Chromosome level genome assembly of the Etruscan shrew Suncus etruscus.

Suncus etruscus is one of the world's smallest mammals, with an average body mass of about 2 grams. The Etruscan shrew's small body is accompanied by a very high energy demand and numerous metabolic adaptations. Here we report a chromosome-level genome assembly using PacBio long read sequencing, 10X Genomics linked short reads, optical mapping, and Hi-C linked reads. The assembly is partially phased, with the 2.472 Gbp primary pseudohaplotype and 1.515 Gbp alternate. We manually curated the primary assembly and identified 22 chromosomes, including X and Y sex chromosomes. The NCBI genome annotation pipeline identified 39,091 genes, 19,819 of them protein-coding. We also identified segmental duplications, inferred GO term annotations, and computed orthologs of human and mouse genes. This reference-quality genome will be an important resource for research on mammalian development, metabolism, and body size control.

A High-Quality Blue Whale Genome, Segmental Duplications, and Historical Demography.

The blue whale, Balaenoptera musculus, is the largest animal known to have ever existed, making it an important case study in longevity and resistance to cancer. To further this and other blue whale-related research, we report a reference-quality, long-read-based genome assembly of this fascinating species. We assembled the genome from PacBio long reads and utilized Illumina/10×, optical maps, and Hi-C data for scaffolding, polishing, and manual curation. We also provided long read RNA-seq data to facilitate the annotation of the assembly by NCBI and Ensembl. Additionally, we annotated both haplotypes using TOGA and measured the genome size by flow cytometry. We then compared the blue whale genome with other cetaceans and artiodactyls, including vaquita (Phocoena sinus), the world's smallest cetacean, to investigate blue whale's unique biological traits. We found a dramatic amplification of several genes in the blue whale genome resulting from a recent burst in segmental duplications, though the possible connection between this amplification and giant body size requires further study. We also discovered sites in the insulin-like growth factor-1 gene correlated with body size in cetaceans. Finally, using our assembly to examine the heterozygosity and historical demography of Pacific and Atlantic blue whale populations, we found that the genomes of both populations are highly heterozygous and that their genetic isolation dates to the last interglacial period. Taken together, these results indicate how a high-quality, annotated blue whale genome will serve as an important resource for biology, evolution, and conservation research.

Engineered Perineural Vascular Plexus for Modeling Developmental Toxicity.

There is a vital need to develop in vitro models of the developing human brain to recapitulate the biological effects that toxic compounds have on the brain. To model perineural vascular plexus (PNVP) in vitro, which is a key stage in embryonic development, human embryonic stem cells (hESC)-derived endothelial cells (ECs), neural progenitor cells, and microglia (MG) with primary pericytes (PCs) in synthetic hydrogels in a custom-designed microfluidics device are cocultured. The formation of a vascular plexus that includes networks of ECs (CD31+, VE-cadherin+), MG (IBA1+), and PCs (PDGFRß+), and an overlying neuronal layer that includes differentiated neuronal cells (ßIII Tubulin+, GFAP+) and radial glia (Nestin+, Notch2NL+), are characterized. Increased brain-derived neurotrophic factor secretion and differential metabolite secretion by the vascular plexus and the neuronal cells over time are consistent with PNVP functionality. Multiple concentrations of developmental toxicants (teratogens, microglial disruptor, and vascular network disruptors) significantly reduce the migration of ECs and MG toward the neuronal layer, inhibit formation of the vascular network, and decrease vascular endothelial growth factor A (VEGFA) secretion. By quantifying 3D cell migration, metabolic activity, vascular network disruption, and cytotoxicity, the PNVP model may be a useful tool to make physiologically relevant predictions of developmental toxicity.

Quantitative Label-Free Imaging of 3D Vascular Networks Self-Assembled in Synthetic Hydrogels.

Vascularization is an important strategy to overcome diffusion limits and enable the formation of complex, physiologically relevant engineered tissues and organoids. Self-assembly is a technique to generate in vitro vascular networks, but engineering the necessary network morphology and function remains challenging. Here, autofluorescence multiphoton microscopy (aMPM), a label-free imaging technique, is used to quantitatively evaluate in vitro vascular network morphology. Vascular networks are generated using human embryonic stem cell-derived endothelial cells and primary human pericytes encapsulated in synthetic poly(ethylene glycol)-based hydrogels. Two custom-built bioreactors are used to generate distinct fluid flow patterns during vascular network formation: recirculating flow or continuous flow. aMPM is used to image these 3D vascular networks without the need for fixation, labels, or dyes. Image processing and analysis algorithms are developed to extract quantitative morphological parameters from these label-free images. It is observed with aMPM that both bioreactors promote formation of vascular networks with lower network anisotropy compared to static conditions, and the continuous flow bioreactor induces more branch points compared to static conditions. Importantly, these results agree with trends observed with immunocytochemistry. These studies demonstrate that aMPM allows label-free monitoring of vascular network morphology to streamline optimization of growth conditions and provide quality control of engineered tissues.

Chromatin architecture reorganization during stem cell differentiation.

Higher-order chromatin structure is emerging as an important regulator of gene expression. Although dynamic chromatin structures have been identified in the genome, the full scope of chromatin dynamics during mammalian development and lineage specification remains to be determined. By mapping genome-wide chromatin interactions in human embryonic stem (ES) cells and four human ES-cell-derived lineages, we uncover extensive chromatin reorganization during lineage specification. We observe that although self-associating chromatin domains are stable during differentiation, chromatin interactions both within and between domains change in a striking manner, altering 36% of active and inactive chromosomal compartments throughout the genome. By integrating chromatin interaction maps with haplotype-resolved epigenome and transcriptome data sets, we find widespread allelic bias in gene expression correlated with allele-biased chromatin states of linked promoters and distal enhancers. Our results therefore provide a global view of chromatin dynamics and a resource for studying long-range control of gene expression in distinct human cell lineages.

Epigenomic analysis of multilineage differentiation of human embryonic stem cells.

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.

Dynamic chromatin states in human ES cells reveal potential regulatory sequences and genes involved in pluripotency.

Pluripotency, the ability of a cell to differentiate and give rise to all embryonic lineages, defines a small number of mammalian cell types such as embryonic stem (ES) cells. While it has been generally held that pluripotency is the product of a transcriptional regulatory network that activates and maintains the expression of key stem cell genes, accumulating evidence is pointing to a critical role for epigenetic processes in establishing and safeguarding the pluripotency of ES cells, as well as maintaining the identity of differentiated cell types. In order to better understand the role of epigenetic mechanisms in pluripotency, we have examined the dynamics of chromatin modifications genome-wide in human ES cells (hESCs) undergoing differentiation into a mesendodermal lineage. We found that chromatin modifications at promoters remain largely invariant during differentiation, except at a small number of promoters where a dynamic switch between acetylation and methylation at H3K27 marks the transition between activation and silencing of gene expression, suggesting a hierarchy in cell fate commitment over most differentially expressed genes. We also mapped over 50 000 potential enhancers, and observed much greater dynamics in chromatin modifications, especially H3K4me1 and H3K27ac, which correlate with expression of their potential target genes. Further analysis of these enhancers revealed potentially key transcriptional regulators of pluripotency and a chromatin signature indicative of a poised state that may confer developmental competence in hESCs. Our results provide new evidence supporting the role of chromatin modifications in defining enhancers and pluripotency.

Chemically defined conditions for human iPSC derivation and culture.

We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.

Somatic coding mutations in human induced pluripotent stem cells.

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, whereas the rest occurred 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 cell safety before clinical use.

Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells.

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.

Distinct epigenomic landscapes of pluripotent and lineage-committed human cells.

Human embryonic stem cells (hESCs) share an identical genome with lineage-committed cells, yet possess the remarkable properties of self-renewal and pluripotency. The diverse cellular properties in different cells have been attributed to their distinct epigenomes, but how much epigenomes differ remains unclear. Here, we report that epigenomic landscapes in hESCs and lineage-committed cells are drastically different. By comparing the chromatin-modification profiles and DNA methylomes in hESCs and primary fibroblasts, we find that nearly one-third of the genome differs in chromatin structure. Most changes arise from dramatic redistributions of repressive H3K9me3 and H3K27me3 marks, which form blocks that significantly expand in fibroblasts. A large number of potential regulatory sequences also exhibit a high degree of dynamics in chromatin modifications and DNA methylation. Additionally, we observe novel, context-dependent relationships between DNA methylation and chromatin modifications. Our results provide new insights into epigenetic mechanisms underlying properties of pluripotency and cell fate commitment.

No evidence for clonal selection due to lentiviral integration sites in human induced pluripotent stem cells.

Derivation of induced pluripotent stem (iPS) cells requires the expression of defined transcription factors (among Oct3/4, Sox2, Klf4, c-Myc, Nanog, and Lin28) in the targeted cells. Lentiviral or standard retroviral gene transfer remains the most robust and commonly used approach. Low reprogramming frequency overall, and the higher efficiency of derivation utilizing integrating vectors compared to more recent nonviral approaches, suggests that gene activation or disruption via proviral integration sites (IS) may play a role in obtaining the pluripotent phenotype. We provide for the first time an extensive analysis of the lentiviral integration profile in human iPS cells. We identified a total of 78 independent IS in eight recently established iPS cell lines derived from either human fetal fibroblasts or newborn foreskin fibroblasts after lentiviral gene transfer of Oct4, Sox2, Nanog, and Lin28. The number of IS ranged from 5 to 15 IS per individual iPS clone, and 75 IS could be assigned to a unique chromosomal location. The different iPS clones had no IS in common. Expression analysis as well as extensive bioinformatic analysis did not reveal functional concordance of the lentiviral targeted genes between the different clones. Interestingly, in six of the eight iPS clones, some of the IS were found in pairs, integrated into the same chromosomal location within six base pairs of each other or in very close proximity. Our study supports recent reports that efficient reprogramming of human somatic cells is not dependent on insertional activation or deactivation of specific genes or gene classes.

Human DNA methylomes at base resolution show widespread epigenomic differences.

DNA cytosine methylation is a central epigenetic modification that has essential roles in cellular processes including genome regulation, development and disease. Here we present the first genome-wide, single-base-resolution maps of methylated cytosines in a mammalian genome, from both human embryonic stem cells and fetal fibroblasts, along with comparative analysis of messenger RNA and small RNA components of the transcriptome, several histone modifications, and sites of DNA-protein interaction for several key regulatory factors. Widespread differences were identified in the composition and patterning of cytosine methylation between the two genomes. Nearly one-quarter of all methylation identified in embryonic stem cells was in a non-CG context, suggesting that embryonic stem cells may use different methylation mechanisms to affect gene regulation. Methylation in non-CG contexts showed enrichment in gene bodies and depletion in protein binding sites and enhancers. Non-CG methylation disappeared upon induced differentiation of the embryonic stem cells, and was restored in induced pluripotent stem cells. We identified hundreds of differentially methylated regions proximal to genes involved in pluripotency and differentiation, and widespread reduced methylation levels in fibroblasts associated with lower transcriptional activity. These reference epigenomes provide a foundation for future studies exploring this key epigenetic modification in human disease and development.

Histone modifications at human enhancers reflect global cell-type-specific gene expression.

The human body is composed of diverse cell types with distinct functions. Although it is known that lineage specification depends on cell-specific gene expression, which in turn is driven by promoters, enhancers, insulators and other cis-regulatory DNA sequences for each gene, the relative roles of these regulatory elements in this process are not clear. We have previously developed a chromatin-immunoprecipitation-based microarray method (ChIP-chip) to locate promoters, enhancers and insulators in the human genome. Here we use the same approach to identify these elements in multiple cell types and investigate their roles in cell-type-specific gene expression. We observed that the chromatin state at promoters and CTCF-binding at insulators is largely invariant across diverse cell types. In contrast, enhancers are marked with highly cell-type-specific histone modification patterns, strongly correlate to cell-type-specific gene expression programs on a global scale, and are functionally active in a cell-type-specific manner. Our results define over 55,000 potential transcriptional enhancers in the human genome, significantly expanding the current catalogue of human enhancers and highlighting the role of these elements in cell-type-specific gene expression.

Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming.

Current DNA methylation assays are limited in the flexibility and efficiency of characterizing a large number of genomic targets. We report a method to specifically capture an arbitrary subset of genomic targets for single-molecule bisulfite sequencing for digital quantification of DNA methylation at single-nucleotide resolution. A set of ~30,000 padlock probes was designed to assess methylation of ~66,000 CpG sites within 2,020 CpG islands on human chromosome 12, chromosome 20, and 34 selected regions. To investigate epigenetic differences associated with dedifferentiation, we compared methylation in three human fibroblast lines and eight human pluripotent stem cell lines. Chromosome-wide methylation patterns were similar among all lines studied, but cytosine methylation was slightly more prevalent in the pluripotent cells than in the fibroblasts. Induced pluripotent stem (iPS) cells appeared to display more methylation than embryonic stem cells. We found 288 regions methylated differently in fibroblasts and pluripotent cells. This targeted approach should be particularly useful for analyzing DNA methylation in large genomes.

NANOG is a direct target of TGFbeta/activin-mediated SMAD signaling in human ESCs.

Self-renewal of human embryonic stem cells (ESCs) is promoted by FGF and TGFbeta/Activin signaling, and differentiation is promoted by BMP signaling, but how these signals regulate genes critical to the maintenance of pluripotency has been unclear. Using a defined medium, we show here that both TGFbeta and FGF signals synergize to inhibit BMP signaling; sustain expression of pluripotency-associated genes such as NANOG, OCT4, and SOX2; and promote long-term undifferentiated proliferation of human ESCs. We also show that both TGFbeta- and BMP-responsive SMADs can bind with the NANOG proximal promoter. NANOG promoter activity is enhanced by TGFbeta/Activin and FGF signaling and is decreased by BMP signaling. Mutation of putative SMAD binding elements reduces NANOG promoter activity to basal levels and makes NANOG unresponsive to BMP and TGFbeta signaling. These results suggest that direct binding of TGFbeta/Activin-responsive SMADs to the NANOG promoter plays an essential role in sustaining human ESC self-renewal.

Induced pluripotent stem cell lines derived from human somatic cells.

Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.