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iPS cell model of LEOPARD syndrome Patient-specific iPS (induced pluripotent stem) cells are seen as key to modelling genetic disorders and developing new treatments for them. Now iPS cell lines have been generated by nuclear reprogramming from patients with LEOPARD syndrome, a rare developmental disorder characterized by skin lesions, heart abnormalities and deafness. Cardiomyocytes derived from the resulting LEOPARD iPS cells have hypertrophic properties resembling those typical of the disease — cardiac hypertrophy occurs in 90% of children with the syndrome. The reprogrammed cells feature extensive alterations in various signal transduction pathway components, including RAS–MAPK, some previously described in association with cardiac hypertrophy. Using these cell lines, together with robust differentiation protocols, it may be possible to identify compounds that reverse diseased cellular phenotypes. The generation of induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders promises to help the basic understanding of complex diseases and the development of therapeutics. Here iPSCs have been generated from patients with LEOPARD syndrome, a developmental disorder with pleiomorphic effects on several tissues and organs. The iPSCs are characterized and the phenotype of cardiomyocytes derived from these cells is investigated. The generation of reprogrammed induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders holds the promise of increased understanding of the aetiologies of complex diseases and may also facilitate the development of novel therapeutic interventions. We have generated iPSCs from patients with LEOPARD syndrome (an acronym formed from its main features; that is, lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitalia, retardation of growth and deafness), an autosomal-dominant developmental disorder belonging to a relatively prevalent class of inherited RAS–mitogen-activated protein kinase signalling diseases, which also includes Noonan syndrome, with pleomorphic effects on several tissues and organ systems 1 , 2 . The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSCs have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro -derived cardiomyocytes from LEOPARD syndrome iPSCs are larger, have a higher degree of sarcomeric organization and preferential localization of NFATC4 in the nucleus when compared with cardiomyocytes derived from human embryonic stem cells or wild-type iPSCs derived from a healthy brother of one of the LEOPARD syndrome patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signalling pathways that may promote the disease phenotype.

The chromatin template imposes an epigenetic barrier during the process of somatic cell reprogramming. Using fibroblasts derived from macroH2A double knockout (dKO) mice, here we show that these histone variants act cooperatively as a barrier to induced pluripotency. Through manipulation of macroH2A isoforms, we further demonstrate that macroH2A2 is the predominant barrier to reprogramming. Genomic analyses reveal that macroH2A1 and macroH2A2, together with H3K27me3, co-occupy pluripotency genes in wild-type (wt) fibroblasts. In particular, we find macroH2A isoforms to be highly enriched at target genes of the K27me3 demethylase, Utx, which are reactivated early in iPS reprogramming. Finally, while macroH2A dKO-induced pluripotent cells are able to differentiate properly in vitro and in vivo , such differentiated cells retain the ability to return to a stem-like state. Therefore, we propose that macroH2A isoforms provide a redundant silencing layer or terminal differentiation ‘lock’ at critical pluripotency genes that presents as an epigenetic barrier when differentiated cells are challenged to reprogram. Chromatin templates can act as barriers against cellular reprogramming. Gaspar-Maia and colleagues use mouse models deficient in the histone variants macroH2A1 and macroH2A2, and find that macroH2A functions as an epigenetic barrier against induced pluripotency by silencing Utx target genes.

Drug-induced gene expression profiles can identify potential mechanisms of toxicity. We focus on obtaining signatures for cardiotoxicity of FDA-approved tyrosine kinase inhibitors (TKIs) in human induced-pluripotent-stem-cell-derived cardiomyocytes, using bulk transcriptomic profiles. We use singular value decomposition to identify drug-selective patterns across cell lines obtained from multiple healthy human subjects. Cellular pathways affected by cardiotoxic TKIs include energy metabolism, contractile, and extracellular matrix dynamics. Projecting these pathways to published single cell expression profiles indicates that TKI responses can be evoked in both cardiomyocytes and fibroblasts. Integration of transcriptomic outlier analysis with whole genomic sequencing of our six cell lines enables us to correctly reidentify a genomic variant causally linked to anthracycline-induced cardiotoxicity and predict genomic variants potentially associated with TKI-induced cardiotoxicity. We conclude that mRNA expression profiles when integrated with publicly available genomic, pathway, and single cell transcriptomic datasets, provide multiscale signatures for cardiotoxicity that could be used for drug development and patient stratification. Using a new computational pipeline for identification of drug-selective transcriptomic responses and FAERS data, the authors identified potential pathways and genomic variants indicative of cancer drug cardiotoxicity in iPSC-derived cardiomyocytes.

The homeodomain transcription factor Nanog plays an important role in embryonic stem cell (ESC) self-renewal and is essential for acquiring ground-state pluripotency during reprogramming. Understanding how Nanog is transcriptionally regulated is important for further dissecting mechanisms of ESC pluripotency and somatic cell reprogramming. Here, we report that Nanog is subjected to a negative autoregulatory mechanism, i.e., autorepression, in ESCs, and that such autorepression requires the coordinated action of the Nanog partner and transcriptional repressor Zfp281. Mechanistically, Zfp281 recruits the NuRD repressor complex onto the Nanog locus and maintains its integrity to mediate Nanog autorepression and, functionally, Zfp281-mediated Nanog autorepression presents a roadblock to efficient somatic cell reprogramming. Our results identify a unique transcriptional regulatory mode of Nanog gene expression and shed light into the mechanistic understanding of Nanog function in pluripotency and reprogramming.

Mechanisms regulating self-renewal and cell fate decisions in mammalian stem cells are poorly understood. We determined global gene expression profiles for mouse and human hematopoietic stem cells and other stages of the hematopoietic hierarchy. Murine and human hematopoietic stem cells share a number of expressed gene products, which define key conserved regulatory pathways in this developmental system. Moreover, in the mouse, a portion of the genetic program of hematopoietic stem cells is shared with embryonic and neural stem cells. This overlapping set of gene products represents a molecular signature of stem cells.

Mesenchymal stromal cells (MSCs) have great value in cell therapies. The MSC therapies have many challenges due to its inconsistent potency and limited quantity. Here, we report a strategy to generate induced MSCs (iMSCs) by directly reprogramming human peripheral blood mononuclear cells (PBMCs) with OCT4, SOX9, MYC, KLF4, and BCL-XL using a nonintegrating episomal vector system. While OCT4 was not required to reprogram PBMCs into iMSCs, omission of OCT4 significantly impaired iMSC functionality. The omission of OCT4 resulted in significantly downregulating MSC lineage specific and mesoderm-regulating genes, including SRPX , COL5A1 , SOX4 , SALL4 , TWIST1 . When reprogramming PBMCs in the absence of OCT4, 67 genes were significantly hypermethylated with reduced transcriptional expression. These data indicate that transient expression of OCT4 may serve as a universal reprogramming factor by increasing chromatin accessibility and promoting demethylation. Our findings represent an approach to produce functional MSCs, and aid in identifying putative function associated MSC markers. Induced MSCs (iMSCs) are generated by direct reprogramming of human PBMCs using 5 factor transfection, including OCT4, SOX9, MYC, KLF4, and BCL-XL.

Background Chemical or small interfering (si) RNA screens measure the effects of many independent experimental conditions, each applied to a population of cells (e.g., all of the cells in a well). High-content screens permit a readout (e.g., fluorescence, luminescence, cell morphology) from each cell in the population. Most analysis approaches compare the average effect on each population, precluding identification of outliers that affect the distribution of the reporter in the population but not its average. Other approaches only measure changes to the distribution with a single parameter, precluding accurate distinction and clustering of interesting outlier distributions. Results We describe a methodology to identify outlier conditions by considering the cell-level measurements from each condition as a sample of an underlying distribution. With appropriate selection of a distance metric, all effects can be embedded in a fixed-dimensionality Euclidean basis, facilitating identification and clustering of biologically interesting outliers. We demonstrate that measurement of distances with the Hellinger distance metric offers substantial computational efficiencies over alternative metrics. We validate this methodology using an RNA interference (RNAi) screen in mouse embryonic stem cells (ESC) with a Nanog reporter. The methodology clusters effects of multiple control siRNAs into their true identities better than conventional approaches describing the median cell fluorescence or the commonly used Kolmogorov-Smirnov distance between the observed fluorescence distribution and the null distribution. It identifies outlier genes with effects on the reporter distribution that would have been missed by other methods. Among them, siRNA targeting Chek1 leads to a wider Nanog reporter fluorescence distribution. Similarly, siRNA targeting Med14 or Med27 leads to a narrower Nanog reporter fluorescence distribution. We confirm the roles of these three genes in regulating pluripotency by mRNA expression and alkaline phosphatase staining using independent short hairpin (sh) RNAs. Conclusions Using our methodology, we describe each experimental condition by a probability distribution. Measuring distances between probability distributions permits a multivariate rather than univariate readout. Clustering points derived from these distances allows us to obtain greater biological insight than methods based solely on single parameters. We find several outliers from a mouse ESC RNAi screen that we confirm to be pluripotency regulators. Many of these outliers would have been missed by other analysis methods.

Cord blood (CB) cells that express CD34 have extensive hematopoietic capacity and rapidly divide ex vivo in the presence of cytokine combinations; however, many of these CB CD34+ cells lose their marrow-repopulating potential. To overcome this decline in function, we treated dividing CB CD34+ cells ex vivo with several histone deacetylase inhibitors (HDACIs). Treatment of CB CD34+ cells with the most active HDACI, valproic acid (VPA), following an initial 16-hour cytokine priming, increased the number of multipotent cells (CD34+CD90+) generated; however, the degree of expansion was substantially greater in the presence of both VPA and cytokines for a full 7 days. Treated CD34+ cells were characterized based on the upregulation of pluripotency genes, increased aldehyde dehydrogenase activity, and enhanced expression of CD90, c-Kit (CD117), integrin α6 (CD49f), and CXCR4 (CD184). Furthermore, siRNA-mediated inhibition of pluripotency gene expression reduced the generation of CD34+CD90+ cells by 89%. Compared with CB CD34+ cells, VPA-treated CD34+ cells produced a greater number of SCID-repopulating cells and established multilineage hematopoiesis in primary and secondary immune-deficient recipient mice. These data indicate that dividing CB CD34+ cells can be epigenetically reprogrammed by treatment with VPA so as to generate greater numbers of functional CB stem cells for use as transplantation grafts.

Anterior foregut endoderm is the precursor of various tissues that might be amenable to cell replacement therapy, including thymus, thyroid, lung and trachea. Green et al . present a method for generating this endoderm subtype from human embryonic stem cells and induced pluripotent stem cells. Directed differentiation of human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells captures in vivo developmental pathways for specifying lineages in vitro , thus avoiding perturbation of the genome with exogenous genetic material. Thus far, derivation of endodermal lineages has focused predominantly on hepatocytes, pancreatic endocrine cells and intestinal cells 1 , 2 , 3 , 4 , 5 . The ability to differentiate pluripotent cells into anterior foregut endoderm (AFE) derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as the thymus; for metabolism, such as thyroid and parathyroid; and for respiratory function, such as trachea and lung. We find that dual inhibition of transforming growth factor (TGF)-β and bone morphogenic protein (BMP) signaling after specification of definitive endoderm from pluripotent cells results in a highly enriched AFE population that is competent to be patterned along dorsoventral and anteroposterior axes. These findings provide an approach for the generation of AFE derivatives.

BackgroundTreatment strategies for Crohn’s disease (CD) suppress diverse inflammatory pathways but many patients remain refractory to treatment. Autologous haematopoietic stem cell transplantation (SCT) is an emerging therapy for medically refractory CD though the mechanisms through which it circumvents refractory pathophysiology are unknown.ObjectiveThe objective of this study is to understand how the immune system reconstitutes post-SCT and whether SCT may function as a cellular therapy restoring appropriately responsive immune cell populations from haematopoietic stem cells (HSCs).DesignAdults with CD with active clinical and endoscopic disease who failed available medical therapies were enrolled in a phase II study of SCT for refractory CD (n=19). Blood and intestinal samples were collected longitudinally and analysed using CyTOF and scRNA-seq. Stem cell autografts were functionally assayed in mouse xenograft models.ResultsscRNA-seq and CyTOF analyses reveal that SCT predominantly affected the intestinal myeloid lineage with loss of inflammatory populations and return of macrophages capable of supporting mucosal healing. Xenograft models using patient HSCs suggested that HSCs support the early reconstitution of the myeloid lineage and reveal an impairment of short and long-term HSC engraftment that may determine SCT outcomes.ConclusionsThis study suggests SCT functions as a myeloid-directed cellular therapy reinforcing the critical role of macrophages in refractory CD pathophysiology and as a target for cellular therapies. Furthermore, we report an unrecognised functional heterogeneity among HSC subpopulations in CD that may be relevant to our understanding of CD treatment and pathophysiology.