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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Gamete failure-derived infertility affects millions of people worldwide; for many patients, gamete donation by unrelated donors is the only available treatment. Embryonic stem cells (ESCs) can differentiate in vitro into germ-like cells, but they are genetically unrelated to the patient. Using an in vitro protocol that aims at recapitulating development, we have achieved, for the first time, complete differentiation of human induced pluripotent stem cells (hiPSCs) to postmeiotic cells. Unlike previous reports using human ESCs, postmeiotic cells arose without the over-expression of germline related transcription factors. Moreover, we consistently obtained haploid cells from hiPSCs of different origin (keratinocytes and cord blood), produced with a different number of transcription factors, and of both genetic sexes, suggesting the independence of our approach from the epigenetic memory of the reprogrammed somatic cells. Our work brings us closer to the production of personalized human gametes in vitro.

Generation of human induced pluripotent stem cells (hiPSCs) by the expression of specific transcription factors depends on successful epigenetic reprogramming to a pluripotent state. Although hiPSCs and human embryonic stem cells (hESCs) display a similar epigenome, recent reports demonstrated the persistence of specific epigenetic marks from the somatic cell type of origin and aberrant methylation patterns in hiPSCs. However, it remains unknown whether the use of different somatic cell sources, encompassing variable levels of selection pressure during reprogramming, influences the level of epigenetic aberrations in hiPSCs. In this work, we characterized the epigenomic integrity of 17 hiPSC lines derived from six different cell types with varied reprogramming efficiencies. We demonstrate that epigenetic aberrations are a general feature of the hiPSC state and are independent of the somatic cell source. Interestingly, we observe that the reprogramming efficiency of somatic cell lines inversely correlates with the amount of methylation change needed to acquire pluripotency. Additionally, we determine that both shared and linespecific epigenetic aberrations in hiPSCs can directly translate into changes in gene expression in both the pluripotent and differentiated states. Significantly, our analysis of different hiPSC lines from multiple cell types of origin allow us to identify a reprogrammingspecific epigenetic signature comprised of nine aberrantly methylated genes that is able to segregate hESC and hiPSC lines regardless of the somatic cell source or differentiation state.I nduction of pluripotency in human somatic cells is an inefficient process that can be achieved by the expression of defined transcription factors (1-5). This reprogramming process involves global epigenetic remodeling and overcoming similar roadblocks present during cell transformation, which might affect genomic and epigenomic integrity (6). In fact, several recent reports have shown that human induced pluripotent stem cells (hiPSCs) contain genetic and epigenetic aberrations throughout their genome compared with their parental somatic cell populations or to human embryonic stem cells (hESCs) (7-12). For example, the analysis of whole-genome DNA methylation profiles at single-nucleotide resolution in hiPSCs, their somatic cells of origin, and hESCs revealed the presence of more than 1,000 differentially methylated regions (DMRs) between hiPSCs and hESCs (11). Moreover, this analysis, and many others, demonstrated both the persistence of specific epigenetic marks from the somatic cell of origin (residual methylation) and the acquisition of unique methylation patterns in mouse iPSCs (miPSCs) and hiPSCs (11,(13)(14)(15)(16)(17)(18)(19)(20)(21). Interestingly, hiPSC lines also show incomplete reprogramming of non-CG methylation in regions proximal to telomeres and centromeres (11). Altogether, these epigenetic aberrations might explain some of the observed transcriptional variation between hESC and hiPSC lines (22)(23)(24). In one of the most co...

IntroductionStem cells are traditionally considered to be either multipotent (eg, embryonic stem [ES] cells) or restricted in their differentiation potential (tissue stem cells). Reports on "transdifferentiation" and the discovery of multipotent adult progenitor cells in bone marrow, brain, and muscle have recently challenged this view. [1][2][3][4][5] One central issue underlying the debate on developmental options of stem cells concerns the molecular mechanisms responsible for establishing and maintaining their transcriptional programs and decisions to differentiate. So far, few of the key regulatory genes active in stem cells and/or multipotent progenitors have been studied at the level of transcriptional regulation. [6][7][8][9][10][11][12][13][14][15][16][17] Identification and comparison of several such genes will provide important insights into the transcriptional programs of stem and multipotent progenitor cells.The Kit (White spotting locus) gene, encoding the transmembrane receptor of the cytokine stem cell factor/Kit ligand (KL), is an important regulator of proliferation/survival and/or migration of several stem cell types such as the primordial germ cells (PGCs), [18][19][20][21][22] the multipotent hematopoietic stem cells (HSCs), 20,23 the neural crest, and the intestinal Cajal cells. 20 Null mutations in the Kit or the KL (Steel) gene result in severe hematopoietic and germ cell defects and in utero or perinatal death, whereas mutations that diminish Kit tyrosine kinase activity or KL production affect mainly hematopoiesis and the development of germ cells, melanocytes, and the intestinal Cajal cells. 20 During mouse developmentKit is expressed at a low level in pluripotent inner cell mass cells (and in epiblast-derived ES cells in culture) 24,25 and, at relatively higher levels, in PGCs, early hematopoietic progenitors, and other cells. 25 In adults, Kit is expressed in a variety of cell types including HSCs, immature hematopoietic progenitors and mast cells, oocytes, a subpopulation of male germ cells, and melanocytes. [18][19][20][21][22][23]25 In the mouse embryo, hematopoietic progenitors are detected both extraembryonically in the yolk sac, after embryonic day 7 (E7), and intraembryonically, first in the para-aortic splanchnopleura and aorta-gonad-mesonephros (AGM) regions, then in fetal liver and vitelline vessels, [26][27][28] and finally in bone marrow. The earlier hematopoietic cells (primitive hematopoietic cells) are morphologically and biologically distinct from the later cells (definitive hematopoietic cells). Definitive hematopoiesis is seeded by HSCs, which arise intraembryonically in the AGM region and in the vitelline vessels around E11. [28][29][30] Immature precursors to definitive HSCs, however, have also been detected both intraembryonically 31,32 and in the yolk sac 32 at earlier stages of development. Murine PGCs first become visible around E7 in the extraembryonic mesoderm, then migrate through the allantois (E8) to the hindgut, from where they move to reach the gonadal ...