Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-b and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays. STEM CELLS

BackgroundDNA methylation at promoters is largely correlated with inhibition of gene expression. However, the role of DNA methylation at enhancers is not fully understood, although a crosstalk with chromatin marks is expected. Actually, there exist contradictory reports about positive and negative correlations between DNA methylation and H3K4me1, a chromatin hallmark of enhancers.ResultsWe investigated the relationship between DNA methylation and active chromatin marks through genome-wide correlations, and found anti-correlation between H3K4me1 and H3K4me3 enrichment at low and intermediate DNA methylation loci. We hypothesized “seesaw” dynamics between H3K4me1 and H3K4me3 in the low and intermediate DNA methylation range, in which DNA methylation discriminates between enhancers and promoters, marked by H3K4me1 and H3K4me3, respectively. Low methylated regions are H3K4me3 enriched, while those with intermediate DNA methylation levels are progressively H3K4me1 enriched. Additionally, the enrichment of H3K27ac, distinguishing active from primed enhancers, follows a plateau in the lower range of the intermediate DNA methylation level, corresponding to active enhancers, and decreases linearly in the higher range of the intermediate DNA methylation. Thus, the decrease of the DNA methylation switches smoothly the state of the enhancers from a primed to an active state. We summarize these observations into a rule of thumb of one-out-of-three methylation marks: “In each genomic region only one out of these three methylation marks {DNA methylation, H3K4me1, H3K4me3} is high. If it is the DNA methylation, the region is inactive. If it is H3K4me1, the region is an enhancer, and if it is H3K4me3, the region is a promoter”. To test our model, we used available genome-wide datasets of H3K4 methyltransferases knockouts. Our analysis suggests that CXXC proteins, as readers of non-methylated CpGs would regulate the “seesaw” mechanism that focuses H3K4me3 to unmethylated sites, while being repulsed from H3K4me1 decorated enhancers and CpG island shores.ConclusionsOur results show that DNA methylation discriminates promoters from enhancers through H3K4me1-H3K4me3 seesaw mechanism, and suggest its possible function in the inheritance of chromatin marks after cell division. Our analyses suggest aberrant formation of promoter-like regions and ectopic transcription of hypomethylated regions of DNA. Such mechanism process can have important implications in biological process in where it has been reported abnormal DNA methylation status such as cancer and aging.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4353-7) contains supplementary material, which is available to authorized users.

Embryonic stem (ES) cells are considered to exist in a ground state if shielded from differentiation triggers. Here we show that FGF4 and TGFβ signaling pathway inhibitors, designated R2i, not only provide the ground state pluripotency in production and maintenance of naïve ES cells from blastocysts of different mouse strains, but also maintain ES cells with higher genomic integrity following long-term cultivation compared with the chemical inhibition of the FGF4 and GSK3 pathways, known as 2i. Global transcriptome analysis of the ES cells highlights augmented BMP4 signaling pathway. The crucial role of the BMP4 pathway in maintaining the R2i ground state pluripotency is demonstrated by BMP4 receptor suppression, resulting in differentiation and cell death. In conclusion, by inhibiting TGFβ and FGF signaling pathways, we introduce a novel defined approach to efficiently establish the ground state pluripotency.

Despite the enormous progress in studying definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs), none of the reported protocols have produced a universal, cost-effective, and competent DE with the capability to further differentiate into endodermal derivatives. In this study, by using a 2-step differentiation strategy, we have treated hESCs for 1 day with "priming" small molecules (SM), [stauprimide, NSC-308848, rapamycin (Rapa), and/or CHIR] and for the next 3 days with "inducing" SM (LY294002, cymarin, IDE1, and/or IDE2) in conjunction with activin A. In the positive control group, we treated hESCs with Wnt3a (25 ng/mL) for 1 day and activin A (100 ng/mL; W/A100-A100) for the next 3 days. Gene expression analysis showed that treatment of hESCs with 100 nM Rapa and 50 ng/mL activin A (Rapa-A50) out of 25 combinations of factors gave rise to higher expressions of 2 DE-specific genes, SOX17 and FOXA2. Similar results were obtained after treating 2 other hESC lines with this regimen. To investigate the competency of Rapa-A50-induced DE for further differentiation into endodermal derivatives, these cells and W/A100-A100-induced DE cells (positive control) were further differentiated into pancreatic progenitors (PP), then into pancreatic endocrine (PE) cells using 5 previously described differentiation protocols. Gene analysis of differentiated cells showed that the established protocols were insufficient to enable universal differentiation into PE, whereas Rapa-A50-induced DE cells were more competent for PP differentiation in a protocol-dependent manner. Additionally, Rapa-A50-induced DE had the capability to differentiate into hepatocyte-like cells (HLCs) as efficiently as W/A100-A100-induced DE. These data have indicated that hESCs primed with Rapa, and induced by a lower concentration of activin A, could lead to DE that had the capability to further differentiate into HLCs and PP cells, but not PE cells. Thus, current protocols for the differentiation of DE into PE still need additional study.

BackgroundPlanarians are non-parasitic Platyhelminthes (flatworms) famous for their regeneration ability and for having a well-organized brain. Dugesia japonica is a typical planarian species that is widely distributed in the East Asia. Extensive cellular and molecular experimental methods have been developed to identify the functions of thousands of genes in this species, making this planarian a good experimental model for regeneration biology and neurobiology. However, no genome-level information is available for D. japonica, and few gene regulatory networks have been identified thus far.ResultsTo obtain whole-genome information on this species and to study its gene regulatory networks, we extracted genomic DNA from 200 planarians derived from a laboratory-bred asexual clonal strain, and sequenced 476 Gb of data by second-generation sequencing. Kmer frequency graphing and fosmid sequence analysis indicated a complex genome that would be difficult to assemble using second-generation sequencing short reads. To address this challenge, we developed a new assembly strategy and improved the de novo genome assembly, producing a 1.56 Gb genome sequence (DjGenome ver1.0, including 202,925 scaffolds and N50 length 27,741 bp) that covers 99.4% of all 19,543 genes in the assembled transcriptome, although the genome is fragmented as 80% of the genome consists of repeated sequences (genomic frequency ≥ 2). By genome comparison between two planarian genera, we identified conserved non-coding elements (CNEs), which are indicative of gene regulatory elements. Transgenic experiments using Xenopus laevis indicated that one of the CNEs in the Djndk gene may be a regulatory element, suggesting that the regulation of the ndk gene and the brain formation mechanism may be conserved between vertebrates and invertebrates.ConclusionThis draft genome and CNE analysis will contribute to resolving gene regulatory networks in planarians. The genome database is available at: http://www.planarian.jp.Electronic supplementary materialThe online version of this article (10.1186/s40851-018-0102-2) contains supplementary material, which is available to authorized users.

SummaryGround-state pluripotency is a cell state in which pluripotency is established and maintained through efficient repression of endogenous differentiation pathways. Self-renewal and pluripotency of embryonic stem cells (ESCs) are influenced by ESC-associated microRNAs (miRNAs). Here, we provide a comprehensive assessment of the “miRNome” of ESCs cultured under conditions favoring ground-state pluripotency. We found that ground-state ESCs express a distinct set of miRNAs compared with ESCs grown in serum. Interestingly, most “ground-state miRNAs” are encoded by an imprinted region on chromosome 12 within the Dlk1-Dio3 locus. Functional analysis revealed that ground-state miRNAs embedded in the Dlk1-Dio3 locus (miR-541-5p, miR-410-3p, and miR-381-3p) promoted pluripotency via inhibition of multi-lineage differentiation and stimulation of self-renewal. Overall, our results demonstrate that ground-state pluripotency is associated with a unique miRNA signature, which supports ground-state self-renewal by suppressing differentiation.

The human protein disulfide isomerase (hPDI), is an essential four-domain multifunctional enzyme. As a result of disulfide shuffling in its terminal domains, hPDI exists in two oxidation states with different conformational preferences which are important for substrate binding and functional activities. Here, we address the redox-dependent conformational dynamics of hPDI through molecular dynamics (MD) simulations. Collective domain motions are identified by the principal component analysis of MD trajectories and redox-dependent opening-closing structure variations are highlighted on projected free energy landscapes. Then, important structural features that exhibit considerable differences in dynamics of redox states are extracted by statistical machine learning methods. Mapping the structural variations to time series of residue interaction networks also provides a holistic representation of the dynamical redox differences. With emphasizing on persistent long-lasting interactions, an approach is proposed that compiled these time series networks to a single dynamic residue interaction network (DRIN). Differential comparison of DRIN in oxidized and reduced states reveals chains of residue interactions that represent potential allosteric paths between catalytic and ligand binding sites of hPDI.

Purpose Whole genome sequencing of SARS-CoV2 is important to find useful information about the viral lineages, variants of interests and variants of concern. As there are not enough data about the circulating SARS-CoV2 variants in Iran, we sequenced 54 SARS-CoV2 genomes during the 5 waves of pandemic in Iran. Methods After viral RNA extraction from clinical samples collected during the COVID-19 pandemic, next generation sequencing was performed using the Nextseq platform. The sequencing data were analyzed and compared with reference sequences. Results During the 1st wave, V and L clades were detected. The second wave was recognized by G, GH and GR clades. Circulating clades during the 3rd wave were GH and GR. In the fourth wave GRY (alpha variant), GK (delta variant) and one GH clade (beta variant) were detected. All viruses in the fifth wave were in clade GK (delta variant). There were different mutations in all parts of the genomes but Spike-D614G, NSP12-P323L, N-R203K and N-G204R were the most frequent mutants in these studied viruses. Conclusions These findings display the significance of SARS-CoV2 monitoring to help on time detection of possible variants for pandemic control and vaccination plans.