Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense, or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, while the rest were newly occurring during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS safety before clinical use.

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Metabolism is vital to every aspect of cell function, yet the metabolome of iPSCs remains largely unexplored. Here we report, using an untargeted metabolomics approach, that human iPSCs share a pluripotent metabolomic signature with ESCs that is distinct from their parental cells, and that is characterized by changes in metabolites involved in cellular respiration. Examination of cellular bioenergetics corroborated with our metabolomic analysis, and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency. Interestingly, the bioenergetics of various somatic cells correlated with their reprogramming efficiencies. We further identified metabolites that differ between iPSCs and ESCs, which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming. Our findings are the first to globally analyze the metabolome of iPSCs, and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency, and in evaluating iPSC and ESC equivalence.

Nuclear architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as aging1-4. It is then plausible that diseases whose manifestations correlate with aging might be connected to the appearance of nuclear aberrations over time. We decided to evaluate nuclear organization in the context of aging-associated disorders by focusing on a Leucine Rich Repeat Kinase 2 (LRRK2) dominant mutation (G2019S) shown to associate with familial and sporadic Parkinson’s Disease (PD), as well as impairment of adult neurogenesis in mice5. Here, we report on the generation of PD patient-derived induced pluripotent stem cells (iPSCs) and the implications of LRRK2(G2019S) in human neural stem cell (NSC) populations. Mutant NSCs showed increased susceptibility to proteasomal stress as well as passage-dependent deficiencies in clonal expansion and neuronal differentiation. Disease phenotypes were rescued by targeted correction of the LRRK2(G2019S) mutation with its wild-type counterpart in PD-iPSCs and recapitulated upon targeted knock-in of LRRK2(G2019S) in human embryonic stem cells (hESCs). Analysis of human brain tissue showed nuclear envelope impairment in clinically diagnosed Parkinson’s patients. Altogether, our results identify the nucleus as a previously unknown cellular organelle in Parkinson’s pathology and may help open new avenues for PD diagnoses as well as potential development of therapeutics targeting this fundamental cell structure.

Summary Human embryonic stem (hES) cells show an atypical cell cycle regulation characterized by a high proliferation rate and a short G1-phase [1, 2]. In fact, a shortened G1-phase might protect ES cells from external signals inducing differentiation, as shown for certain stem cells [3]. It has been suggested that self-renewal and pluripotency are intimately linked to cell cycle regulation in ES cells [4–6] although little is known about the overall importance of the cell cycle machinery in maintaining ES cell identity. An appealing model to address whether the acquisition of stem cell properties are linked to cell cycle regulation emerged with the ability to generate induced pluripotent stem (iPS) cells by expression of defined transcription factors [7–11]. Here, we show that the characteristic cell cycle signature of hES cells is acquired as an early event in cell reprogramming. We demonstrate that induction of cell proliferation increases reprogramming efficiency whereas cell cycle arrest inhibits successful reprogramming. Furthermore, we show that cell cycle arrest is sufficient to drive hES cells towards irreversible differentiation. Our results establish a link that intertwines the mechanisms of cell cycle control to the mechanisms underlying the acquisition and maintenance of ES cell identity.