AKT1 induces Nanog promoter in a SUMOylation-dependent manner in different pluripotent contexts.

AKT/PKB is a kinase crucial for pluripotency maintenance in pluripotent stem cells. Multiple post-translational modifications modulate its activity. We have previously demonstrated that AKT1 induces the expression of the pluripotency transcription factor Nanog in a SUMOylation-dependent manner in mouse embryonic stem cells. Here, we studied different cellular contexts and main candidates that could mediate this induction. Our results strongly suggest the pluripotency transcription factors OCT4 and SOX2 are not essential mediators. Additionally, we concluded that this induction takes place in different pluripotent contexts but not in terminally differentiated cells. Finally, the cross-matching analysis of ESCs, iPSCs and MEFs transcriptomes and AKT1 phosphorylation targets provided new clues about possible factors that could be involved in the SUMOylation-dependent Nanog induction by AKT.

The dynamical organization of the core pluripotency transcription factors responds to differentiation cues in early S-phase.

DNA replication in stem cells is a major challenge for pluripotency preservation and cell fate decisions. This process involves massive changes in the chromatin architecture and the reorganization of many transcription-related molecules in different spatial and temporal scales. Pluripotency is controlled by the master transcription factors (TFs) OCT4, SOX2 and NANOG that partition into condensates in the nucleus of embryonic stem cells. These condensates are proposed to play relevant roles in the regulation of gene expression and the maintenance of pluripotency. Here, we asked whether the dynamical distribution of the pluripotency TFs changes during the cell cycle, particularly during DNA replication. Since the S phase is considered to be a window of opportunity for cell fate decisions, we explored if differentiation cues in G1 phase trigger changes in the distribution of these TFs during the subsequent S phase. Our results show a spatial redistribution of TFs condensates during DNA replication which was not directly related to chromatin compaction. Additionally, fluorescence fluctuation spectroscopy revealed TF-specific, subtle changes in the landscape of TF-chromatin interactions, consistent with their particularities as key players of the pluripotency network. Moreover, we found that differentiation stimuli in the preceding G1 phase triggered a relatively fast and massive reorganization of pluripotency TFs in early-S phase. Particularly, OCT4 and SOX2 condensates dissolved whereas the lifetimes of TF-chromatin interactions increased suggesting that the reorganization of condensates is accompanied with a change in the landscape of TF-chromatin interactions. Notably, NANOG showed impaired interactions with chromatin in stimulated early-S cells in line with its role as naïve pluripotency TF. Together, these findings provide new insights into the regulation of the core pluripotency TFs during DNA replication of embryonic stem cells and highlight their different roles at early differentiation stages.

Sumoylation and the oncogenic E17K mutation affect AKT1 subcellular distribution and impact on Nanog-binding dynamics to chromatin in embryonic stem cells.

AKT/PKB is a kinase involved in the regulation of a plethora of cell processes. Particularly, in embryonic stem cells (ESCs), AKT is crucial for the maintenance of pluripotency. Although the activation of this kinase relies on its recruitment to the cellular membrane and subsequent phosphorylation, multiple other post-translational modifications (PTMs), including SUMOylation, fine-tune its activity and target specificity. Since this PTM can also modify the localization and availability of different proteins, in this work we explored if SUMOylation impacts on the subcellular compartmentalization and distribution of AKT1 in ESCs. We found that this PTM does not affect AKT1 membrane recruitment, but it modifies the AKT1 nucleus/cytoplasm distribution, increasing its nuclear presence. Additionally, within this compartment, we found that AKT1 SUMOylation also impacts on the chromatin-binding dynamics of NANOG, a central pluripotency transcription factor. Remarkably, the oncogenic E17K AKT1 mutant produces major changes in all these parameters increasing the binding of NANOG to its targets, also in a SUMOylation dependent manner. These findings demonstrate that SUMOylation modulates AKT1 subcellular distribution, thus adding an extra layer of regulation of its function, possibly by affecting the specificity and interaction with its downstream targets.

Extracellular vesicles from pluripotent stem cell-derived mesenchymal stem cells acquire a stromal modulatory proteomic pattern during differentiation.

Mesenchymal stem/stromal cells (MSCs) obtained from pluripotent stem cells (PSCs) constitute an interesting alternative to classical MSCs in regenerative medicine. Among their many mechanisms of action, MSC extracellular vesicles (EVs) are a potential suitable substitute for MSCs in future cell-free-based therapeutic approaches. Unlike cells, EVs do not elicit acute immune rejection, and they can be produced in large quantities and stored until ready to use. Although the therapeutic potential of MSC EVs has already been proven, a thorough characterization of MSC EVs is lacking. In this work, we used a label-free liquid chromatography tandem mass spectrometry proteomic approach to identify the most abundant proteins in EVs that are secreted from MSCs derived from PSCs (PD-MSCs) and from their parental induced PSCs (iPSCs). Next, we compared both datasets and found that while iPSC EVs enclose proteins that modulate RNA and microRNA stability and protein sorting, PD-MSC EVs are rich in proteins that organize extracellular matrix, regulate locomotion, and influence cell-substrate adhesion. Moreover, compared to their respective cells, iPSCs and iPSC EVs share a greater proportion of proteins, while the PD-MSC proteome appears to be more specific. Correlation and principal component analysis consistently aggregate iPSCs and iPSC EVs but segregate PD-MSC and their EVs. Altogether, these findings suggest that during differentiation, compared with their parental iPSC EVs, PD-MSC EVs acquire a more specific set of proteins; arguably, this difference might confer their therapeutic properties.

AKT/GSK3ß signaling pathway is critically involved in human pluripotent stem cell survival.

Human embryonic and induced pluripotent stem cells are self-renewing pluripotent stem cells (PSC) that can differentiate into a wide range of specialized cells. Basic fibroblast growth factor is essential for PSC survival, stemness and self-renewal. PI3K/AKT pathway regulates cell viability and apoptosis in many cell types. Although it has been demonstrated that PI3K/AKT activation by bFGF is relevant for PSC stemness maintenance its role on PSC survival remains elusive. In this study we explored the molecular mechanisms involved in the regulation of PSC survival by AKT. We found that inhibition of AKT with three non-structurally related inhibitors (GSK690693, AKT inhibitor VIII and AKT inhibitor IV) decreased cell viability and induced apoptosis. We observed a rapid increase in phosphatidylserine translocation and in the extent of DNA fragmentation after inhibitors addition. Moreover, abrogation of AKT activity led to Caspase-9, Caspase-3, and PARP cleavage. Importantly, we demonstrated by pharmacological inhibition and siRNA knockdown that GSK3ß signaling is responsible, at least in part, of the apoptosis triggered by AKT inhibition. Moreover, GSK3ß inhibition decreases basal apoptosis rate and promotes PSC proliferation. In conclusion, we demonstrated that AKT activation prevents apoptosis, partly through inhibition of GSK3ß, and thus results relevant for PSC survival.

Generation of iPSC line iPSC-FH2.1 in hypoxic conditions from human foreskin fibroblasts.

Human foreskin fibroblasts were used to generate the iPSC line iPSC-FH2.1 using the EF1a-hSTEMCCA-loxP vector expressing OCT4, SOX2, c-MYC and KLF4, in 5% O2 culture conditions. Stemness was confirmed, as was pluripotency both in vivo and in vitro, in normoxia and hypoxia. Human Embryonic Stem Cell (hESC) line WA-09 and reprogrammed fibroblast primary culture HFF-FM were used as controls.