BackgroundHuman pluripotent stem cells have the ability to generate all cell types present in the adult organism, therefore harboring great potential for the in vitro study of differentiation and for the development of cell-based therapies. Nonetheless their use may prove challenging as incomplete differentiation of these cells might lead to tumoregenicity. Interestingly, many cancer types have been reported to display metabolic modifications with features that might be similar to stem cells. Understanding the metabolic properties of human pluripotent stem cells when compared to their differentiated counterparts can thus be of crucial importance. Furthermore recent data has stressed distinct features of different human pluripotent cells lines, namely when comparing embryo-derived human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs) reprogrammed from somatic cells.Methodology/Principal FindingsWe compared the energy metabolism of hESCs, IPSCs, and their somatic counterparts. Focusing on mitochondria, we tracked organelle localization and morphology. Furthermore we performed gene expression analysis of several pathways related to the glucose metabolism, including glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. In addition we determined oxygen consumption rates (OCR) using a metabolic extracellular flux analyzer, as well as total intracellular ATP levels by high performance liquid chromatography (HPLC). Finally we explored the expression of key proteins involved in the regulation of glucose metabolism.Conclusions/FindingsOur results demonstrate that, although the metabolic signature of IPSCs is not identical to that of hESCs, nonetheless they cluster with hESCs rather than with their somatic counterparts. ATP levels, lactate production and OCR revealed that human pluripotent cells rely mostly on glycolysis to meet their energy demands. Furthermore, our work points to some of the strategies which human pluripotent stem cells may use to maintain high glycolytic rates, such as high levels of hexokinase II and inactive pyruvate dehydrogenase (PDH).

Summary Human embryonic (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia nor haploid spermatocytes or spermatids. Here we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages including post-meiotic, spermatid-like cells in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating pluripotent stem cells into UTF1, PLZF and CDH1-positive spermatogonia-like cells, HIWI and HILI-positive spermatocyte-like cells, and haploid cells expressing acrosin, transition protein 1 and protamine 1, proteins found uniquely in either spermatids and/or sperm. These spermatids show uniparental genomic imprints similar to human sperm on two loci: H19 and IGF2. These results demonstrate that male pluripotent stem cells have the capability to directly differentiate into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro.

BackgroundInduced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming, which uses a defined set of transcription factors, iPS cells represent important sources of patient-specific cells for clinical applications. However, before these cells can be used in therapeutic designs, it is essential to understand their genetic stability.Methodology/Principal FindingsHere, we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation, iPS cells activate checkpoint signaling, evidenced by phosphorylation of ATM, NBS1, CHEK2, and TP53, localization of ATM to the double strand breaks (DSB), and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G2 phase of the cell cycle, displaying a lack of the G1/S cell cycle arrest similar to human embryonic stem (ES) cells. Furthermore, both cell types remove DSB within six hours of γ-irradiation, form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally, we report elevated expression of genes involved in DNA damage signaling, checkpoint function, and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts.Conclusions/SignificanceHigh degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage, dramatic changes in cell cycle structure, including a high percentage of cells in the S phase, increased radiosensitivity and loss of DNA damage-induced G1/S cell cycle arrest, were observed in stem cells generated by induced pluripotency.

Deciding to exit pluripotency and undergo differentiation is of singular importance for pluripotent cells, including embryonic stem cells (ESCs). The molecular mechanisms for these decisions to differentiate, as well as reversing those decisions during induced pluripotency (iPS), have focused largely on transcriptomic controls. Here, we explore the role of translational control for the maintenance of pluripotency and the decisions to differentiate. Global protein translation is significantly reduced in hESCs compared to their differentiated progeny. Furthermore, p70 S6K activation is restricted in hESCs compared to differentiated fibroblast-like cells. Disruption of p70 S6K-mediated translation by rapamycin or siRNA knockdown in undifferentiated hESCs does not alter cell viability or expression of the pluripotency markers Oct4 and Nanog. However, expression of constitutively active p70 S6K, but not wild-type p70 S6K, induces differentiation. Additionally, hESCs exhibit high levels of the mTORC1/p70 S6K inhibitory complex TSC1/TSC2 and preferentially express more rapamycin insensitive mTORC2 compared to differentiated cells. siRNA-mediated knockdown of both TSC2 and Rictor elevates p70 S6K activation and induces differentiation of hESCs. These results suggest that hESCs tightly regulate mTORC1/p70 S6K-mediated protein translation to maintain a pluripotent state as well as implicate a novel role for protein synthesis as a driving force behind hESC differentiation.