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Embryonic tissues undergoing shape change draw mechanical input from extraembryonic substrates. In avian eggs, the early blastoderm disk is under the tension of the vitelline membrane (VM). Here we report that the chicken VM characteristically downregulates tension and stiffness to facilitate stage-specific embryo morphogenesis. Experimental relaxation of the VM early in development impairs blastoderm expansion, while maintaining VM tension in later stages resists the convergence of the posterior body causing stalled elongation, failure of neural tube closure, and axis rupture. Biochemical and structural analysis shows that VM weakening is associated with the reduction of outer-layer glycoprotein fibers, which is caused by an increasing albumen pH due to CO2 release from the egg. Our results identify a previously unrecognized potential cause of body axis defects through mis-regulation of extraembryonic tissue tension.

In the central nervous system, the type I transmembrane glycoprotein NG2 (nerve-glia antigen 2) is only expressed by pericytes and oligodendrocyte precursor cells (OPCs). Therefore, OPCs are also termed NG2 glia. Their fate during development has been investigated systematically in several genetically modified mouse models. Consensus exists that postnatal NG2 glia are restricted to the oligodendrocyte (OL) lineage, while, at least in the forebrain, embryonic NG2 glia could also generate astrocytes. In addition, experimental evidence for a neurogenic potential of NG2 glia in the early embryonic brain (before E16.5) has been provided. However, this observation is still controversial. Here, we took advantage of reliable transgene expression in NG2-EYFP and NG2-CreERT2 knock-in mice to study the fate of early embryonic NG2 glia. While pericytes were the main cells with robust NG2 gene activity at E12.5, only a few OPCs expressed NG2 at this early stage of embryogenesis. Subsequently, this proportion of OPCs increased from 3% (E12.5) to 11% and 25% at E14.5 and E17.5, respectively. When Cre DNA recombinase activity was induced at E12.5 and E14.5 and pups were analyzed at postnatal day 0 (P0) and P10, the vast majority of recombined cells, besides pericytes, belonged to the OL lineage cells, with few astrocytes in the ventral forebrain. In other brain regions such as brain stem, cerebellum, and olfactory bulb only OL lineage cells were detected. Therefore, we conclude that NG2 glia from early embryonic brain are restricted to a gliogenic fate and do not differentiate into neurons after birth.

Musculoskeletal injuries are common in humans. The cascade of cellular and molecular events following such injuries results either in healing with functional recovery or scar formation. While fibrotic scar tissue serves to bridge between injured planes, it undermines functional integrity. Hence, faithful regeneration is the most desired outcome; however, the potential to regenerate is limited in humans. In contrast, various non-mammalian vertebrates have fascinating capabilities of regenerating even an entire appendage following amputation. Among them, zebrafish is an important and accessible laboratory model organism, sharing striking similarities with mammalian embryonic musculoskeletal development. Moreover, clinically relevant muscle and skeletal injury zebrafish models recapitulate mammalian regeneration. Upon muscle injury, quiescent stem cells - known as satellite cells - become activated, proliferate, differentiate and fuse to form new myofibres, while bone fracture results in a phased response involving hematoma formation, inflammation, fibrocartilaginous callus formation, bony callus formation and remodelling. These models are well suited to testing gene- or pharmaco-therapy for the benefit of conditions like muscle tears and fractures. Insights from further studies on whole body part regeneration, a hallmark of the zebrafish model, have the potential to complement regenerative strategies to achieve faster and desired healing following injuries without any scar formation and, in the longer run, drive progress towards the realisation of large-scale regeneration in mammals. Here, we provide an overview of the basic mechanisms of musculoskeletal regeneration, highlight the key features of zebrafish as a regenerative model and outline the relevant studies that have contributed to the advancement of this field. AKKP is supported by a Nanyang Technological University (Lee Kong Chian School of Medicine) Research Scholarship. PWI is supported by the Toh Kian Chui Foundation.

Induced pluripotent stem cells (iPSCs) can enhance the efficiency of buffalo genetic improvements because of their differentiation potential and proliferation ability, which are similar to those of embryonic stem cells. However, very few studies have focussed on buffalo iPSCs, and a stable induction system has not been established for buffalo somatic cell reprogramming. In this study, we constructed a PiggyBac transposon vector co-expressing buffalo OCT4, C-MYC, KLF4 and SOX2 genes (PB_OMKS) separated by the nucleotide sequence of three 2A peptides and established the buffalo foetal skin fibroblast (BFSF) cell line BFSF_OMKS. RNA-seq technology and bioinformatics analysis methods were mainly employed to perform a transcriptome analysis between BFSF and BFSF_OMKS. The results revealed that over-expression of OCT4, C-MYC, KLF4 and SOX2 in BFSFs led to the activation of reprogramming-related LIF, activin, BMP4, SMAD1/5/9 and Wnt signals. These results increased our understanding of buffalo somatic cell reprogramming mechanisms and could provide a possible theory for the selection of small-molecule cocktails to promote reprogramming.

Embryonic dorsal-ventral (DV) patterning by Bone Morphogenetic Proteins (BMPs) is a conserved feature of Bilateria, based on graded BMP activity set up by diffusible BMP ligands and Chordin/Sog antagonists. In the fly Drosophila melanogaster BMP function is secondary to patterning by the Toll pathway, suggesting a more restricted role for BMPs in insects. With widespread genome sequencing technologies allied to functional analysis in a growing number of species, recent work has shown that BMP's role in DV patterning relative to Toll varies among insect orders. Further, the role of BMP antagonists to set up BMP gradients is also greatly diversified. Here we review the recent findings concerning the role of BMP in the DV patterning of insects and address the potential aspects that may have co-evolved with BMPs to attain this functional divergence.

Chromatin modification contributes to pluripotency maintenance in embryonic stem cells (ESCs). However, the related mechanisms remain obscure. Here, we show that Npac, a "reader" of histone H3 lysine 36 trimethylation (H3K36me3), is required to maintain mouse ESC (mESC) pluripotency since knockdown of Npac causes mESC differentiation. Depletion of Npac in mouse embryonic fibroblasts (MEFs) inhibits reprogramming efficiency. Furthermore, our chromatin immunoprecipitation followed by sequencing (ChIP-seq) results of Npac reveal that Npac co-localizes with histone H3K36me3 in gene bodies of actively transcribed genes in mESCs. Interestingly, we find that Npac interacts with positive transcription elongation factor b (p-TEFb), Ser2-phosphorylated RNA Pol II (RNA Pol II Ser2P), and Ser5-phosphorylated RNA Pol II (RNA Pol II Ser5P). Furthermore, depletion of Npac disrupts transcriptional elongation of the pluripotency genes Nanog and Rif1. Taken together, we propose that Npac is essential for the transcriptional elongation of pluripotency genes by recruiting p-TEFb and interacting with RNA Pol II Ser2P and Ser5P.

SUMMARYThe formation of the primitive streak (PS) and the subsequent induction of neuroectoderm are hallmarks of gastrulation. Combining an in vitro reconstitution of this process based on mouse embryonic stem cells (mESCs) with a collection of knockouts in reporter mESC lines, we reassessed the contribution of retinoic acid (RA) signaling at early stages of neural commitment and its cross-talk with TGFβ and Wnt signaling inhibition. Single-cell RNA sequencing analysis captured the temporal unfolding of cell type diversification from epiblast and primitive streak-like cells up to the emergence of anterior and posterior neural fates. In conditions thought to lack RA synthesis, we discovered a hitherto unidentified residual RA production via a sensitive RA reporter. Genetic perturbations proved that the RA-degrading enzyme Cyp26a1 safeguard the developmental capabilities of the PS-like cells, limiting neural differentiation in wild type and in Chrd-/-Nog-/- or Dkk1-/- cells. Finally, the knockout of the three RAR receptors highlighted their function as negative regulators of loci critical for neural induction. Overall, we identified two mechanisms whereby components of the RA pathway can control the formation of neural progenitors in our PS-like context: a RA-dependent neural induction gated by RARs, and a receptor-mediated repression in the absence of ligand.

Summary The genetic modification of stem cells (SCs) is typically achieved using integrating vectors, whose potential integrative genotoxicity and propensity for epigenetic silencing during differentiation limit their application. The genetic modification of cells should provide sustainable levels of transgene expression, without compromising the viability of a cell or its progeny. We developed nonviral, nonintegrating, and autonomously replicating minimally sized DNA nanovectors to persistently genetically modify SCs and their differentiated progeny without causing any molecular or genetic damage. These DNA vectors are capable of efficiently modifying murine and human pluripotent SCs with minimal impact and without differentiation-mediated transgene silencing or vector loss. We demonstrate that these vectors remain episomal and provide robust and sustained transgene expression during self-renewal and targeted differentiation of SCs both in vitro and in vivo through embryogenesis and differentiation into adult tissues, without damaging their phenotypic characteristics., Graphical abstract, Highlights • Nanovectors are used to engineer SCs efficiently, safely, and persistently • Isogenic SC lines retain their capacity for self-renewal and pluripotency • Nanovectors survive reprogramming and differentiation without loss or silencing • Nanovectors are a universal genetic tool for the modification of any cell, In this study, Harbottle and colleagues show that nonviral, nonintegrating, and autonomously replicating minimally sized DNA nanovectors can be used to persistently modify SCs genetically without causing any molecular or genetic damage. The DNA vectors remain episomal during self-renewal and differentiation in vitro and in vivo, while providing robust and sustained transgene expression.

Vertebrate organs are arranged in a stereotypic, species-specific position along the animal body plan. Substantial morphological variation exists between related species, especially so in the vastly diversified teleost clade. It is still unclear how tissues, organs and systems can accommodate such diverse scaffolds. Here, we use the distinctive arrangement of neuromasts in the posterior lateral line (pLL) system of medaka fish to address the tissue-interactions defining a pattern. We show that patterning in this peripheral nervous system is established by autonomous organ precursors independent of neuronal wiring. In addition, we target the keratin 15 gene to generate stuck-in-the-midline (siml) mutants, which display epithelial lesions and a disrupted pLL patterning. By using siml/wt chimeras, we determine that the aberrant siml pLL pattern depends on the mutant epithelium, since a wild type epithelium can rescue the siml phenotype. Inducing epithelial lesions by 2-photon laser ablation during pLL morphogenesis phenocopies siml genetic mutants and reveals that epithelial integrity defines the final position of the embryonic pLL neuromasts. Our results using the medaka pLL disentangle intrinsic from extrinsic properties during the establishment of a sensory system. We speculate that intrinsic programs guarantee proper organ morphogenesis, while instructive interactions from surrounding tissues facilitates the accommodation of sensory organs to the diverse body plans found among teleosts.

Salivary gland hypofunction due to radiation therapy for head and neck cancer or Sjogren syndrome may cause various oral diseases, which can lead to a decline in the quality of life. Cell therapy using salivary gland stem cells is a promising method for restoring hypofunction. Herein, we show that salivary gland-like cells can be induced from epithelial tissues that were transdifferentiated from mouse embryonic fibroblasts (MEFs). We introduced four genes, Dnp63a, Tfap2a, Grhl2, and Myc (PTMG) that are known to transdifferentiate fibroblasts into oral mucosa-like epithelium in vivo into MEFs. MEFs overexpressing these genes showed epithelial cell characteristics, such as cobblestone appearance and E-cadherin positivity, and formed oral epithelial-like tissue under air–liquid interface culture conditions. The epithelial sheet detached from the culture dish was infected with adenoviruses encoding Sox9 and Foxc1, which we previously identified as essential factors to induce salivary gland formation. The cells detached from the cell sheet formed spheres 10 days after infection and showed a branching morphology. The spheres expressed genes encoding basal/myoepithelial markers, cytokeratin 5, cytokeratin 14, acinar cell marker, aquaporin 5, and the myoepithelial marker α-smooth muscle actin. The dissociated cells of these primary spheres had the ability to form secondary spheres. Taken together, our results provide a new strategy for cell therapy of salivary glands and hold implications in treating patients with dry mouth.

Parkinson’s disease (PD) is the second most common neurodegenerative disease and a prime target of cell therapies. In fact, aborted fetal tissue has been used as donor material for such therapies since the 1980s. These cell therapies, however, suffer from several problems, such as a short supply of donor materials, quality instability of the tissues, and ethical restrictions. The advancement of stem cell technologies has enabled the production of donor cells from pluripotent stem cells with unlimited scale, stable quality, and less ethical problems. Several research groups have established protocols to induce dopamine neural progenitors from pluripotent stem cells in a clinically compatible manner and confirmed efficacy and safety in non-clinical studies. Based on the results from these non-clinical studies, several clinical trials of pluripotent stem cell-based therapies for PD have begun. In the context of immune rejection, there are several modes of stem cell-based therapies: autologous transplantation, allogeneic transplantation without human leukocyte antigen-matching, and allogeneic transplantation with matching. In this mini-review, several practical points of stem cell-based therapies for PD are discussed.

In the past several decades, the establishment of in vitro models of pluripotency has ushered in a golden era for developmental and stem cell biology. Research in this arena has led to profound insights into the regulatory features that shape early embryonic development. Nevertheless, an integrative theory of the epigenetic principles that govern the pluripotent nucleus remains elusive. Here, we summarize the epigenetic characteristics that define the pluripotent state. We cover what is currently known about the epigenome of pluripotent stem cells and reflect on the use of embryonic stem cells as an experimental system. In addition, we highlight insights from super-resolution microscopy, which have advanced our understanding of the form and function of chromatin, particularly its role in establishing the characteristically "open chromatin" of pluripotent nuclei. Further, we discuss the rapid improvements in 3C-based methods, which have given us a means to investigate the 3D spatial organization of the pluripotent genome. This has aided the adaptation of prior notions of a "pluripotent molecular circuitry" into a more holistic model, where hotspots of co-interacting domains correspond with the accumulation of pluripotency-associated factors. Finally, we relate these earlier hypotheses to an emerging model of phase separation, which posits that a biophysical mechanism may presuppose the formation of a pluripotent-state-defining transcriptional program.

The regenerative potential of Muller glia (MG) is extraordinary in fish, poor in chick and terrible in mammals. In the chick model, MG readily reprogram into proliferating Muller glia-derived progenitor cells (MGPCs), but neuronal differentiation is very limited. The factors that suppress the neurogenic potential of MGPCs in the chick are slowly being revealed. Isoforms of Nuclear Factor I (NFI) are cell-intrinsic factors that limit neurogenic potential; these factors are required for the formation of MG in the developing mouse retina (Clark et al., 2019) and deletion of these factors reprograms MG into neuron-like cells in mature mouse retina (Hoang et al., 2020). Accordingly, we sought to characterize the patterns of expression NFIs in the developing, mature and damaged chick retina. In addition, we characterized patterns of expression of NFIs in the retinas of large mammals, pigs and monkeys. Using a combination of single cell RNA-sequencing (scRNA-seq) and immunolabeling we probed for patterns of expression. In embryonic chick, levels of NFIs are very low in early E5 (embryonic day 5) retinal progenitor cells (RPCs), up-regulated in E8 RPCs, further up-regulated in differentiating MG at E12 and E15. NFIs are maintained in mature resting MG, microglia and neurons. Levels of NFIs are reduced in activated MG in retinas treated with NMDA and/or insulin+FGF2, and further down-regulated in proliferating MGPCs. However, levels of NFIs in MGPCs were significantly higher than those seen in RPCs. Immunolabeling for NFIA and NFIB closely matched patterns of expression revealed in different types of retinal neurons and glia, consistent with findings from scRNA-seq. In addition, we find expression of NFIA and NFIB through progenitors in the circumferential marginal zone at the far periphery of the retina. We find similar patterns of expression for NFIs in scRNA-seq databases for pig and monkey retinas. Patterns of expression of NFIA and NFIB were validated with immunofluorescence in pig and monkey retinas wherein these factors were predominantly detected in MG and a few types of inner retinal neurons. In summary, NFIA and NFIB are prominently expressed in developing chick retina and by mature neurons and glia in the retinas of chicks, pigs and monkeys. Although levels of NFIs are decreased in chick, in MGPCs these levels remain higher than those seen in neurogenic RPCs. We propose that the neurogenic potential of MGPCs in the chick retina is suppressed by NFIs. This article is protected by copyright. All rights reserved.

RNA-binding proteins (RBPs) play diverse roles in regulating co-transcriptional RNA-processing and chromatin functions, but our knowledge of the repertoire of chromatin-associated RBPs (caRBPs) and their interactions with chromatin remains limited. Here, we developed SPACE (Silica Particle Assisted Chromatin Enrichment) to isolate global and regional chromatin components with high specificity and sensitivity, and SPACEmap to identify the chromatin-contact regions in proteins. Applied to mouse embryonic stem cells, SPACE identified 1459 chromatin-associated proteins, ∼48% of which are annotated as RBPs, indicating their dual roles in chromatin and RNA-binding. Additionally, SPACEmap stringently verified chromatin-binding of 403 RBPs and identified their chromatin-contact regions. Notably, SPACEmap showed that about 40% of the caRBPs bind chromatin by intrinsically disordered regions (IDRs). Studying SPACE and total proteome dynamics from mES cells grown in 2iL and serum medium indicates significant correlation (R = 0.62). One of the most dynamic caRBPs is Dazl, which we find co-localized with PRC2 at transcription start sites of genes that are distinct from Dazl mRNA binding. Dazl and other PRC2-colocalised caRBPs are rich in intrinsically disordered regions (IDRs), which could contribute to the formation and regulation of phase-separated PRC condensates. Together, our approach provides an unprecedented insight into IDR-mediated interactions and caRBPs with moonlighting functions in native chromatin., Graphical Abstract Graphical AbstractSPACE, SPACEmap and ChIP-SPACE are highly sensitive and stringent approaches for identification of chromatin-associated proteins and their chromatin-contact regions.

Morphogenesis drives the formation of functional living shapes. Gene expression patterns and signaling pathways define the body plans of the animal and control the morphogenetic processes shaping the embryonic tissues. During embryogenesis, a tissue can undergo composite morphogenesis resulting from multiple concomitant shape changes. While previous studies have unraveled the mechanisms that drive simple morphogenetic processes, how a tissue can undergo multiple and simultaneous changes in shape is still not known and not much explored. In this chapter, we focus on the process of concomitant tissue folding and extension that is vital for the animal since it is key for embryo gastrulation and neurulation. Recent pioneering studies focus on this problem highlighting the roles of different spatially coordinated cell mechanisms or of the synergy between different patterns of gene expression to drive composite morphogenesis.

2,2′,4,4′-Tetrabromodiphenyl ether (BDE47) poses potential risks to reproduction and development, but the mechanism of its toxicity has not yet been elucidated. To explore the developmental toxicity of BDE47, mouse embryonic stem cells (mESCs), which are ideal models for testing the developmental toxicity of environmental contaminants in vitro, were exposed to BDE47 (0.04 μM, 1 μM, 25 μM, or 100 μM) for 24 h or 48 h in this study. Our results indicated that BDE47 treatment changed the morphology of mESCs, inhibited cell viability and increased apoptosis. In addition, alkaline phosphatase (AP) staining in mESCs was significantly decreased after BDE47 treatment (25 μM and 100 μM), indicating that BDE47 treatment affected the pluripotency of mESCs. Through a cell immunofluorescence assay, we found that the fluorescence intensities of Oct4, Sox2 and Nanog were all significantly lower in the group treated with the highest BDE47 concentration (100 μM) than in the control group, consistent with the qRT-PCR and Western blot results. The levels of miR-145 and miR-34a, which regulate genes related to cell differentiation, were significantly increased in BDE47-treated mESCs, further clarifying the potential mechanism. Overall, our findings demonstrate that BDE47 exposure upregulates the expression of miR-145 and miR-34a and in turn downregulates the expression of Oct4, Sox2 and Nanog, thereby affecting apoptosis and pluripotency and causing toxicity during embryonic development.

Microglia, which migrate into the central nervous system (CNS) during the early embryonic stages, are considered to play various roles in CNS development. However, their embryonic roles are largely unknown, partly due to the lack of an effective microglial ablation system in the embryo. Here, we show a microglial ablation model by injecting diphtheria toxin (DT) into the amniotic fluid of Siglechdtr mice, in which the gene encoding DT receptor is knocked into the microglia-specific gene locus Siglech. We revealed that embryonic microglia were depleted for several days throughout the CNS, including some regions where microglia transiently accumulated, at any embryonic time point from embryonic day 10.5, when microglia colonize the CNS. This ablation system was specific for microglia because CNS-associated macrophages, which are a distinct population from microglia that reside in the CNS interfaces such as meninges, were unaffected. Therefore, this microglial ablation system is highly effective for studying the embryonic functions of microglia.

Summary RYBP (Ring1 and YY1 binding protein), an essential component of the Polycomb repressive complex 1 (PRC1), plays pivotal roles in development and diseases. However, the roles of Rybp in neuronal development remains completely unknown. In the present study, we have shown that the depletion of Rybp inhibits proliferation and promotes neuronal differentiation of embryonic neural progenitor cells (eNPCs). In addition, Rybp deficiency impairs the morphological development of neurons. Mechanistically, Rybp deficiency does not affect the global level of ubiquitination of H2A, but it inhibits Notch signaling pathway in eNPCs. The direct interaction between RYBP and CIR1 facilitates the binding of RBPJ to Notch intracellular domain (NICD) and consequently activated Notch signaling. Rybp loss promotes CIR1 competing with RBPJ to bind with NICD, and inhibits Notch signaling. Furthermore, ectopic Hes5, Notch signaling downstream target, rescues Rybp-deficiency-induced deficits. Collectively, our findings show that RYBP regulates embryonic neurogenesis and neuronal development through modulating Notch signaling in a PRC1-independent manner., Highlights • Rybp deficiency inhibits the proliferation and promotes the neuronal differentiation of embryonic neural progenitor cells (eNPCs) • Rybp deficiency impairs the morphological development of hippocampal neuron • Rybp loss does not affect the global level of ubiquitination of H2A in eNPCs, but promotes CIR1 competing with RBPJ to bind with NICD • The inhibited Notch signaling in Rybp-deficient eNPCs can be rescued by ectopic Hes5, In this article, Li and colleagues explore the function of RYBP in embryonic neurogenesis. They demonstrate that Rybp deficiency results in aberrant neurogenesis by inhibiting the proliferation and promoting neuronal differentiation of embryonic neural progenitor cells (eNPCs). In addition, they show that RYBP regulates embryonic neurogenesis through affecting Notch signaling in a Polycomb repressive complex 1 (PRC1)-independent manner.

The positioning of the nucleus, the central organelle of the cell, is an active and regulated process crucially linked to cell cycle, differentiation, migration, and polarity. Alterations in positioning have been correlated with cell and tissue function deficiency and genetic or chemical manipulation of nuclear position is embryonic lethal. Nuclear positioning is a precursor for symmetric or asymmetric cell division which is accompanied by fate determination of the daughter cells. Nuclear positioning also plays a key role during early embryonic developmental stages in insects, such as Drosophila, where hundreds of nuclei divide without cytokinesis and are distributed within the large syncytial embryo at roughly regular spacing. While the cytoskeletal elements and the linker proteins to the nucleus are fairly well characterised, including some of the force generating elements driving nuclear movement, there is considerable uncertainty about the biophysical mechanism of nuclear positioning, while the field is debating different force models. In this review, we highlight the current body of knowledge, discuss cell context dependent models of nuclear positioning, and outline open questions.

The transcription factor NANOG regulates self-renewal and pluripotency in embryonic cells, and its downregulation leads to cell differentiation. Recent studies have linked upregulation of NANOG in various cancers and the regulation of expression of different molecules, and vice versa, to induce proliferation, metastasis, invasion and chemoresistance. Thus NANOG is an oncogene that functions by inducing stem cells’ circuitries and heterogeneity in cancers. Understanding NANOG’s role in various cancers may lead to it becoming a therapeutic target to halt cancer progression. The NANOG network can also be targeted to resensitize resistant cancer cells to conventional therapies. The current review focuses on NANOG regulation in the various signaling networks leading to cancer progression and chemoresistance, and highlights the therapeutic aspect of targeting NANOG in various cancers.

Pig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.

The placenta regulates maternal-fetal communication, and its defect leads to significant pregnancy complications. The maternal and embryonic circulations are primitively connected in early placentation, but the function of the placenta during this developmentally essential period is relatively unknown. We thus performed a comparative proteomic analysis of the placenta before and after primary placentation and found that the metabolism and transport of lipids were characteristically activated in this period. The placental fatty acid (FA) carriers in specific placental compartments were upregulated according to gestational age, and metabolomic analysis also showed that the placental transport of FAs increased in a time-dependent manner. Further analysis of two mutant mice models with embryonic lethality revealed that lipid-related signatures could reflect the functional state of the placenta. Our findings highlight the importance of the nutrient transport function of the primary placenta in the early gestational period and the role of lipids in embryonic development. Summary Sentence The placenta is activated characteristically in terms of lipid transport during primary placentation, and the lipid-related signatures closely reflect the functional state of the placenta.

Multifaceted microglial functions in the developing brain, such as promoting the differentiation of neural progenitors and contributing to the positioning and survival of neurons, have been progressively revealed. Although previous studies have noted the relationship between vascular endothelial cells and microglia in the developing brain, little attention has been given to the importance of pericytes, the mural cells surrounding endothelial cells. In this study, we attempted to dissect the role of pericytes in microglial distribution and function in developing mouse brains. Our immunohistochemical analysis showed that approximately half of the microglia attached to capillaries in the cerebral walls. Notably, a magnified observation of the position of microglia, vascular endothelial cells and pericytes demonstrated that microglia were preferentially associated with pericytes that covered 79.8% of the total capillary surface area. Throughin vivopericyte depletion induced by the intraventricular administration of a neutralizing antibody against platelet-derived growth factor receptor (PDGFR)β (clone APB5), we found that microglial density was markedly decreased compared with that in control antibody-treated brains because of their low proliferative capacity. Moreover,in vitrococulture of isolated CD11b+microglia and NG2+PDGFRα–cells, which are mostly composed of pericytes, from parenchymal cells indicated that pericytes promote microglial proliferation via the production of soluble factors. Furthermore, pericyte depletion by APB5 treatment resulted in a failure of microglia to promote the differentiation of neural stem cells into intermediate progenitors. Taken together, our findings suggest that pericytes facilitate microglial homeostasis in the developing brains, thereby indirectly supporting microglial effects on neural progenitors.SIGNIFICANCE STATEMENTThis study highlights the novel effect of pericytes on microglia in the developing mouse brain. Through multiple analyses using anin vivopericyte depletion mouse model and anin vitrococulture study of isolated pericytes and microglia from parenchymal cells, we demonstrated that pericytes contribute to microglial proliferation and support microglia in efficiently promoting the differentiation of neural stem cells into intermediate progenitors. Our present data provide evidence that pericytes function not only in the maintenance of cerebral microcirculation and blood brain barrier (BBB) integrity but also in microglial homeostasis in the developing cerebral walls. These findings will expand our knowledge and help elucidate the mechanism of brain development both in healthy and disease conditions.

Hypoxia is involved in the regulation of various cell functions in the body, including the regulation of stem cells. The hypoxic microenvironment is indispensable from embryonic development to the regeneration and repair of adult cells. In addition to embryonic stem cells, which need to maintain their self‐renewal properties and pluripotency in a hypoxic environment, adult stem cells, including neural stem cells (NSCs), also exist in a hypoxic microenvironment. The subventricular zone (SVZ) and hippocampal dentate gyrus (DG) are the main sites of adult neurogenesis in the brain. Hypoxia can promote the proliferation, migration, and maturation of NSCs in these regions. Also, because most neurons in the brain are non‐regenerative, stem cell transplantation is considered as a promising strategy for treating central nervous system (CNS) diseases. Hypoxic treatment also increases the effectiveness of stem cell therapy. In this review, we firstly describe the role of hypoxia in different stem cells, such as embryonic stem cells, NSCs, and induced pluripotent stem cells, and discuss the role of hypoxia‐treated stem cells in CNS diseases treatment. Furthermore, we highlight the role and mechanisms of hypoxia in regulating adult neurogenesis in the SVZ and DG and adult proliferation of other cells in the CNS., Hypoxia promotes stem cell proliferation, differentiation, and survival. Hypoxia promotes the pluripotency, proliferation, and directed differentiation of embryonic stem cells (ESCs) and neural stem cells (NSCs) and helps induced pluripotent stem cells (iPSCs) to develop into different types of cells in vitro. Hypoxia is also involved in adult neurogenesis, angiogenesis, and gliogenesis. DG, dentate gyrus; LV, lateral ventricle; SVZ, subventricular zone.

Reactive oxygen species (ROS) play important roles as second messengers in a wide array of cellular processes including differentiation of stem cells. We identified Nox4 as the major ROS-generating enzyme whose expression is induced during differentiation of embryoid body (EB) into cells of all three germ layers. The role of Nox4 was examined using induced pluripotent stem cells (iPSCs) generated from Nox4 knockout (Nox4−/−) mouse. Differentiation markers showed significantly reduced expression levels consistent with the importance of Nox4-generated ROS during this process. From transcriptomic analyses, we found insulin-like growth factor 2 (IGF2), a member of a gene family extensively involved in embryonic development, as one of the most down-regulated genes in Nox4−/− cells. Indeed, addition of IGF2 to culture partly restored the differentiation competence of Nox4−/− iPSCs. Our results reveal an important signaling axis mediated by ROS in control of crucial events during differentiation of pluripotent stem cells. Graphical Abstract

Hippo signaling pathway is involved in many biological processes including the fate decision of embryonic stem cells (ESCs). Yes-associated protein (Yap) function as a key effector of Hippo pathway, but its role in ESCs is still controversial. So far, only two isoforms of Yap have been identified and they have both overlapping and distinct functions. Here, we identify six novel isoforms of mouse Yap, bringing the total number of isoforms to eight. According to the differences in the first exon, they are divided into two subtypes (a and b). Isoform-a and isoform-b exhibit different subcellular localizations. Moreover, isoform-a can fully reverse the impaired self-renewal phenotype induced by Yap knockout (KO). Upon overexpression, isoform-a moderately promotes mESCs self-renewal and markedly delays differentiation. On the contrary, no significant pro-self-renewal phenotype is observed when isoform-b overexpressed in wildtype (WT) mESCs or re-expressed in Yap KO cell lines. These finding not only help to clarify the role of Yap in mESCs, but also lay the foundation for advancing functional researches of Yap in other processes.

[Background]: Fibroblast growth factors (Fgfs) are required for survival and organ formation during embryogenesis. Fgfs often execute their functions redundantly. Previous analysis of Fgf3 mutants revealed effects on inner ear formation and embryonic survival with incomplete penetrance. [Results]: Here, we show that presence of a neomycin resistance gene (neo) replacing the Fgf3 coding region leads to reduced survival during embryogenesis and an increased penetrance of inner ear defects. Fgf3neo/neo mutants showed reduced expression of Fgf4, which is positioned in close proximity to the Fgf3 locus in the mouse genome. Conditional inactivation of Fgf4 during inner ear development on a Fgf3 null background using Fgf3/4 cis mice revealed a redundant requirement between these Fgfs during otic placode induction. In contrast, inactivation of Fgf3 and Fgf4 in the pharyngeal region where both Fgfs are also co-expressed using a Foxg1-Cre driver did not affect development of the pharyngeal arches. However, these mutants showed reduced perinatal survival. [Conclusions]: These results highlight the importance of Fgf signaling during development. In particular, different members of the Fgf family act redundantly to guarantee inner ear formation and embryonic survival., Consejería de Educación, Junta de Castilla y León, Grant/Award Number: CSI143P20; Programa Estratégico Instituto de Biología y Genética Molecular (IBGM), Escalera de Excelencia, Junta de Castilla y León, Grant/Award Numbers: CCVC8485, CLU-2019-02; MEC, Grant/Award Number: BFU2004-00860/BFI

The axon initial segment (AIS) is a specialized structure that controls neuronal excitability via action potential (AP) generation. Currently, AIS plasticity with regard to changes in length and location in response to neural activity has been extensively investigated, but how AIS diameter is regulated remains elusive. Here we report that COUP-TFI (chicken ovalbumin upstream promotor-transcription factor 1) is an essential regulator of AIS diameter in both developing and adult mouse neocortex. Either embryonic or adult ablation of COUP-TFI results in reduced AIS diameter and impaired AP generation. Although COUP-TFI ablations in sparse single neurons and in populations of neurons have similar impacts on AIS diameter and AP generation, they strengthen and weaken, respectively, the receiving spontaneous network in mutant neurons. In contrast, overexpression of COUP-TFI in sparse single neurons increases the AIS diameter and facilitates AP generation, but decreases the receiving spontaneous network. Our findings demonstrate that COUP-TFI is indispensable for both the expansion and maintenance of AIS diameter and that AIS diameter fine-tunes action potential generation and synaptic inputs in mammalian cortical neurons. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12264-021-00792-8.

The harmful consumption of ethanol is associated with significant health problems and social burdens. This drug activates a complex network of reward mechanisms and habit formation learning that is supposed to contribute to the consumption of increasingly high and frequent amounts, ultimately leading to addiction. In the context of fetal alcohol spectrum disorders, fetal alcohol syndrome (FAS) is a consequence of the harmful use of alcohol during pregnancy, which affects the embryonic development of the fetus. FAS can be easily reproduced in zebrafish by exposing the embryos to different concentrations of ethanol in water. In this regard, the aim of the present review is to discuss the late pathological implications in zebrafish exposed to ethanol at the embryonic stage, providing information in the context of human fetal alcoholic spectrum disorders. Experimental FAS in zebrafish is associated with impairments in the metabolic, morphological, neurochemical, behavioral, and cognitive domains. Many of the pathways that are affected by ethanol in zebrafish have at least one ortholog in humans, collaborating with the wider adoption of zebrafish in studies on alcohol disorders. In fact, zebrafish present validities required for the study of these conditions, which contributes to the use of this species in research, in addition to studies with rodents.

Premature ovarian failure (POF) is defined by amenorrhea, hypoestrogenism, elevated gonadotropin levels, and infertility. Chemotherapeutic agents are the most gonadotoxic agents that lead to POF. Although some previous studies have presented that mesenchymal stem cells (MSCs) transplantation could rescue the ovary function of POF animal models through the paracrine pathway, these mechanisms require further investigation. However, mechanisms of embryonic stem cell-derived MSCs (ES-MSCs) therapeutic effects on POF animal models have not been fully investigated yet. This study aimed to evaluate the migration and distribution of ES-MSCs in a model of chemotherapy-induced POF. Female mice received intraperitoneal injections of cyclophosphamide (Cy) to induce POF. Then, MSCs were labeled with green fluorescent protein (GFP) in vitro and injected intravenously into POF mice, and the distribution of MSCs was dynamically monitored at 1 week after transplantation. We harvested the lungs, liver, spleen, ovaries, heart, and kidneys 1 week after transplantation. The sections of these tissues were observed under the fluorescent microscope. More than 70% MSCs were successfully labeled with GFP at 72 h after labeling. MSCs were uniformly distributed in multiple organs and tissues including lungs, liver, spleen, ovaries, heart, and kidneys of POF mice. In mice, at 1week after intravenous transplantation, GFP labeled ES-MSCs were observed in the lungs, liver, spleen, ovaries, heart, and kidneys of POF mice, and the number of GFP labeled ES-MSCs in lungs, ovaries, and heart were higher than that in the spleen, kidneys, and liver. Our results revealed intravenously implanted ES-MSCs could migrate into the various tissues in chemotherapy-induced damaged POF mice.

Macrophages are the dominant immune cell types in the adipose tissue, the liver or the aortic wall and they were originally believed to mainly derived from monocytes to fuel tissue inflammation in cardiometabolic diseases. However, over the last decade the identification of tissue resident macrophages (trMacs) from embryonic origin in these metabolic tissues has provided a breakthrough in the field forcing to better comprehend macrophage diversity during pathological states. Infiltrated monocyte-derived macrophages (moMacs), similar to trMacs, adapt to the local metabolic environment that eventually shapes their functions. In this review, we will summarize the emerging versatility of macrophages in cardiometabolic diseases with a focus in the control of adipose tissue, liver and large vessels homeostasis.

Summary p53 alterations occur during culture of pluripotent stem cells (PSCs), but the significance of these events on epigenetic control of PSC fate determination remains poorly understood. Wdr5 deletion in p53-null (DKO) mouse ESCs (mESCs) leads to impaired self-renewal, defective retinal neuroectoderm differentiation, and de-repression of germ cell/meiosis (GCM)-specific genes. Re-introduction of a WDR5 mutant with defective H3K4 methylation activity into DKO ESCs restored self-renewal and suppressed GCM gene expression but failed to induce retinal neuroectoderm differentiation. Mechanistically, mutant WDR5 targets chromatin that is largely devoid of H3K4me3 and regulates gene expression in p53-null mESCs. Furthermore, MAX and WDR5 co-target lineage-specifying chromatin and regulate chromatin accessibility of GCM-related genes. Importantly, MAX and WDR5 are core subunits of a non-canonical polycomb repressor complex 1 responsible for gene silencing. This function, together with canonical, pro-transcriptional WDR5-dependent MLL complex H3K4 methyltransferase activity, highlight how WDR5 mediates crosstalk between transcription and repression during mESC fate choice., Graphical abstract, Highlights • H3K4me defective WDR5 supports self-renewal and GCM differentiation in p53-null mESCs • WDR5 regulates H3K4me-independent stemness and GCM gene expression in p53-null mESCs • MAX and WDR5 repress GCM-related gene chromatin accessibility upon differentiation, In this article, Rao and colleagues show that an H3K4 methylation-independent, WDR5-driven mechanism maintains self-renewal and represses germ cell/meiosis (GCM)-specific transcription in p53-null mESCs. WDR5 inactivation triggers enhanced chromatin accessibility on most gene targets bound by MAX and WDR5. Thus, WDR5, a component of polycomb repressive complex 1.6, controls cell type-specific gene repression during mESC fate determination in a non-canonical fashion.

RASSF6 is a member of the tumor suppressor Ras-association domain family (RASSF) proteins. We have reported using human cancer cell lines that RASSF6 induces apoptosis and cell cycle arrest via p53 and plays tumor-suppressive roles. In this study, we generated Rassf6 knockout mice by CRISPR/Cas technology. Contrary to our expectation, Rassf6 knockout mice were apparently healthy. However, Rassf6-null mouse embryonic fibroblasts (MEF) were resistant against ultraviolet (UV)-induced apoptosis/cell cycle arrest and senescence. UV-induced p53-target gene expression was compromised and DNA repair was delayed in Rassf6-null MEF. More importantly, KRAS active mutant promoted the colony formation of Rassf6-null MEF but not the wild-type MEF. RNA sequencing analysis revealed that NF-κB signaling was enhanced in Rassf6-null MEF. Consistently, 7,12-Dimethylbenz(a)anthracene (DMBA) induced skin inflammation in Rassf6 knockout mice more remarkably than in the wild-type mice. Hence, Rassf6 deficiency not only compromises p53 function but also enhances NF-κB signaling to lead to oncogenesis.

The Notch pathway is an ancient intercellular signaling system with crucial roles in numerous cell-fate decision processes across species. While the canonical pathway is activated by ligand-induced cleavage and nuclear localization of membrane-bound Notch, Notch can also exert its activity in a ligand/transcription-independent fashion, which is conserved in Drosophila, Xenopus, and mammals. However, the noncanonical role remains poorly understood in in vivo processes. Here we show that increased levels of the Notch intracellular domain (NICD) in the early mesoderm inhibit heart development, potentially through impaired induction of the second heart field (SHF), independently of the transcriptional effector RBP-J. Similarly, inhibiting Notch cleavage, shown to increase noncanonical Notch activity, suppressed SHF induction in embryonic stem cell (ESC)-derived mesodermal cells. In contrast, NICD overexpression in late cardiac progenitor cells lacking RBP-J resulted in an increase in heart size. Our study suggests that noncanonical Notch signaling has stage-specific roles during cardiac development.

The corpus callosum (CC) is a major interhemispheric commissure of placental mammals. Early steps of CC formation rely on guidance strategies, such as axonal branching and collateralization. Here we analyze the time-course dynamics of axonal bifurcation during typical cortical development or in a CC dysgenesis mouse model. We use Swiss mice as a typical CC mouse model and find that axonal bifurcation rates rise in the cerebral cortex from embryonic day (E)17 and are reduced by postnatal day (P)9. Since callosal neurons populate deep and superficial cortical layers, we compare the axon bifurcation ratio between those neurons by electroporating ex vivo brains at E13 and E15, using eGFP reporter to label the newborn neurons on organotypic slices. Our results suggest that deep layer neurons bifurcate 32% more than superficial ones. To investigate axonal bifurcation in CC dysgenesis, we use BALB/c mice as a spontaneous CC dysgenesis model. BALB/c mice present a typical layer distribution of SATB2 callosal cells, despite the occurrence of callosal anomalies. However, using anterograde DiI tracing, we find that BALB/c mice display increased rates of axonal bifurcations during early and late cortical development in the medial frontal cortex. Midline guidepost cells adjacent to the medial frontal cortex are significant reduced in the CC dysgenesis mouse model. Altogether these data suggest that callosal collateral axonal exuberance is maintained in the absence of midline guidepost signaling and might facilitate aberrant connections in the CC dysgenesis mouse model.

Objective To demonstrate that functional spermatids can be derived in vitro from nonhuman primate pluripotent stem cells. Design Green fluorescent protein-labeled, rhesus macaque nonhuman primate embryonic stem cells (nhpESCs) were differentiated into advanced male germ cell lineages using a modified serum-free spermatogonial stem cell culture medium. In vitro-derived round spermatid-like cells (rSLCs) from differentiated nhpESCs were assessed for their ability to fertilize rhesus oocytes by intracytoplasmic sperm(atid) injection. Setting Multiple academic laboratory settings. Patient(s) Not applicable. Intervention(s) Intracytoplasmic sperm(atid) injection of in vitro-derived spermatids from nhpESCs into rhesus macaque oocytes. Main Outcome Measure(s) Differentiation into spermatogenic cell lineages was measured through multiple assessments including ribonucleic acid sequencing and immunocytochemistry for various spermatogenic markers. In vitro spermatids were assessed for their ability to fertilize oocytes by intracytoplasmic sperm(atid) injection by assessing early fertilization events such as spermatid deoxyribonucleic acid decondensation and pronucleus formation/apposition. Preimplantation embryo development from the one-cell zygote stage to the blastocyst stage was also assessed. Result(s) Nonhuman primate embryonic stem cells can be differentiated into advanced germ cell lineages, including haploid rSLCs. These rSLCs undergo deoxyribonucleic acid decondensation and pronucleus formation/apposition when microinjected into rhesus macaque mature oocytes, which, after artificial activation and coinjection of ten-eleven translocation 3 protein, undergo embryonic divisions with approximately 12% developing successfully into expanded blastocysts. Conclusion(s) This work demonstrates that rSLCs, generated in vitro from primate pluripotent stem cells, mimic many of the capabilities of in vivo round spermatids and perform events essential for preimplantation development. To our knowledge, this work represents, for the first time, that functional spermatid-like cells can be derived in vitro from primate pluripotent stem cells.

Summary Mouse embryonic stem cells (ESCs) show cell-to-cell heterogeneity. A small number of two-cell-like cells (2CLCs) marked by endogenous retrovirus activation emerge spontaneously. The 2CLCs are unstable and they are prone to transiting back to the pluripotent state without extrinsic stimulus. To understand how this bidirectional transition takes place, we performed single-cell RNA sequencing on isolated 2CLCs that underwent 2C-like state exit and re-entry, and revealed a step-by-step transitional process between 2C-like and pluripotent states. Mechanistically, we found that cell cycle played an important role in mediating these transitions by regulating assembly of the nucleolus and peri-nucleolar heterochromatin to influence 2C gene Dux expression. Collectively, our findings provide a roadmap of the 2C-like state entry and exit in ESCs and also a causal role of the cell cycle in promoting these transitions., Highlights • The entry to and exit from the 2C-like state showed a step-by-step roadmap • Cell cycle participates in mediating dynamic transitions between ESCs and 2CLCs • G1/S phase arrest facilitates the Dux locus escape from heterochromatin • Nucleolus-heterochromatin remodeling is involved in 2C activation, In this article, Jin Zhang and colleagues show that cell-cycle variations distinguish 2CLCs, ESCs, and the intermediate states using single-cell RNA-seq. They found that cell-cycle-associated remodeling of nucleoli and peri-nucleolar heterochromatin was involved in the regulation of the 2C program. They provide novel advances in the fundamental field of 2C activation and stem cell fate decision.

Plasma circulating extracellular vesicles (EVs) have been utilized as a potential therapeutic strategy to treat ischemic disease through intramyocardial injection (efficient but invasive) or tail vein injection (noninvasive but low cardiac retention). An effective and noninvasive delivery of EVs for future clinical use is necessary. The large animal (canine) model was complemented with a murine ischemia-reperfusion injury (IRI) model, as well as H9 human embryonic stem cell–induced cardiomyocytes or neonatal rat cardiomyocytes to investigate the effective delivery method and the role of plasma EVs in the IRI model. We further determine the crucial molecule within EVs that confers the cardioprotective role in vivo and in vitro and investigate the efficiency of CHP (cardiac homing peptide)-linked EVs in alleviating IRI. D-SPECT imaging showed that percutaneous intracoronary delivery of EVs reduced infarct extent in dogs. CHP-EVs further reduced IRI-induced cardiomyocyte apoptosis in mice and neonatal rat cardiomyocytes. Mechanistically, administration of EVs by percutaneous intracoronary delivery (in dog) and myocardial injection (in mice) just before reperfusion reduced infarct size of IRI by increasing miR-486 levels. miR-486–deleted EVs exacerbated oxygen-glucose deprivation/reoxygenation–induced human embryonic stem cell–induced cardiomyocytes and neonatal rat cardiomyocyte apoptosis. EV-miR-486 inhibited the PTEN (phosphatase and tensin homolog deleted on chromosome ten) expression and then promoted AKT (protein kinase B) activation in human embryonic stem cell–induced cardiomyocytes and neonatal rat cardiomyocytes. In conclusion, plasma-derived EVs convey miR-486 to the myocardium and attenuated IRI-induced infarction and cardiomyocyte apoptosis. CHP strategy was effective to improve cardiac retention of EVs in mice (in vivo) and dogs (ex vivo).

The nucleolus is the organelle for ribosome biogenesis and sensing various types of stress. However, its role in regulating stem cell fate remains unclear. Here, we present evidence that nucleolar stress induced by interfering rRNA biogenesis can drive the 2-cell stage embryo-like (2C-like) program and induce an expanded 2C-like cell population in mouse embryonic stem (mES) cells. Mechanistically, nucleolar integrity maintains normal liquid-liquid phase separation (LLPS) of the nucleolus and the formation of peri-nucleolar heterochromatin (PNH). Upon defects in rRNA biogenesis, the natural state of nucleolus LLPS is disrupted, causing dissociation of the NCL/TRIM28 complex from PNH and changes in epigenetic state and reorganization of the 3D structure of PNH, which leads to release of Dux, a 2C program transcription factor, from PNH to activate a 2C-like program. Correspondingly, embryos with rRNA biogenesis defect are unable to develop from 2-cell (2C) to 4-cell embryos, with delayed repression of 2C/ERV genes and a transcriptome skewed toward earlier cleavage embryo signatures. Our results highlight that rRNA-mediated nucleolar integrity and 3D structure reshaping of the PNH compartment regulates the fate transition of mES cells to 2C-like cells, and that rRNA biogenesis is a critical regulator during the 2-cell to 4-cell transition of murine pre-implantation embryo development., The role of the nucleolus in cell fate is not well known. Here the authors show nucleolar integrity maintained by rRNA-mediated liquid-liquid phase separation and 3D chromatin structure of perinucleolar heterochromatin regulates the fate transition of mouse embryonic stem cells to 2 cell-stage embryo-like cells, and that rRNA biogenesis regulates the 2-cell-to-4-cell transition of development.

Embryonic stem cells (ESCs) maintain a pluripotent state and genome integrity in long-term culture. A rare population of ESCs showing 2-cell embryo-specific gene expression is believed to play critical roles in sustainable pluripotency and genome stability. However, the molecular mechanism controlling this transition to a 2-cell embryo-like (2CL) state remains unclear. We carried out screening to search for the factors involved in 2CL state induction and found a ribosomal RNA processing factor, Pum3 to be a candidate. Increased 2CL state population accompanied with an accumulation of pre-ribosomal RNA and activated p53 in the Pum3-KO ESC. Furthermore, the increase of 2CL state cells in the Pum3-KO ESCs was completely abrogated by the deletion of p53. The DNA damage induced by the Ultraviolet light (UV) irradiation and Zeocin promoted the transition to a 2CL state in a p53-dependent manner. Thus, our study provides new insights into a 2CL state transition mechanism through stress-dependent p53 activation of ESCs.

Nicotinamide, the amide form of Vitamin B3, is a common nutrient supplement that plays important role in human fetal development. Nicotinamide has been widely used in clinical treatments, including the treatment of diseases during pregnancy. However, its impacts during embryogenesis have not been fully understood. In this study, we show that nicotinamide plays multiplex roles in mesoderm differentiation of human embryonic stem cells (hESCs). Nicotinamide promotes cardiomyocyte fate from mesoderm progenitor cells, and suppresses the emergence of other cell types. Independent of its functions in PARP and Sirtuin pathways, nicotinamide modulates differentiation through kinase inhibition. A KINOMEscan assay identifies 14 novel nicotinamide targets among 468 kinase candidates. We demonstrate that nicotinamide promotes cardiomyocyte differentiation through p38 MAP kinase inhibition. Furthermore, we show that nicotinamide enhances cardiomyocyte survival as a Rho-associated protein kinase (ROCK) inhibitor. This study reveals nicotinamide as a pleiotropic molecule that promotes the derivation and survival of cardiomyocytes, and it could become a useful tool for cardiomyocyte production for regenerative medicine. It also provides a theoretical foundation for physicians when nicotinamide is considered for treatments for pregnant women.