TEAD2 initiates ground-state pluripotency by mediating chromatin looping.

The transition of mouse embryonic stem cells (ESCs) between serum/LIF and 2i(MEK and GSK3 kinase inhibitor)/LIF culture conditions serves as a valuable model for exploring the mechanisms underlying ground and confused pluripotent states. Regulatory networks comprising core and ancillary pluripotency factors drive the gene expression programs defining stable naïve pluripotency. In our study, we systematically screened factors essential for ESC pluripotency, identifying TEAD2 as an ancillary factor maintaining ground-state pluripotency in 2i/LIF ESCs and facilitating the transition from serum/LIF to 2i/LIF ESCs. TEAD2 exhibits increased binding to chromatin in 2i/LIF ESCs, targeting active chromatin regions to regulate the expression of 2i-specific genes. In addition, TEAD2 facilitates the expression of 2i-specific genes by mediating enhancer-promoter interactions during the serum/LIF to 2i/LIF transition. Notably, deletion of Tead2 results in reduction of a specific set of enhancer-promoter interactions without significantly affecting binding of chromatin architecture proteins, CCCTC-binding factor (CTCF), and Yin Yang 1 (YY1). In summary, our findings highlight a novel prominent role of TEAD2 in orchestrating higher-order chromatin structures of 2i-specific genes to sustain ground-state pluripotency.

Conductive Polymer PEDOT:PSS-Based Platform for Embryonic Stem-Cell Differentiation.

Organic semiconductors are constantly gaining interest in regenerative medicine. Their tunable physico-chemical properties, including electrical conductivity, are very promising for the control of stem-cell differentiation. However, their use for combined material-based and electrical stimulation remains largely underexplored. Therefore, we carried out a study on whether a platform based on the conductive polymer poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) can be beneficial to the differentiation of mouse embryonic stem cells (mESCs). The platform was prepared using the layout of a standard 24-well cell-culture plate. Polyethylene naphthalate foil served as the substrate for the preparation of interdigitated gold electrodes by physical vapor deposition. The PEDOT:PSS pattern was fabricated by precise screen printing over the gold electrodes. The PEDOT:PSS platform was able to produce higher electrical current with the pulsed-direct-current (DC) electrostimulation mode (1 Hz, 200 mV/mm, 100 ms pulse duration) compared to plain gold electrodes. There was a dominant capacitive component. In proof-of-concept experiments, mESCs were able to respond to such electrostimulation by membrane depolarization and elevation of cytosolic calcium. Further, the PEDOT:PSS platform was able to upregulate cardiomyogenesis and potentially inhibit early neurogenesis per se with minor contribution of electrostimulation. Hence, the present work highlights the large potential of PEDOT:PSS in regenerative medicine.

YY2 in Mouse Preimplantation Embryos and in Embryonic Stem Cells.

Yin Yang 2 encodes a mammalian-specific transcription factor (YY2) that shares high homology in the zinc finger region with both YY1 and REX1/ZFP42, encoded by the Yin Yang 1 and Reduced Expression Protein 1/Zinc Finger Protein 42 gene, respectively. In contrast to the well-established roles of the latter two in gene regulation, X chromosome inactivation and binding to specific transposable elements (TEs), much less is known about YY2, and its presence during mouse preimplantation development has not been described. As it has been reported that mouse embryonic stem cells (mESC) cannot be propagated in the absence of Yy2, the mechanistic understanding of how Yy2 contributes to mESC maintenance remains only very partially characterized. We describe Yy2 expression studies using RT-PCR and staining with a high-affinity polyclonal serum in mouse embryos and mESC. Although YY2 is expressed during preimplantation development, its presence appears dispensable for developmental progress in vitro until formation of the blastocyst. Attenuation of Yy2 levels failed to alter either Zscan4 levels in two-cell embryos or IAP and MERVL levels at later preimplantation stages. In contrast to previous claims that constitutively expressed shRNA against Yy2 in mESC prohibited the propagation of mESC in culture, we obtained colonies generated from mESC with attenuated Yy2 levels. Concomitant with a decreased number of undifferentiated colonies, Yy2-depleted mESC expressed higher levels of Zscan4 but no differences in the expression of TEs or other pluripotency markers including Sox2, Oct4, Nanog and Esrrb were observed. These results confirm the contribution of Yy2 to the maintenance of mouse embryonic stem cells and show the preimplantation expression of YY2. These functions are discussed in relation to mammalian-specific functions of YY1 and REX1.

Small molecule TCS21311 can replace BMP7 and facilitate cell proliferation in in vitro expansion culture of nephron progenitor cells.

Several groups have developed in vitro expansion cultures for mouse metanephric nephron progenitor cells (NPCs) using cocktails of small molecules and growth factors including BMP7. However, the detailed mechanisms by which BMP7 acts in the NPC expansion remain to be elucidated. Here, by performing chemical screening for BMP substitutes, we identified a small molecule, TCS21311, that can replace BMP7 and revealed a novel inhibitory role of BMP7 in JAK3-STAT3 signaling in NPC expansion culture. Further, we found that TCS21311 facilitates the proliferation of mouse embryonic NPCs and human induced pluripotent stem cell-derived NPCs when added to the expansion culture. These results will contribute to understanding the mechanisms of action of BMP7 in NPC proliferation in vitro and in vivo and to the stable supply of NPCs for regenerative therapy, disease modeling and drug discovery for kidney diseases.

Supplement of nitric oxide through calcium carbonate-based nanoparticles contributes osteogenic differentiation of mouse embryonic stem cells.

This study investigated the delivery of S-nitrosothiol (GSNO) as a nitric oxide (NO) donor loaded into calcium carbonate-based mineralized nanoparticles (GSNO-MNPs) to regulate cell signaling pathways for the osteogenic differentiation of mouse embryonic stem cells (ESCs). GSNO-MNPs were prepared by an anionic block copolymer template-mediated calcium carbonate (CaCO3) mineralization process in the presence of GSNO. GSNO-MNPs were spherical and had a narrow size distribution. GSNO was stably loaded within the MNPs without denaturation. TEM analysis also demonstrated the localization of GSNO-MNPs within membrane-bound structures in the cell, indicating the successful introduction of GSNO-MNPs into the cytosol of ESCs. Intracellular levels of NO and cGMP were significantly increased upon treatment with GSNO-MNPs, compared with the control group. When cells were exposed to GSNO-MNPs, the effects of nanoparticles on cell viability were not statistically significant. GSNO-MNPs treatment increased ALP activity assay and intracellular calcium levels. Real-time RT-PCR also revealed highly increased expression levels of the osteogenic target genes ALP, osteocalcin (OCN), and osterix (OSX) in GSNO-MNP-treated ESCs. The protein levels of OSX and Runt-related transcription factor 2 (RUNX2) showed similar patterns of expression based on real-time RT-PCR. These results indicate that GSNO-MNPs influenced the osteogenic differentiation of ESCs. Transcriptome profiling identified several significantly enriched and involved biological networks, such as RAP1, RAS, PI3K-AKT, and MAPK signaling pathways. These findings suggest that GSNO-MNPs can modulate osteogenic differentiation in ESCs via complex molecular pathways.

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Evaluation of in vitro models of stem cell-derived cardiomyocytes to screen for potential cardiotoxicity of chemicals.

Cardiotoxicity is an important toxicological endpoint for chemical and drug safety assessment. The present study aims to evaluate two stemcell-based in vitro models for cardiotoxicity screening of chemicals. Eleven model compounds were used to evaluate responses of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) using beating arrest as a readout and the analysis of electrophysiological parameters measured with a multi-electrode array (MEA) platform of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Results revealed that the hiPSC-CM MEA assay responded to all compounds. The mESC-CM beating arrest assay was not responsive to potassium channel blockers and showed a lower sensitivity to sodium channel blockers and Na+/K+ ATPase inhibitors compared to the hiPSC-CM MEA assay. Calcium channel blockers and a ß-adrenergic receptor agonist showed comparable potencies in both models. The in vitro response concentrations from hiPSC-CMs were highly concordant with human effective serum concentrations of potassium and sodium channel blockers. It is concluded that both in vitro models enable the cardiotoxicity screening with different applicability domains. The mESC-CM beating arrest assay may be used as a first step in a tiered approach while the hiPSC-CM MEA assay may be the best starting point for quantitative in vitro to in vivo extrapolations.

Template Activating Factor-I α Regulates Retroviral Silencing during Reprogramming.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is accompanied by dramatic changes in epigenetic programs, including silencing of endogenous and exogenous retroviruses. Here, we utilized replication-defective and persistent Sendai virus (SeVdp)-based vectors to monitor retroviral silencing during reprogramming. We observed that retroviral silencing occurred at an early reprogramming stage without a requirement for KLF4 or the YY1-binding site in the retroviral genome. Insertional chromatin immunoprecipitation (iChIP) enabled us to isolate factors assembled on the silenced provirus, including components of inhibitor of histone acetyltransferase (INHAT), which includes the SET/TAF-I oncoprotein. Knockdown of SET/TAF-I in mouse embryonic fibroblasts (MEFs) diminished retroviral silencing during reprogramming, and overexpression of template activating factor-I α (TAF-Iα), a SET/TAF-I isoform predominant in embryonic stem cells (ESCs), reinforced retroviral silencing by an SeVdp-based vector that is otherwise defective in retroviral silencing. Our results indicate an important role for TAF-Iα in retroviral silencing during reprogramming.

HCN4-Overexpressing Mouse Embryonic Stem Cell-Derived Cardiomyocytes Generate a New Rapid Rhythm in Rats with Bradycardia.

A biological pacemaker is expected to solve the persisting problems of an artificial cardiac pacemaker including short battery life, lead breaks, infection, and electromagnetic interference. We previously reported HCN4 overexpression enhances pacemaking ability of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) in vitro. However, the effect of these cells on bradycardia in vivo has remained unclear. Therefore, we transplanted HCN4-overexpressing mESC-CMs into bradycardia model animals and investigated whether they could function as a biological pacemaker. The rabbit Hcn4 gene was transfected into mouse embryonic stem cells and induced HCN4-overexpressing mESC-CMs. Non-cardiomyocytes were removed under serum/glucose-free and lactate-supplemented conditions. Cardiac balls containing 5 × 103 mESC-CMs were made by using the hanging drop method. One hundred cardiac balls were injected into the left ventricular free wall of complete atrioventricular block (CAVB) model rats. Heart beats were evaluated using an implantable telemetry system 7 to 30 days after cell transplantation. The result showed that ectopic ventricular beats that were faster than the intrinsic escape rhythm were often observed in CAVB model rats transplanted with HCN4-overexpressing mESC-CMs. On the other hand, the rats transplanted with non-overexpressing mESC-CMs showed sporadic single premature ventricular contraction but not sustained ectopic ventricular rhythms. These results indicated that HCN4-overexpressing mESC-CMs produce rapid ectopic ventricular rhythms as a biological pacemaker.

Inducible knockdown of procollagen I protects mice from liver fibrosis and leads to dysregulated matrix genes and attenuated inflammation.

Organ fibrosis is characterized by a chronic wound-healing response, with excess deposition of extracellular matrix components. Here, collagen type I represents the most abundant scar component and a primary target for antifibrotic therapies. Liver fibrosis can progress to cirrhosis and primary liver cancer, which are the major causes of liver related morbidity and mortality. However, a (pro-)collagen type I specific therapy remains difficult and its therapeutic abrogation may incur unwanted side effects. We therefore designed tetracycline-regulated procollagen alpha1(I) short hairpin (sh)RNA expressing mice that permit a highly efficient inducible knockdown of the procollagen alpha1(I) gene in activated (myo-)fibroblasts, to study the effect of induced procollagen type I deficiency. Transgenic mice were generated using recombinase-mediated integration in embryonic stem cells or zinc-finger nuclease-aided genomic targeting combined with miR30-shRNA technology. Liver fibrosis was induced in transgenic mice by carbon tetrachloride, either without or with doxycycline supplementation. Doxycycline treated mice showed an 80-90% suppression of procollagen alpha1(I) transcription and a 40-50% reduction in hepatic collagen accumulation. Procollagen alpha1(I) knockdown also downregulated procollagens type III, IV and VI and other fibrosis related parameters. Moreover, this was associated with an attenuation of chronic inflammation, suggesting that collagen type I serves not only as major scar component, but also as modulator of other collagens and promoter of chronic inflammation.

Methionine metabolism is essential for SIRT1-regulated mouse embryonic stem cell maintenance and embryonic development.

Methionine metabolism is critical for epigenetic maintenance, redox homeostasis, and animal development. However, the regulation of methionine metabolism remains unclear. Here, we provide evidence that SIRT1, the most conserved mammalian NAD+-dependent protein deacetylase, is critically involved in modulating methionine metabolism, thereby impacting maintenance of mouse embryonic stem cells (mESCs) and subsequent embryogenesis. We demonstrate that SIRT1-deficient mESCs are hypersensitive to methionine restriction/depletion-induced differentiation and apoptosis, primarily due to a reduced conversion of methionine to S-adenosylmethionine. This reduction markedly decreases methylation levels of histones, resulting in dramatic alterations in gene expression profiles. Mechanistically, we discover that the enzyme converting methionine to S-adenosylmethionine in mESCs, methionine adenosyltransferase 2a (MAT2a), is under control of Myc and SIRT1. Consistently, SIRT1 KO embryos display reduced Mat2a expression and histone methylation and are sensitive to maternal methionine restriction-induced lethality, whereas maternal methionine supplementation increases the survival of SIRT1 KO newborn mice. Our findings uncover a novel regulatory mechanism for methionine metabolism and highlight the importance of methionine metabolism in SIRT1-mediated mESC maintenance and embryonic development.

Collagen-alginate microspheres as a 3D culture system for mouse embryonic stem cells differentiation to primordial germ cells.

Germ cells differentiation of stem cells will aid treatment of adults with infertility. Biopolymers utilization provided synthetic extracellular matrix (ECM) and desired attributes in in vitro to improve conditions for stem cells attachment, proliferation and differentiation. Mixture of alginate as a biocompatible hydrogel, with collagen IV, could establish an in vitro 3 dimensional (3D) culture model. The objective of this study was investigation of the mouse ESCs differentiation capacity to putative primordial germ cells (PGCs) in the alginate and alginate-collagen IV microspheres (CAM). ESCs aggregated together to form embryoid bodies (EB) in CAM under basal medium supplemented with bone morphogenetic protein-4 (BMP4) as a differentiation factor. Viability and PGC differentiation of the stem cells in microspheres was evaluated by apoptosis and PGC related gene markers. Flow cytometry analysis was also used to detect of Mvh endogenous protein as a specific PGC marker. PGC gene and protein expression revealed that differentiation potential of ESCs to putative PGCs in CAM is significantly higher than control groups. Taking together, it was concluded that CAM demonstrated a great potential to use in PGCs differentiation and treatment of adults with infertility and may be a reliable means of producing mature germ cells.

Generation of thalamic neurons from mouse embryonic stem cells.

The thalamus is a diencephalic structure that plays crucial roles in relaying and modulating sensory and motor information to the neocortex. The thalamus develops in the dorsal part of the neural tube at the level of the caudal forebrain. However, the molecular mechanisms that are essential for thalamic differentiation are still unknown. Here, we have succeeded in generating thalamic neurons from mouse embryonic stem cells (mESCs) by modifying the default method that induces the most-anterior neural type in self-organizing culture. A low concentration of the caudalizing factor insulin and a MAPK/ERK kinase inhibitor enhanced the expression of the caudal forebrain markers Otx2 and Pax6. BMP7 promoted an increase in thalamic precursors such as Tcf7l2+/Gbx2+ and Tcf7l2+/Olig3+ cells. mESC thalamic precursors began to express the glutamate transporter vGlut2 and the axon-specific marker VGF, similar to mature projection neurons. The mESC thalamic neurons extended their axons to cortical layers in both organotypic culture and subcortical transplantation. Thus, we have identified the minimum elements sufficient for in vitro generation of thalamic neurons. These findings expand our knowledge of thalamic development.

Fingerprinting of neurotoxic compounds using a mouse embryonic stem cell dual luminescence reporter assay.

Identification of neurotoxic drugs and environmental chemicals is an important challenge. However, only few tools to address this topic are available. The aim of this study was to develop a neurotoxicity/developmental neurotoxicity (DNT) test system, using the pluripotent mouse embryonic stem cell line CGR8 (ESCs). The test system uses ESCs at two differentiation stages: undifferentiated ESCs and ESC-derived neurons. Under each condition, concentration-response curves were obtained for three parameters: activity of the tubulin alpha 1 promoter (typically activated in early neurons), activity of the elongation factor 1 alpha promoter (active in all cells), and total DNA content (proportional to the number of surviving cells). We tested 37 compounds from the ESNATS test battery, which includes polypeptide hormones, environmental pollutants (including methylmercury), and clinically used drugs (including valproic acid and tyrosine kinase inhibitors). Different classes of compounds showed distinct concentration-response profiles. Plotting of the lowest observed adverse effect concentrations (LOAEL) of the neuronal promoter activity against the general promoter activity or against cytotoxicity, allowed the differentiation between neurotoxic/DNT substances and non-neurotoxic controls. Reporter activity responses in neurons were more susceptible to neurotoxic compounds than the reporter activities in ESCs from which they were derived. To relate the effective/toxic concentrations found in our study to relevant in vivo concentrations, we used a reverse pharmacokinetic modeling approach for three exemplary compounds (teriflunomide, geldanamycin, abiraterone). The dual luminescence reporter assay described in this study allows high-throughput, and should be particularly useful for the prioritization of the neurotoxic potential of a large number of compounds.

Differentiation of Spermatogonia Stem Cells into Functional Mature Neurons Characterized with Differential Gene Expression.

Transplantation of embryonic stem cells (ESCs) is a promising therapeutic approach for the treatment of neurodegenerative diseases. However, ESCs are not usable clinically due to immunological and ethical limitations. The identification of an alternative safe cell source opens novel options via autologous transplantation in neuro-regeneration circumventing these problems. Here, we examined the neurogenic capacity of embryonic stem-like cells (ES-like cells) derived from the testis using neural growth factor inducers and utilized them to generate functional mature neurons. The neuronal differentiation of ES-like cells is induced in three stages. Stage 1 is related to embryoid body (EB) formation. To induce neuroprogenitor cells, EBs were cultured in the presence of retinoic acid, N2 supplement and fibroblast growth factor followed by culturing in a neurobasal medium containing B27, N2 supplements for additional 10 days, to allow the maturation and development of neuronal progenitor cells. The neurogenic differentiation was confirmed by immunostaining for markers of mature neurons. The differentiated neurons were positive for Tuj1 and Tau1. Real-time PCR dates indicated the expression of Nestin and Neuro D (neuroprogenitor markers) in induced cells at the second stage of the differentiation protocol. The differentiated mature neurons exhibited the specific neuron markers Map2 and ß-tubulin. The functional maturity of neurons was confirmed by an electrophysiological analysis of passive and active neural membrane properties. These findings indicated a differentiation capacity of ES-like cells derived from the testis to functionally mature neurons, which proposes them as a novel cell source for neuroregenerative medicine.

MicroRNA-29a promotes smooth muscle cell differentiation from stem cells by targeting YY1.

MicroRNA-29a (miR-29a) has been extensively studied in tumor biology and fibrotic diseases, but little is known about its functional roles in vascular smooth muscle cell (VSMC) differentiation from embryonic stem cells (ESCs). Using well-established VSMC differentiation models, we have observed that miR-29a induces VSMC differentiation from mouse ESCs by negatively regulating YY1, a transcription factor that inhibits muscle cell differentiation and muscle-specific gene expression. Moreover, gene expression levels of three VSMC specific transcriptional factors were up-regulated by miR-29a over-expression, but down-regulated by miR-29a inhibition or YY1 over-expression. Taken together, our data demonstrate that miR-29a and its target gene, YY1, play a regulatory role in VSMC differentiation from ESCs in vitro and in vivo.

Wnt5a Supports Osteogenic Lineage Decisions in Embryonic Stem Cells.

The specification of pluripotent stem cells into the bone-forming osteoblasts has been explored in a number of studies. However, the current body of literature has yet to adequately address the role of Wnt glycoproteins in the differentiation of pluripotent stem cells along the osteogenic lineage. During mouse embryonic stem cell (ESC) in vitro osteogenesis, the noncanonical WNT5a is expressed early on. Cells either sorted by their positive WNT5a expression or when supplemented with recombinant WNT5a (rWNT5a) during a 2-day window showed significantly enhanced osteogenic yield. Mechanistically, rWNT5a supplementation upregulated protein kinase C (PKC), calcium/calmodulin-dependent kinase II (CamKII) and c-Jun N-terminal kinase (JNK) activity while antagonizing the key effector of canonical Wnt signaling: ß-catenin. Conversely, when recombinant WNT3a (rWNT3a) or other positive regulators of ß-catenin were employed during this same time window there was a decrease in osteogenic marker expression. However, if rWNT3a was supplemented during a time window following rWNT5a treatment, osteogenic differentiation was enhanced both in murine and human ESCs. Elucidating the role of these WNT ligands in directing the early stages of osteogenesis has the potential to considerably improve tissue engineering protocols and applications for regenerative medicine.

Amino Acid Transport Associated to Cluster of Differentiation 98 Heavy Chain (CD98hc) Is at the Cross-road of Oxidative Stress and Amino Acid Availability.

CD98hc functions as an amino acid (AA) transporter (together with another subunit) and integrin signaling enhancer. It is overexpressed in highly proliferative cells in both physiological and pathological conditions. CD98hc deletion induces strong impairment of cell proliferation in vivo and in vitro Here, we investigate CD98hc-associated AA transport in cell survival and proliferation. By using chimeric versions of CD98hc, the two functions of the protein can be uncoupled. Although recovering the CD98hc AA transport capacity restores the in vivo and in vitro proliferation of CD98hc-null cells, reconstitution of the integrin signaling function of CD98hc is unable to restore in vitro proliferation of those cells. CD98hc-associated transporters (i.e. xCT, LAT1, and y(+)LAT2 in wild-type cells) are crucial to control reactive oxygen species and intracellular AA levels, thus sustaining cell survival and proliferation. Moreover, in CD98hc-null cells the deficiency of CD98hc/xCT cannot be compensated, leading to cell death by ferroptosis. Supplementation of culture media with ß-mercaptoethanol rescues CD98hc-deficient cell survival. Under such conditions null cells show oxidative stress and intracellular AA imbalance and, consequently, limited proliferation. CD98hc-null cells also present reduced intracellular levels of branched-chain and aromatic amino acids (BCAAs and ARO AAs, respectively) and induced expression of peptide transporter 1 (PEPT1). Interestingly, external supply of dipeptides containing BCAAs and ARO AAs rescues cell proliferation and compensates for impaired uptake of CD98hc/LAT1 and CD98hc/y(+)LAT2. Our data establish CD98hc as a master protective gene at the cross-road of redox control and AA availability, making it a relevant therapeutic target in cancer.

Beat Rate Variability in Murine Embryonic Stem Cell-Derived Cardiomyocytes: Effect of Antiarrhythmic Drugs.

BACKGROUND/AIMS: Heart rate variability (HRV) refers to the fluctuation of the time interval between consecutive heartbeats in humans. It has recently been discovered that cardiomyocytes derived from human embryonic and induced pluripotent stem cells show beat rate variability (BRV) that is similar to the HRV in humans. In the present study, clinical aspects of HRV were transferred to an in vitro model. The aims of the study were to explore the BRV in murine embryonic stem cell (mESC)-derived cardiomyocytes and to demonstrate the influence of antiarrhythmic drugs on BRV as has been shown in clinical trials previously. METHODS: The Microelectrode Array (MEA) technique was used to perform short-term recordings of extracellular field potentials (FPs) of spontaneously beating cardiomyocytes derived from mESCs (D3 cell line, αPig-44). Offline analysis was focused on time domain and nonlinear methods. RESULTS: The Poincaré-Plot analysis of measurements without pharmacological intervention revealed that three different shapes of scatter plots occurred most frequently. Comparable shapes have been described in clinical studies before. The antiarrhythmic drugs Ivabradine, Verapamil and Sotalol augmented BRV, whereas Flecainide decreased BRV parameters at low concentrations (SDSD 79.0 ± 8.7% of control at 10(-9) M, p < 0.05) and increased variability measures at higher concentrations (SDNN 258.8 ± 42.7% of control at 10(-5) M, p < 0.05). Amiodarone and Metoprolol did not alter BRV significantly. CONCLUSIONS: Spontaneously beating cardiomyocytes derived from mESCs showed BRV that appears to be similar to the HRV known from humans. Antiarrhythmic drugs affected BRV parameters similar to clinical observations. Therefore, our study demonstrates that this in vitro model can contribute to a better understanding of electrophysiological properties of mESC-derived cardiomyocytes and might serve as a valuable tool for drug safety screening.

Actl6a protects embryonic stem cells from differentiating into primitive endoderm.

Actl6a (actin-like protein 6A, also known as Baf53a or Arp4) is a subunit shared by multiple complexes including esBAF, INO80, and Tip60-p400, whose main components (Brg1, Ino80, and p400, respectively) are crucial for the maintenance of embryonic stem cells (ESCs). However, whether and how Actl6a functions in ESCs has not been investigated. ESCs originate from the epiblast (EPI) that is derived from the inner cell mass (ICM) in blastocysts, which also give rise to primitive endoderm (PrE). The molecular mechanisms for EPI/PrE specification remain unclear. In this study, we provide the first evidence that Actl6a can protect mouse ESCs (mESCs) from differentiating into PrE. While RNAi knockdown of Actl6a, which appeared highly expressed in mESCs and downregulated during differentiation, induced mESCs to differentiate towards the PrE lineage, ectopic expression of Actl6a was able to repress PrE differentiation. Our work also revealed that Actl6a could interact with Nanog and Sox2 and promote Nanog binding to pluripotency genes such as Oct4 and Sox2. Interestingly, cells depleted of p400, but not of Brg1 or Ino80, displayed similar PrE differentiation patterns. Mutant Actl6a with impaired ability to bind Tip60 and p400 failed to block PrE differentiation induced by Actl6a dysfunction. Finally, we showed that Actl6a could target to the promoters of key PrE regulators (e.g., Sall4 and Fgf4), repressing their expression and inhibiting PrE differentiation. Our findings uncover a novel function of Actl6a in mESCs, where it acts as a gatekeeper to prevent mESCs from entering into the PrE lineage through a Yin/Yang regulating pattern.