Comparative computational analysis of pluripotency in human and mouse stem cells

Pluripotent cells can be subdivided into two distinct states, the naïve and the primed state, the latter being further advanced on the path of differentiation. There are substantial differences in the regulation of pluripotency between human and mouse, and in humans only stem cells that resemble the primed state in mouse are readily available. Reprogramming of human stem cells into a more naïve-like state is an important research focus. Here, we developed a pipeline to reanalyze transcriptomics data sets that describe both states, naïve and primed pluripotency, in human and mouse. The pipeline consists of identifying regulated start-ups/shut-downs in terms of molecular interactions, followed by functional annotation of the genes involved and aggregation of results across conditions, yielding sets of mechanisms that are consistently regulated in transitions towards similar states of pluripotency. Our results suggest that one published protocol for naïve human cells gave rise to human cells that indeed share putative mechanisms with the prototypical naïve mouse pluripotent cells, such as DNA damage response and histone acetylation. However, cellular response and differentiation-related mechanisms are similar between the naïve human state and the primed mouse state, so the naïve human state did not fully reflect the naïve mouse state

Sirtuin 1 regulation of developmental genes during differentiation of stem cells

The longevity-promoting NAD+-dependent class III histone deacetylase Sirtuin 1 (SIRT1) is involved in stem cell function by controlling cell fate decision and/or by regulating the p53-dependent expression of NANOG. We show that SIRT1 is down-regulated precisely during human embryonic stem cell differentiation at both mRNA and protein levels and that the decrease in Sirt1 mRNA is mediated by a molecular pathway that involves the RNA-binding protein HuR and the arginine methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). SIRT1 down-regulation leads to reactivation of key developmental genes such as the neuroretinal morphogenesis effectors DLL4, TBX3, and PAX6, which are epigenetically repressed by this histone deacetylase in pluripotent human embryonic stem cells. Our results indicate that SIRT1 is regulated during stem cell differentiation in the context of a yet-unknown epigenetic pathway that controls specific developmental genes in embryonic stem cells

Human Embryonic Stem Cells and Embryonal Carcinoma Cells Have Overlapping and Distinct Metabolic Signatures

While human embryonic stem cells (hESCs) and human embryonal carcinoma cells (hECCs) have been studied extensively at the levels of the genome, transcriptome, proteome and epigenome our knowledge of their corresponding metabolomes is limited. Here, we present the metabolic signatures of hESCs and hESCs obtained by untargeted gas chromatography coupled to mass spectrometry (GC-MS). Whilst some metabolites are common to both cell types, representing the self-renewal and house-keeping signatures, others were either higher (e.g., octadecenoic acid, glycerol-3-phosphate, 4-hydroxyproline) or lower (e.g., glutamic acid, mannitol, malic acid, GABA) in hESCs (H9) compared to hECCs (NTERA2), these represent cell type specific signatures. Further, our combined results of GC-MS and microarray based gene expression profiling of undifferentiated and OCT4-depleted hESCs are consistent with the Warburg effect which is increased glycolysis in embryonic cells and tumor cells in the presence of O2 while oxidative phosphorylation (OXPHOS) is impaired or even shut down. RNAi-based OCT4 knock down mediated differentiation resulted in the activation of the poised OXPHOS machinery by expressing missing key proteins such as NDUFC1, UQCRB and COX, increase in TCA cycle activity and decreased lactate metabolism. These results shed light on the metabolite layer of pluripotent stem cells and could potentially establish novel metabolic markers of self renewal and pluripotency

studies of DPPA4 in Human Pluripotent Stem Cells

Pluripotent human embryonic stem cells (hES) can proliferate indefinitely in vitro and differentiate in all three germ layers and represent therefore a valuable tool for drug discovery, cell replacement therapy and regenerative medicine. Therefore, it is fundamental to understand the genetic network that regulates' pluripotency. A microarray experiment carried out in our laboratory compared the expression profile of undifferentiated versus differentiated hES cells. The results highlighted the down regulation upon differentiation of both already known master genes of pluripotency, like OCT3/4 and NANOG, and others, some of which were so far not associated with the undifferentiated state. We focused investigations on DPPA4, a SAP domain protein that has been associated with pluripotency, but remains poorly understood. SAP domain proteins are mainly implicated in chromosomal organisation, DNA repair and RNA metabolism. The aim of this study was to determine whether DPPA4 maintains pluripotency. Using RNAi we demonstrated that DPPA4 is not regulating the pluripotent state. Morphological differentiation and an accompanied reduced growth curve were induced, however, key markers of pluripotency, eg the transcription factors NANOG, OCT3/4 and SOX2 were unaffected. Unexpectedly, CDX2 was upregulated, but not hCG, both are markers of the trophoectodermal lineage. The cell surface antigens SSEA3 and TRA1-60, which are expressed in the undifferentiated state and downregulated upon differentiation were unaffected upon DPPA4 knock down. However, SSEA1 another cell. surface antigen, that has a - reciprocal expression pattern to SSEA3, was upregulated. Expression analysis in human cell lines, including hES and hEC cells, using a generated polyclonal peptide anti-DPPA4 antibody showed that all pluirpotent stem cells expressed DPPA4. However, it was also expressed in nullipotent hEC cells and on lower levels in choriocarcinoma cells, indicating that DPPA4 does not induce pluripotency. In localization studies using a DPPA4-EGFP fusion protein and immunohistochemistry experiments we found that DPPA4 is associated with chromatin, thus narrowing the potential role of this SAP domain protein. Taken together, we hypothesize that DPPA4 is playing a role in chromatin remodelling of pluripotent cells and some germ cell tumours.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Heat map representing the relative quantities of significantly changed metabolites from polar extracts of hESC versus hECCs.

<p>Statistical analysis (T-Test) was done with MeV (p-value <0.05).</p

Scatter-plot of normalized peak areas of metabolites detected in hESCs versus hECCs.

<p>Only metabolites with more than 3-fold difference are named. Error bars are the standard error.</p

Metabolites more than 2-fold higher in hESC compared to hECC cells.

<p>Metabolites present in DMEM medium written in bold.</p>1<p>Statistical significance was determined by a T-test with adjusted Bonferroni correction in MeV. P-values <0.05 marked as *, P-values <0.01 marked as **.</p

Identified metabolites present in hESCs and hECCs.

<p>Metabolites present in DMEM medium written in bold.</p>1<p>Statistical significance was determined by a T-test with adjusted Bonferroni correction in MeV. P-values <0.05 marked as *, p-values <0.01 marked as **.</p

List of metabolite associated pathways in OCT4 depleted hESCs compared to EGFP depleted control cells.

<p>Shown are the numbers of genes and their difference enriched for the specific pathways.</p

Qualitative modeling identifies IL-11 as a novel regulator in maintaining self-renewal in human pluripotent stem cells

Pluripotency in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is regulated by three transcription factors—OCT3/4, SOX2, and NANOG. To fully exploit the therapeutic potential of these cells it is essential to have a good mechanistic understanding of the maintenance of self-renewal and pluripotency. In this study, we demonstrate a powerful systems biology approach in which we first expand literature-based network encompassing the core regulators of pluripotency by assessing the behavior of genes targeted by perturbation experiments. We focused our attention on highly regulated genes encoding cell surface and secreted proteins as these can be more easily manipulated by the use of inhibitors or recombinant proteins. Qualitative modeling based on combining boolean networks and in silico perturbation experiments were employed to identify novel pluripotency-regulating genes. We validated Interleukin-11 (IL-11) and demonstrate that this cytokine is a novel pluripotency-associated factor capable of supporting self-renewal in the absence of exogenously added bFGF in culture. To date, the various protocols for hESCs maintenance require supplementation with bFGF to activate the Activin/Nodal branch of the TGFβ signaling pathway. Additional evidence supporting our findings is that IL-11 belongs to the same protein family as LIF, which is known to be necessary for maintaining pluripotency in mouse but not in human ESCs. These cytokines operate through the same gp130 receptor which interacts with Janus kinases. Our finding might explain why mESCs are in a more naïve cell state compared to hESCs and how to convert primed hESCs back to the naïve state. Taken together, our integrative modeling approach has identified novel genes as putative candidates to be incorporated into the expansion of the current gene regulatory network responsible for inducing and maintaining pluripotency