 serves a pivotal regulatory role in nuclear activities such as transcription regulation and histone modification. In that regard, co-immunoprecipitation experiments showed that binding partners of Reptin52 (Pontin52, beta-catenin and ATF-2) associate with this regulatory protein in hESC-derived neuronal precursors. Moreover, expression of two of these proteins (beta-catenin - the end product of the Wnt signaling pathway - and ATF-2) is coordinately regulated with Reptin52.

Human embryonic and mesenchymal stem cells express different nuclear proteomes.

Human embryonic stem cells (hESCs) are characterized by their immortality and pluripotency. Human mesenchymal stem cells (hMSC), on the other hand, have limited self-renewal and differentiation capabilities. The underlying molecular differences that account for this characteristic self-renewal and plasticity are, however, poorly understood. This study reports a nuclear proteomic analysis of human embryonic and bone marrow-derived mesenchymal stem cells. Our proteomic screen highlighted a 5-fold difference in the expression of Reptin52. We show, using two-dimensional difference gel electrophoresis (2-DIGE), western analysis, and quantitative reverse transcriptase polymerase chain reaction, that Reptin52 is more abundantly expressed in hESC than hMSC. Moreover, we observed differential expression of Pontin52 and beta-catenin-proteins known to interact with Reptin52. This difference in the expression of Reptin52 and Pontin52 (known regulators of beta-catenin) further supports a role for Wnt signaling in stem cell self-renewal and proliferation.

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

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.

Enhanced nuclear proteomics.

Nuclear proteomics provides an opportunity to examine protein effectors that contribute to cellular phenotype. Both the quality and sensitivity of gel-based nuclear proteomics are limited, however, by the over-representation of histones in the protein mixture. These highly charged proteins overshadow rare species and interfere with IEF. A nuclear isolation and protein extraction procedure, tested on human embryonic stem cells, is reported that effectively isolates intact nuclei and then depletes the sample of histones by taking advantage of their ability to form an insoluble complex with DNA at lower pH (even under denaturing conditions). Ubiquitous histones and abundant nuclear actin, are depleted up to 99 +/- 0.02 and 42 +/- 5%, respectively. This technique greatly improves electrofocusing efficacy and nearly doubles the number of detected protein spots. This approach to nuclear protein isolation for 2-D PAGE opens the door to better investigation of nuclear protein dynamics.

Nuclear proteomics and directed differentiation of embryonic stem cells.

During the past decade, regenerative medicine has been the subject of intense interest due, in large part, to our growing knowledge of embryonic stem (ES) cell biology. ES cells give rise to cell lineages from the three primordial germ layers--endoderm, mesoderm, and ectoderm. This process needs to be channeled if these cells are to be differentiated efficiently and used subsequently for therapeutic purposes. Indeed, an important area of investigation involves directed differentiation to influence the lineage commitment of these pluripotent cells in vitro. Various strategies involving timely growth factor supplementation, cell co-cultures, and gene transfection are used to drive lineage specific emergence. The underlying goal is to control directly the center of gene expression and cellular programming--the nucleus. Gene expression is enabled, managed, and sustained by the collective actions and interactions of proteins found in the nucleus--the nuclear proteome--in response to extracellular signaling. Nuclear proteomics can inventory these nuclear proteins in differentiating cells and decipher their dynamics during cellular phenotypic commitment. This review details what is currently known about nuclear effectors of stem cell differentiation and describes emerging techniques in the discovery of nuclear proteomics that will illuminate new transcription factors and modulators of gene expression.