An open chromatin largely devoid of heterochromatin is a hallmark of stem cells. It remains unknown whether an open chromatin is necessary for the differentiation potential of stem cells, and which molecules are needed to maintain open chromatin. Here we show that the chromatin remodelling factor Chd1 is required to maintain the open chromatin of pluripotent mouse embryonic stem cells. Chd1 is a euchromatin protein that associates with the promoters of active genes, and downregulation of Chd1 leads to accumulation of heterochromatin. Chd1-deficient embryonic stem cells are no longer pluripotent, because they are incapable of giving rise to primitive endoderm and have a high propensity for neural differentiation. Furthermore, Chd1 is required for efficient reprogramming of fibroblasts to the pluripotent stem cell state. Our results indicate that Chd1 is essential for open chromatin and pluripotency of embryonic stem cells, and for somatic cell reprogramming to the pluripotent state.

Among the three embryonic germ layers, the mesoderm is a major source of the mesenchymal precursors giving rise to skeletal and connective tissues, but these precursors have not previously been identified and characterized. Using human embryonic stem cells directed toward mesendodermal differentiation, we show that mesenchymal stem/stromal cells (MSCs) originate from a population of mesodermal cells identified by expression of apelin receptor. In semisolid medium, these precursors form FGF2-dependent compact spheroid colonies containing mesenchymal cells with a transcriptional profile representative of mesoderm-derived embryonic mesenchyme. When transferred to adherent cultures, individual colonies give rise to MSC lines with chondro-, osteo-, and adipogenic differentiation potentials. Although the MSC lines lacked endothelial potential, endothelial cells could be derived from the mesenchymal colonies, suggesting that, similar to hematopoietic cells, MSCs arise from precursors with angiogenic potential. Together, these studies identified a common precursor of mesenchymal and endothelial cells, mesenchymoangioblast, as the source of mesoderm-derived MSCs.

The recent identification of hemogenic endothelium (HE) in human pluripotent stem cell (hPSC) cultures presents opportunities to investigate signaling pathways that are essential for blood development from endothelium and provides an exploratory platform for de novo generation of hematopoietic stem cells (HSCs). However, the use of poorly defined human or animal components limits the utility of the current differentiation systems for studying specific growth factors required for HE induction and manufacturing clinical-grade therapeutic blood cells. Here, we identified chemically defined conditions required to produce HE from hPSCs growing in Essential 8 (E8) medium and showed that Tenascin C (TenC), an extracellular matrix protein associated with HSC niches, strongly promotes HE and definitive hematopoiesis in this system. hPSCs differentiated in chemically defined conditions undergo stages of development similar to those previously described in hPSCs cocultured on OP9 feeders, including the formation of VE-Cadherin(+)CD73(-)CD235a/CD43(-) HE and hematopoietic progenitors with myeloid and T lymphoid potential.

The salamander has the remarkable ability to regenerate its limb after amputation. Cells at the site of amputation form a blastema and then proliferate and differentiate to regrow the limb. To better understand this process, we performed deep RNA sequencing of the blastema over a time course in the axolotl, a species whose genome has not been sequenced. Using a novel comparative approach to analyzing RNA-seq data, we characterized the transcriptional dynamics of the regenerating axolotl limb with respect to the human gene set. This approach involved de novo assembly of axolotl transcripts, RNA-seq transcript quantification without a reference genome, and transformation of abundances from axolotl contigs to human genes. We found a prominent burst in oncogene expression during the first day and blastemal/limb bud genes peaking at 7 to 14 days. In addition, we found that limb patterning genes, SALL genes, and genes involved in angiogenesis, wound healing, defense/immunity, and bone development are enriched during blastema formation and development. Finally, we identified a category of genes with no prior literature support for limb regeneration that are candidates for further evaluation based on their expression pattern during the regenerative process.

Advancing pluripotent stem cell technologies for modelling haematopoietic stem cell development and blood therapies requires identifying key regulators of haematopoietic commitment from human pluripotent stem cells (hPSCs). Here, by screening the effect of 27 candidate factors, we reveal two groups of transcriptional regulators capable of inducing distinct haematopoietic programs from hPSCs: pan-myeloid (ETV2 and GATA2) and erythro-megakaryocytic (GATA2 and TAL1). In both cases, these transcription factors directly convert hPSCs to endothelium, which subsequently transform into blood cells with pan-myeloid or erythro-megakaryocytic potential. These data demonstrate that two distinct genetic programs regulate the haematopoietic development from hPSCs and that both of these programs specify hPSCs directly to haemogenic endothelial cells. In addition, this study provides a novel method for the efficient induction of blood and endothelial cells from hPSCs via the overexpression of modified mRNA for the selected transcription factors.

During the reprogramming of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells, the activation of pluripotency genes such as NANOG occurs after the mesenchymal to epithelial transition. Here we report that both adult stem cells (neural stem cells) and differentiated cells (astrocytes) of the neural lineage can activate NANOG in the absence of cadherin expression during reprogramming. Gene expression analysis revealed that only the NANOG+E-cadherin+ populations expressed stabilization markers, had upregulated several cell cycle genes; and were transgene independent. Inhibition of DOT1L activity enhanced both the numbers of NANOG+ and NANOG+E-cadherin+ colonies in neural stem cells. Expressing SOX2 in MEFs prior to reprogramming did not alter the ratio of NANOG colonies that express E-cadherin. Taken together these results provide a unique pathway for reprogramming taken by cells of the neural lineage.

The ability to quantify cellular heterogeneity is a major advantage of single-cell technologies. However, statistical methods often treat cellular heterogeneity as a nuisance. We present a novel method to characterize differences in expression in the presence of distinct expression states within and among biological conditions. We demonstrate that this framework can detect differential expression patterns under a wide range of settings. Compared to existing approaches, this method has higher power to detect subtle differences in gene expression distributions that are more complex than a mean shift, and can characterize those differences. The freely available R package scDD implements the approach.

Long range regulatory interactions among distal enhancers and target genes are important for tissue-specific gene expression. Genome-scale identification of these interactions in a cell line-specific manner, especially using the fewest possible datasets, is a significant challenge. We develop a novel computational approach, Regulatory Interaction Prediction for Promoters and Long-range Enhancers (RIPPLE), that integrates published Chromosome Conformation Capture (3C) data sets with a minimal set of regulatory genomic data sets to predict enhancer-promoter interactions in a cell line-specific manner. Our results suggest that CTCF, RAD21, a general transcription factor (TBP) and activating chromatin marks are important determinants of enhancer-promoter interactions. To predict interactions in a new cell line and to generate genome-wide interaction maps, we develop an ensemble version of RIPPLE and apply it to generate interactions in five human cell lines. Computational validation of these predictions using existing ChIA-PET and Hi-C data sets showed that RIPPLE accurately predicts interactions among enhancers and promoters. Enhancer-promoter interactions tend to be organized into subnetworks representing coordinately regulated sets of genes that are enriched for specific biological processes and cis-regulatory elements. Overall, our work provides a systematic approach to predict and interpret enhancer-promoter interactions in a genome-wide cell-type specific manner using a few experimentally tractable measurements.

Elucidating the mechanism of reprogramming is confounded by heterogeneity due to the low efficiency and differential kinetics of obtaining induced pluripotent stem cells (iPSCs) from somatic cells. Therefore, we increased the efficiency with a combination of epigenomic modifiers and signaling molecules and profiled the transcriptomes of individual reprogramming cells. Contrary to the established temporal order, somatic gene inactivation and upregulation of cell cycle, epithelial, and early pluripotency genes can be triggered independently such that any combination of these events can occur in single cells. Sustained co-expression of Epcam, Nanog, and Sox2 with other genes is required to progress toward iPSCs. Ehf, Phlda2, and translation initiation factor Eif4a1 play functional roles in robust iPSC generation. Using regulatory network analysis, we identify a critical role for signaling inhibition by 2i in repressing somatic expression and synergy between the epigenomic modifiers ascorbic acid and a Dot1L inhibitor for pluripotency gene activation.

Defects in somitogenesis result in vertebral malformations at birth known as spondylocostal dysostosis (SCDO). Somites are formed with a species-specific periodicity controlled by the "segmentation clock," which comprises a group of oscillatory genes in the presomitic mesoderm. Here, we report that a segmentation clock model derived from human embryonic stem cells shows many hallmarks of the mammalian segmentation clock in vivo, including a dependence on the NOTCH and WNT signaling pathways. The gene expression oscillations are highly synchronized, displaying a periodicity specific to the human clock. Introduction of a point of mutation into HES7, a specific mutation previously associated with clinical SCDO, eliminated clock gene oscillations, successfully reproducing the defects in the segmentation clock. Thus, we provide a model for studying the previously inaccessible human segmentation clock to better understand the mechanisms contributing to congenital skeletal defects.

During vertebrate development, progenitor cells give rise to tissues and organs through a complex choreography that commences at gastrulation. A hallmark event of gastrulation is the formation of the primitive streak, a linear assembly of cells along the anterior-posterior (AP) axis of the developing organism. To examine the primitive streak at a single-cell resolution, we measured the transcriptomes of individual chick cells from the streak or the surrounding tissue (the rest of the area pellucida) in Hamburger-Hamilton stage 4 embryos. The single-cell transcriptomes were then ordered by the statistical method Wave-Crest to deduce both the relative position along the AP axis and the prospective lineage of single cells. The ordered transcriptomes reveal intricate patterns of gene expression along the primitive streak.

Comparative time series transcriptome analysis is a powerful tool to study development, evolution, aging, disease progression and cancer prognosis. We develop TimeMeter, a statistical method and tool to assess temporal gene expression similarity, and identify differentially progressing genes where one pattern is more temporally advanced than the other. We apply TimeMeter to several datasets, and show that TimeMeter is capable of characterizing complicated temporal gene expression associations. Interestingly, we find: (i) the measurement of differential progression provides a novel feature in addition to pattern similarity that can characterize early developmental divergence between two species; (ii) genes exhibiting similar temporal patterns between human and mouse during neural differentiation are under strong negative (purifying) selection during evolution; (iii) analysis of genes with similar temporal patterns in mouse digit regeneration and axolotl blastema differentiation reveals common gene groups for appendage regeneration with potential implications in regenerative medicine.

Human pluripotent stem cells hold significant promise for regenerative medicine. However, long differentiation protocols and immature characteristics of stem cell-derived cell types remain challenges to the development of many therapeutic applications. In contrast to the slow differentiation of human stem cells in vitro that mirrors a nine-month gestation period, mouse stem cells develop according to a much faster three-week gestation timeline. Here, we tested if co-differentiation with mouse pluripotent stem cells could accelerate the differentiation speed of human embryonic stem cells. Following a six-week RNA-sequencing time course of neural differentiation, we identified 929 human genes that were upregulated earlier and 535 genes that exhibited earlier peaked expression profiles in chimeric cell cultures than in human cell cultures alone. Genes with accelerated upregulation were significantly enriched in Gene Ontology terms associated with neurogenesis, neuron differentiation and maturation, and synapse signaling. Moreover, chimeric mixed samples correlated with in utero human embryonic samples earlier than human cells alone, and acceleration was dose-dependent on human-mouse co-culture ratios. The altered gene expression patterns and developmental rates described in this report have implications for accelerating human stem cell differentiation and the use of interspecies chimeric embryos in developing human organs for transplantation.

Inhibiting the histone 3 lysine 79 (H3K79) methyltransferase, disruptor of telomeric silencing 1-like (DOT1L), increases the efficiency of reprogramming somatic cells to induced pluripotent stem cells (iPSCs). Here, we find that, despite the enrichment of H3K79 methylation on thousands of actively transcribed genes in somatic cells, DOT1L inhibition (DOT1Li) does not immediately cause the shutdown of the somatic transcriptional profile to enable transition to pluripotency. Contrary to the prevalent view, DOT1Li promotes iPSC generation beyond the mesenchymal to epithelial transition and even from already epithelial cell types. DOT1Li is most potent at the midpoint of reprogramming in part by repressing Nfix that persists at late stages of reprogramming. Importantly, regulation of single genes cannot substitute for DOT1Li, demonstrating that H3K79 methylation has pleiotropic effects in maintaining cell identity.

Changes in transcriptional regulatory networks can significantly alter cell fate. To gain insight into transcriptional dynamics, several studies have profiled bulk multi-omic data sets with parallel transcriptomic and epigenomic measurements at different stages of a developmental process. However, integrating these data to infer cell type-specific regulatory networks is a major challenge. We present dynamic regulatory module networks (DRMNs), a novel approach to infer cell type-specific cis-regulatory networks and their dynamics. DRMN integrates expression, chromatin state, and accessibility to predict cis-regulators of context-specific expression, where context can be cell type, developmental stage, or time point, and uses multitask learning to capture network dynamics across linearly and hierarchically related contexts. We applied DRMNs to study regulatory network dynamics in three developmental processes, each showing different temporal relationships and measuring a different combination of regulatory genomic data sets: cellular reprogramming, liver dedifferentiation, and forward differentiation. DRMN identified known and novel regulators driving cell type-specific expression patterns, showing its broad applicability to examine dynamics of gene regulatory networks from linearly and hierarchically related multi-omic data sets.

With improvements in next-generation sequencing technologies and reductions in price, ordered RNA-seq experiments are becoming common. Of primary interest in these experiments is identifying genes that are changing over time or space, for example, and then characterizing the specific expression changes. A number of robust statistical methods are available to identify genes showing differential expression among multiple conditions, but most assume conditions are exchangeable and thereby sacrifice power and precision when applied to ordered data.

Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) represents a profound change in cell fate. Here, we show that combining ascorbic acid (AA) and 2i (MAP kinase and GSK inhibitors) increases the efficiency of reprogramming from fibroblasts and synergistically enhances conversion of partially reprogrammed intermediates to the iPSC state. AA and 2i induce differential transcriptional responses, each leading to the activation of specific pluripotency loci. A unique cohort of pluripotency genes including Esrrb require both stimuli for activation. Temporally, AA-dependent histone demethylase effects are important early, whereas Tet enzyme effects are required throughout the conversion. 2i function could partially be replaced by depletion of components of the epidermal growth factor (EGF) and insulin growth factor pathways, indicating that they act as barriers to reprogramming. Accordingly, reduction in the levels of the EGF receptor gene contributes to the activation of Esrrb. These results provide insight into the rewiring of the pluripotency network at the late stage of reprogramming.

De novo RNA-Seq assembly facilitates the study of transcriptomes for species without sequenced genomes, but it is challenging to select the most accurate assembly in this context. To address this challenge, we developed a model-based score, RSEM-EVAL, for evaluating assemblies when the ground truth is unknown. We show that RSEM-EVAL correctly reflects assembly accuracy, as measured by REF-EVAL, a refined set of ground-truth-based scores that we also developed. Guided by RSEM-EVAL, we assembled the transcriptome of the regenerating axolotl limb; this assembly compares favorably to a previous assembly. A software package implementing our methods, DETONATE, is freely available at http://deweylab.biostat.wisc.edu/detonate.

Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However, it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines, along with methylomes of ES cells, somatic cells, and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability, including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation, and differences in CG methylation and histone modifications. Lastly, differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency, providing an iPSC reprogramming signature that is maintained after differentiation.

Epigenetic mechanisms have been proposed to play crucial roles in mammalian development, but their precise functions are only partially understood. To investigate epigenetic regulation of embryonic development, we differentiated human embryonic stem cells into mesendoderm, neural progenitor cells, trophoblast-like cells, and mesenchymal stem cells and systematically characterized DNA methylation, chromatin modifications, and the transcriptome in each lineage. We found that promoters that are active in early developmental stages tend to be CG rich and mainly engage H3K27me3 upon silencing in nonexpressing lineages. By contrast, promoters for genes expressed preferentially at later stages are often CG poor and primarily employ DNA methylation upon repression. Interestingly, the early developmental regulatory genes are often located in large genomic domains that are generally devoid of DNA methylation in most lineages, which we termed DNA methylation valleys (DMVs). Our results suggest that distinct epigenetic mechanisms regulate early and late stages of ES cell differentiation.