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Post-translational modifications （PTMs） are known to be essential mechanisms used by eukaryotic cells to diversify their protein functions and dynamically coordinate their signaling networks. Defects in PTMs have been linked to numerous developmental disorders and human diseases, highlighting the importance of PTMs in maintaining nor- mal cellular states. Human pluripotent stem ceils （hPSCs）, including embryonic stem ceils （hESCs） and induced plu- ripotent stem cells （hiPSCs）, are capable of self-renewal and differentiation into a variety of functional somatic cells; these cells hold a great promise for the advancement of biomedical research and clinical therapy. The mechanisms underlying cellular pluripotency in human cells have been extensively explored in the past decade. In addition to the vast amount of knowledge obtained from the genetic and transcriptional research in hPSCs, there is a rapidly growing interest in the stem cell biology field to examine pluripotency at the protein and PTM level. This review addresses recent progress toward understanding the role of PTMs （glycosylation, phosphorylation, acetylation and methylation） in the regulation of cellular pluripotency.

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. In addition to cognitive deficits, FXS patients exhibit hyperactivity, attention deficits, social difficulties, anxiety, and other autistic-like behaviors. FXS is caused by an expanded CGG trinucleotide repeat in the 5' untranslated region of the Fragile X Mental Retardation (FMR1) gene leading to epigenetic silencing and loss of expression of the Fragile X Mental Retardation protein (FMRP). Despite the known relationship between FMR1 CGG repeat expansion and FMR1 silencing, the epigenetic modifications observed at the FMR1 locus, and the consequences of the loss of FMRP on human neurodevelopment and neuronal function remain poorly understood. To address these limitations, we report on the generation of induced pluripotent stem cell (iPSC) lines from multiple patients with FXS and the characterization of their differentiation into post-mitotic neurons and glia. We show that clones from reprogrammed FXS patient fibroblast lines exhibit variation with respect to the predominant CGG-repeat length in the FMR1 gene. In two cases, iPSC clones contained predominant CGG-repeat lengths shorter than measured in corresponding input population of fibroblasts. In another instance, reprogramming a mosaic patient having both normal and pre-mutation length CGG repeats resulted in genetically matched iPSC clonal lines differing in FMR1 promoter CpG methylation and FMRP expression. Using this panel of patient-specific, FXS iPSC models, we demonstrate aberrant neuronal differentiation from FXS iPSCs that is directly correlated with epigenetic modification of the FMR1 gene and a loss of FMRP expression. Overall, these findings provide evidence for a key role for FMRP early in human neurodevelopment prior to synaptogenesis and have implications for modeling of FXS using iPSC technology. By revealing disease-associated cellular phenotypes in human neurons, these iPSC models will aid in the discovery of novel therapeutics for FXS and other autism-spectrum disorders sharing common pathophysiology.

RNA-Seq of single cells has been limited by biases in transcript coverage and unknown technical variability. Ramsköld et al . describe a protocol to reproducibly recover full-length transcripts and use it to quantitatively analyze splice isoforms in single cells. Genome-wide transcriptome analyses are routinely used to monitor tissue-, disease- and cell type–specific gene expression, but it has been technically challenging to generate expression profiles from single cells. Here we describe a robust mRNA-Seq protocol (Smart-Seq) that is applicable down to single cell levels. Compared with existing methods, Smart-Seq has improved read coverage across transcripts, which enhances detailed analyses of alternative transcript isoforms and identification of single-nucleotide polymorphisms. We determined the sensitivity and quantitative accuracy of Smart-Seq for single-cell transcriptomics by evaluating it on total RNA dilution series. We found that although gene expression estimates from single cells have increased noise, hundreds of differentially expressed genes could be identified using few cells per cell type. Applying Smart-Seq to circulating tumor cells from melanomas, we identified distinct gene expression patterns, including candidate biomarkers for melanoma circulating tumor cells. Our protocol will be useful for addressing fundamental biological problems requiring genome-wide transcriptome profiling in rare cells.

Increasing evidence has demonstrated that epigenetic factors can profoundly influence gene expression and, in turn, influence resistance or susceptibility to disease. Epigenetic drugs, such as histone deacetylase (HDAC) inhibitors, are finding their way into clinical practice, although their exact mechanisms of action are unclear. To identify mechanisms associated with HDAC inhibition, we performed microarray analysis on brain and muscle samples treated with the HDAC1/3-targeting inhibitor, HDACi 4b. Pathways analyses of microarray datasets implicate DNA methylation as significantly associated with HDAC inhibition. Further assessment of DNA methylation changes elicited by HDACi 4b in human fibroblasts from normal controls and patients with Huntington’s disease (HD) using the Infinium HumanMethylation450 BeadChip revealed a limited, but overlapping, subset of methylated CpG sites that were altered by HDAC inhibition in both normal and HD cells. Among the altered loci of Y chromosome-linked genes, KDM5D , which encodes Lys (K)-specific demethylase 5D, showed increased methylation at several CpG sites in both normal and HD cells, as well as in DNA isolated from sperm from drug-treated male mice. Further, we demonstrate that first filial generation (F1) offspring from drug-treated male HD transgenic mice show significantly improved HD disease phenotypes compared with F1 offspring from vehicle-treated male HD transgenic mice, in association with increased Kdm5d expression, and decreased histone H3 Lys4 (K4) (H3K4) methylation in the CNS of male offspring. Additionally, we show that overexpression of Kdm5d in mutant HD striatal cells significantly improves metabolic deficits. These findings indicate that HDAC inhibitors can elicit transgenerational effects, via cross-talk between different epigenetic mechanisms, to have an impact on disease phenotypes in a beneficial manner. Significance We demonstrate that histone deacetylase (HDAC) inhibition can elicit changes in DNA methylation in Huntington’s disease (HD) human fibroblasts, as well as in sperm from HD transgenic mice, in association with DNA methylation-related gene expression changes. We suggest that alterations in sperm DNA methylation lead to transgenerational effects, and, accordingly, we show that first filial generation (F1) offspring of HDAC inhibitor-treated male HD transgenic mice show improved HD disease phenotypes compared with F1 offspring from vehicle-treated male HD transgenic mice. These findings have significant implications for human health because they enforce the concept that ancestral drug exposure may be a major molecular factor that can affect disease phenotypes, yet in a positive manner. Further, we implicate Lys (K)-specific demethylase 5d expression in this phenomenon.

Neural stem cell (NSC) transplantation represents an unexplored approach for treating neurodegenerative disorders associated with cognitive decline such as Alzheimer disease (AD). Here, we used aged triple transgenic mice (3xTg-AD) that express pathogenic forms of amyloid precursor protein, presenilin, and tau to investigate the effect of neural stem cell transplantation on AD-related neuropathology and cognitive dysfunction. Interestingly, despite widespread and established Ass plaque and neurofibrillary tangle pathology, hippocampal neural stem cell transplantation rescues the spatial learning and memory deficits in aged 3xTg-AD mice. Remarkably, cognitive function is improved without altering Ass or tau pathology. Instead, the mechanism underlying the improved cognition involves a robust enhancement of hippocampal synaptic density, mediated by brain-derived neurotrophic factor (BDNF). Gain-of-function studies show that recombinant BDNF mimics the beneficial effects of NSC transplantation. Furthermore, loss-of-function studies show that depletion of NSC-derived BDNF fails to improve cognition or restore hippocampal synaptic density. Taken together, our findings demonstrate that neural stem cells can ameliorate complex behavioral deficits associated with widespread Alzheimer disease pathology via BDNF.

Down’s syndrome (DS), caused by trisomy of human chromosome 21, is the most common genetic cause of intellectual disability. Here we use induced pluripotent stem cells (iPSCs) derived from DS patients to identify a role for astrocytes in DS pathogenesis. DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules. Astrocyte-conditioned medium collected from DS astroglia causes toxicity to neurons, and fails to promote neuronal ion channel maturation and synapse formation. Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo . We also observed abnormal gene expression profiles from DS astroglia. Finally, we show that the FDA-approved antibiotic drug, minocycline, partially corrects the pathological phenotypes of DS astroglia by specifically modulating the expression of S100B, GFAP, inducible nitric oxide synthase, and thrombospondins 1 and 2 in DS astroglia. Our studies shed light on the pathogenesis and possible treatment of DS by targeting astrocytes with a clinically available drug. Down’s syndrome is characterized by intellectual disability and other neuropathological symptoms. Here, the authors show that astroglia derived from induced pluripotent stem cells from Down’s syndrome patients impair the development of neurons, and that this can be attenuated with the drug minocycline.

There is concern that the stresses of inducing pluripotency may lead to deleterious DNA mutations in induced pluripotent stem cell (iPSC) lines, which would compromise their use for cell therapies. Here we report comparative genomic analysis of nine isogenic iPSC lines generated using three reprogramming methods: integrating retroviral vectors, non-integrating Sendai virus and synthetic mRNAs. We used whole-genome sequencing and de novo genome mapping to identify single-nucleotide variants, insertions and deletions, and structural variants. Our results show a moderate number of variants in the iPSCs that were not evident in the parental fibroblasts, which may result from reprogramming. There were only small differences in the total numbers and types of variants among different reprogramming methods. Most importantly, a thorough genomic analysis showed that the variants were generally benign. We conclude that the process of reprogramming is unlikely to introduce variants that would make the cells inappropriate for therapy. It is feared that reprogramming may introduce DNA mutations. Here Bhutani et al . take three different reprogramming methods and using comparative whole genome analyses do identify nucleotide variations that are different in reprogrammed cells from the original fibroblasts, but none convey oncogenic potential.

The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies.

Pluripotent stem cells (PSCs) express a distinctive set of microRNAs (miRNAs). Many of these miRNAs have similar targeting sequences and are predicted to regulate downstream targets cooperatively. These enriched miRNAs are involved in the regulation of the unique PSC cell cycle, and there is increasing evidence that they also influence other important characteristics of PSCs, including their morphology, epigenetic profile and resistance to apoptosis. Detailed studies of miRNAs and their targets in PSCs should help to parse the regulatory networks that underlie developmental processes and cellular reprogramming.

Classifiers trained on a large microarray gene expression dataset can assess pluripotency in query samples of human cells. Pluripotent stem cells (PSCs) are defined by their potential to generate all cell types of an organism. The standard assay for pluripotency of mouse PSCs is cell transmission through the germline, but for human PSCs researchers depend on indirect methods such as differentiation into teratomas in immunodeficient mice. Here we report PluriTest, a robust open-access bioinformatic assay of pluripotency in human cells based on their gene expression profiles.