Inhibition of BMP-mediated SMAD pathway supports the pluripotency of pig embryonic stem cells in the absence of feeder cells.

Here, we examined the effects of the BMP signaling pathway inhibitor LDN-193189 on the pluripotency of porcine embryonic stem cells (ESCs) in the absence of feeder cells using molecular and transcriptomic techniques. Additionally, the effects of some extracellular matrix components on porcine ESC pluripotency were evaluated to develop an optimized and sustainable feeder-free culture system for porcine ESCs. Feeder cells were found to play an important role in supporting the pluripotency of porcine ESCs by blocking trophoblast and mesodermal differentiation through the inhibition of the BMP pathway. Additionally, treatment with LDN-193189, an inhibitor of the BMP pathway, maintained the pluripotency and homogeneity of porcine ESCs for an extended period in the absence of feeder cells by stimulating the secretion of chemokines and suppressing differentiation, based on transcriptome analysis. Conclusively, these results suggest that LDN-193189 could be a suitable replacement for feeder cells in the maintenance of porcine ESC pluripotency during culture. Additionally, these findings contribute to the understanding of pluripotency gene networks and comparative embryogenesis.

Size-Optimized Layered Double Hydroxide Nanoparticles Promote Neural Progenitor Cells Differentiation of Embryonic Stem Cells Through the Regulation of M6A Methylation.

Purpose: The committed differentiation fate regulation has been a difficult problem in the fields of stem cell research, evidence showed that nanomaterials could promote the differentiation of stem cells into specific cell types. Layered double hydroxide (LDH) nanoparticles possess the regulation function of stem cell fate, while the underlying mechanism needs to be investigated. In this study, the process of embryonic stem cells (ESCs) differentiate to neural progenitor cells (NPCs) by magnesium aluminum LDH (MgAl-LDH) was investigated. Methods: MgAl-LDH with diameters of 30, 50, and 100 nm were synthesized and characterized, and their effects on the cytotoxicity and differentiation of NPCs were detected in vitro. Dot blot and MeRIP-qPCR were performed to detect the level of m6A RNA methylation in nanoparticles-treated cells. Results: Our work displayed that LDH nanoparticles of three different sizes were biocompatible with NPCs, and the addition of MgAl-LDH could significantly promote the process of ESCs differentiate to NPCs. 100 nm LDH has a stronger effect on promoting NPCs differentiation compared to 30 nm and 50 nm LDH. In addition, dot blot results indicated that the enhanced NPCs differentiation by MgAl-LDH was closely related to m6A RNA methylation process, and the major modification enzyme in LDH controlled NPCs differentiation may be the m6A RNA methyltransferase METTL3. The upregulated METTL3 by LDH increased the m6A level of Sox1 mRNA, enhancing its stability. Conclusion: This work reveals that MgAl-LDH nanoparticles can regulate the differentiation of ESCs into NPCs by increasing m6A RNA methylation modification of Sox1.

Equine Embryonic Stem Cell-Derived Tenocytes are Insensitive to a Combination of Inflammatory Cytokines and Have Distinct Molecular Responses Compared to Primary Tenocytes.

Tissue fibrosis following tendon injury is a major clinical problem due to the increased risk of re-injury and limited treatment options; however, its mechanism remains unclear. Evidence suggests that insufficient resolution of inflammation contributes to fibrotic healing by disrupting tenocyte activity, with the NF-κB pathway being identified as a potential mediator. Equine embryonic stem cell (ESC) derived tenocytes may offer a potential cell-based therapy to improve tendon regeneration, but how they respond to an inflammatory environment is largely unknown. Our findings reveal for the first time that, unlike adult tenocytes, ESC-tenocytes are unaffected by IFN-γ, TNFα, and IL-1ß stimulation; producing minimal changes to tendon-associated gene expression and generating 3-D collagen gel constructs indistinguishable from unstimulated controls. Inflammatory pathway analysis found these inflammatory cytokines failed to activate NF-κB in the ESC-tenocytes. However, NF-κB could be activated to induce changes in gene expression following stimulation with NF-κB pharmaceutical activators. Transcriptomic analysis revealed differences between cytokine and NF-κB signalling components between adult and ESC-tenocytes, which may contribute to the mechanism by which ESC-tenocytes escape inflammatory stimuli. Further investigation of these molecular mechanisms will help guide novel therapies to reduce fibrosis and encourage superior tendon healing.

ISRIB facilitates the co-culture of human trophoblast stem cells and embryonic stem cells.

The embryo-like structures (embryoids) constructed by aggregating embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) have provided revolutionary tools for studying the intricate interaction between embryonic and extra-embryonic tissues during early embryonic development, which has been achieved in mice. However, due to the opposite dependence on some signalling pathways for in vitro culture of human ESCs (hESCs) and TSCs (hTSCs), particularly WNT and TGFß signalling pathways, which limits the construction of human post-implantation embryoids by aggregating hESCs and hTSCs. To overcome this challenge, here, by screening 1639 chemicals, we found that an inhibitor of integrated stress response, ISRIB, can replace WNT agonists and TGFß inhibitors to maintain the stemness and differentiation capacity of hTSCs. Thus, we developed an ISRIB-dependent in vitro culture medium for hTSCs, namely nTSM. Furthermore, we demonstrated that ISRIB could also maintain the hESC stemness. Using a 3D co-culture system (hESCs and hTSCs aggregate, ETA), we demonstrated that a 1:1 mixture of hESC culture medium (ESM) and nTSM improved the cell proliferation and organisation of both hESC- and hTSC-compartments and the lumenogenesis of hESC-compartment in ETAs. Overall, our study provided an ISRIB-dependent system for co-culturing hESCs and hTSCs, which facilitated the construction of human embryoids by aggregating hESCs and hTSCs.

The role of L-carnitine in bovine embryo metabolism. A review of the effect of supplementation with a metabolic modulator on in vitro embryo production.

Early embryo development is driven first by the maternal RNAs and proteins accumulated during the oocyte's cytoplasmic maturation and then after the embryo genome activation. In mammalian cells, ATP generation occurs via oxidative pathways or by glycolysis, whereas in embryonic stem cells, the consumption of glucose, pyruvate, lipids, and amino acids results in ATP synthesis. Although the bovine embryo has energy reserves in glycogen and lipids, the glycogen concentration is deficient. Conversely, lipids represent the most abundant energy reservoir of bovine embryos, where lipid droplets-containing triacylglycerols are the main fatty acid stores. Oocytes of many mammalian species contain comparatively high amounts of lipids stored as droplets in the ooplasm. L-carnitine has been described as a cofactor that facilitates the mobilization of fatty acids present in the oocyte's cytoplasm into the mitochondria to facilitate ß-oxidation processes. However, the L-carnitine effects by addition to media in the in vitro produced embryos on the quality are highly disputed and contradictory by different researchers. This review's objective was to explore the effect that the addition of L-carnitine on culture media could have on the overall bovine embryo production in vitro, from the oocyte metabolism to the modulation of gene expression in the developing embryos.

Induction of mouse totipotent stem cells by a defined chemical cocktail.

In mice, only the zygotes and blastomeres from 2-cell embryos are authentic totipotent stem cells (TotiSCs) capable of producing all the differentiated cells in both embryonic and extraembryonic tissues and forming an entire organism1. However, it remains unknown whether and how totipotent stem cells can be established in vitro in the absence of germline cells. Here we demonstrate the induction and long-term maintenance of TotiSCs from mouse pluripotent stem cells using a combination of three small molecules: the retinoic acid analogue TTNPB, 1-azakenpaullone and the kinase blocker WS6. The resulting chemically induced totipotent stem cells (ciTotiSCs), resembled mouse totipotent 2-cell embryo cells at the transcriptome, epigenome and metabolome levels. In addition, ciTotiSCs exhibited bidirectional developmental potentials and were able to produce both embryonic and extraembryonic cells in vitro and in teratoma. Furthermore, following injection into 8-cell embryos, ciTotiSCs contributed to both embryonic and extraembryonic lineages with high efficiency. Our chemical approach to totipotent stem cell induction and maintenance provides a defined in vitro system for manipulating and developing understanding of the totipotent state and the development of multicellular organisms from non-germline cells.

NACA and LRP6 Are Part of a Common Genetic Pathway Necessary for Full Anabolic Response to Intermittent PTH.

PTH induces phosphorylation of the transcriptional coregulator NACA on serine 99 through Gαs and PKA. This leads to nuclear translocation of NACA and expression of the target gene Lrp6, encoding a coreceptor of the PTH receptor (PTH1R) necessary for full anabolic response to intermittent PTH (iPTH) treatment. We hypothesized that maintaining enough functional PTH1R/LRP6 coreceptor complexes at the plasma membrane through NACA-dependent Lrp6 transcription is important to ensure maximal response to iPTH. To test this model, we generated compound heterozygous mice in which one allele each of Naca and Lrp6 is inactivated in osteoblasts and osteocytes, using a knock-in strain with a Naca99 Ser-to-Ala mutation and an Lrp6 floxed strain (test genotype: Naca99S/A; Lrp6+/fl;OCN-Cre). Four-month-old females were injected with vehicle or 100 µg/kg PTH(1-34) once daily, 5 days a week for 4 weeks. Control mice showed significant increases in vertebral trabecular bone mass and biomechanical properties that were abolished in compound heterozygotes. Lrp6 expression was reduced in compound heterozygotes vs. controls. The iPTH treatment increased Alpl and Col1a1 mRNA levels in the control but not in the test group. These results confirm that NACA and LRP6 form part of a common genetic pathway that is necessary for the full anabolic effect of iPTH.

Hormesis and embryonic stem cells.

This paper provides an identification and detailed assessment of hormetic dose responses of embryonic stem cells (ESCs) with particular emphasis on cell renewal (proliferation) and differentiation, underlying mechanistic foundations and potential therapeutic implications. Hormetic dose responses were commonly reported, being induced by a broad range of chemicals, including pharmaceuticals (e.g., atorvastatin, isoproterenol, lithium, nicotine, ouabain), dietary supplements (e.g., curcumin, multiple ginsenosides, resveratrol), endogenous agents (e.g., estrogen, hydrogen peroxide, melatonin), and physical stressor agents (e.g., hypoxia, ionizing radiation). ESC-hormetic dose responses are similar for other stem cell types (e.g., adipose-derived stem cells, apical papilla, bone marrow stem cells, dental pulp stem cells, endothelial stem cells, muscle stem cells, periodontal ligament stem cells, neural stem cells), indicating a high degree of generality for the hormetic-stem cells response. The widespread occurrence of hormetic dose responses shown by ESCs and other stem cells suggests that the hormetic dose response may represent a fundamental and highly conserved evolutionary strategy.

MCPH1 inhibits Condensin II during interphase by regulating its SMC2-Kleisin interface.

Dramatic change in chromosomal DNA morphology between interphase and mitosis is a defining features of the eukaryotic cell cycle. Two types of enzymes, namely cohesin and condensin confer the topology of chromosomal DNA by extruding DNA loops. While condensin normally configures chromosomes exclusively during mitosis, cohesin does so during interphase. The processivity of cohesin's loop extrusion during interphase is limited by a regulatory factor called WAPL, which induces cohesin to dissociate from chromosomes via a mechanism that requires dissociation of its kleisin from the neck of SMC3. We show here that a related mechanism may be responsible for blocking condensin II from acting during interphase. Cells derived from patients affected by microcephaly caused by mutations in the MCPH1 gene undergo premature chromosome condensation. We show that deletion of Mcph1 in mouse embryonic stem cells unleashes an activity of condensin II that triggers formation of compact chromosomes in G1 and G2 phases, accompanied by enhanced mixing of A and B chromatin compartments, and this occurs even in the absence of CDK1 activity. Crucially, inhibition of condensin II by MCPH1 depends on the binding of a short linear motif within MCPH1 to condensin II's NCAPG2 subunit. MCPH1's ability to block condensin II's association with chromatin is abrogated by the fusion of SMC2 with NCAPH2, hence may work by a mechanism similar to cohesin. Remarkably, in the absence of both WAPL and MCPH1, cohesin and condensin II transform chromosomal DNAs of G2 cells into chromosomes with a solenoidal axis.

Gallic acid inhibits celecoxib-induced mitochondrial permeability transition and reduces its toxicity in isolated cardiomyocytes and mitochondria.

BACKGROUND: Mitochondria are the main target organelles through which drugs and chemicals exert their toxic effect on cardiomyocytes. The mitochondria-related mechanisms of celecoxib-induced cardiotoxicity have been extensively studied. Accumulated evidence shows natural molecules targeting mitochondria have proven to be effective in preventing cardiotoxicity. PURPOSE: In the present study, we examined the ameliorative effect of gallic acid (GA) against celecoxib-induced cellular and mitochondrial toxicity in isolated cardiomyocytes and mitochondria. RESEARCH DESIGN: The isolated cardiomyocytes and mitochondria were divided into various group, namely, control, celecoxib, celecoxib + GA (10, 50, and 100 µM). Several cellular and mitochondrial parameters such as cell viability, lipid peroxidation, succinate dehydrogenase (SDH) activity, reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) collapse, and mitochondrial swelling were assessed in isolated cardiomyocytes and mitochondria. RESULTS: Our results showed that administration of celecoxib (16 µg/ml) induced cytotoxicity and mitochondrial dysfunction at 6 h and 1 h, respectively, which is associated with lipid peroxidation intact cardiomyocytes, mitochondrial ROS formation, MMP collapse, and mitochondrial swelling. The cardiomyocytes and mitochondria treated with celecoxib + GA (10, 50, and 100 µM) significantly and dose-dependently restore the altered levels of cellular and mitochondrial parameters. CONCLUSIONS: We concluded that GA through antioxidant potential and inhibition of mitochondrial permeability transition (MPT) pore exerted ameliorative role in celecoxib-induced toxicity in isolated cardiomyocytes and mitochondria. The data of the current study suggested that GA supplementation may reduce celecoxib-induced cellular and mitochondrial toxicity during exposure and may provide a potential prophylactic and defensive candidate for coxibs-induced mitochondrial dysfunction, oxidative stress, and cardiotoxicity.

Magnetic Fe3 O4 @graphene oxide improves the therapeutic effects of embryonic stem cells on acute liver damage.

OBJECTIVE: Acute liver failure is usually associated with inflammation and oxidation of hepatocytes and has high mortality and resource costs. Mesenchymal stem cell (MSCs) has occasionally been reported to have no beneficial effect due to poor transplantation and the survival of implanted cells. Recent studies showed that embryonic stem cell (ESC)-derived MSCs are an alternative for regenerative medicine. On the other hand, graphene-based nanostructures have proven useful in biomedicine. In this study, we investigated whether magnetic graphene oxide (MGO) improved the effects of ESC-MSC conditioned medium (CM) on protecting hepatocytes and stimulating the regeneration of damaged liver cells. MATERIALS AND METHODS: To provide a rat model of acute liver failure, male rats were injected intraperitoneally with carbon tetrachloride (CCl4 ). The rats were randomly divided into six groups, namely control, sham, CCl4 , ESC-MSC-CM, MGO and ESC-MSC-CM + MGO. In the experimental groups, the rats received, depending on the group, 2 ml/kg body weight CCl4 and either ESC-MSC-CM with 5 × 106 MSCs or 300 µg/kg body weight MGO or both. Symptoms of acute liver failure appeared 4 days after the injection. All groups were compared and analysed both histologically and biochemically 4 days after the injection. Finally, the results of ESC-MSC-CM and MSC-CM were compared. RESULTS: The results indicated that the use of MGO enhanced the effect of ESC-MSC-CM on reducing necrosis, inflammation, aspartate transaminase, alanine aminotransferase and alkaline phosphatase in the CCl4 -induced liver failure of the rat model. Also, the expression of vascular endothelial growth factor and matrix metalloproteinase-9 (MMP-9) was significantly upregulated after treatment with MGO. Also, the results showed that the ESC-MSC-CM has more efficient effective compared to MSC-CM. CONCLUSION: Magnetic graphene oxide improved the hepatoprotective effects of ESC-MSC-CM on acute liver damage, probably by suppressing necrosis, apoptosis and inflammation of hepatocytes.

Construction of extracellular matrix-based 3D hydrogel and its effects on cardiomyocytes.

Some discoveries resulted from 2-dimensional (2D) cultured cardiac cells have been disqualified in animal testing and later clinical trials. Extracellular matrix (ECM) plays a vital role in cardiac homeostasis, cardiac ECM (cECM)-based 3D cell cultures can mimics the physiological and pathological conditions in vivo closely, it is hopeful of addressing this challenge. Construction of cECM-based 3-dimensional (3D) hydrogel (cECM3DH) and its effects on cell behaviors were studied here. The results indicated that cellular compartments could be efficiently removed from heart tissue via sodium dodecyl sulfonate (SDS)- and Triton X-100-mediated decellularization, remaining the natural fibrous network structure and major proteins. 3D hydrogel consisted of 1 × 107 cells/mL cells and 75% cECM could promote the proliferation and anti-apoptosis ability of human embryonic kidney (HEK)-293T cells. 0.25% trypsin or 0.20% collagenase was suitable to retrieve these cells from 3D hydrogel for further researches. Compared with 2D culture system, cECM3DH could significantly increase the proportion of GATA 4+ cardiomyocytes (CMs) derived from heart tissue of neonatal mouse or induced differentiation of embryonic stem cells (ESCs) (P < 0.05) The expression levels of mature genes including cTnT, JCN, CaV1.2, MYL2, CASQ2, NCX1, and Cx43 of these CMs in adult pig cECM-based 3D hydrogel (APcECM3DH) were significantly higher than that in 2D culture system and in newborn piglet cECM-based 3D hydrogel (NPcECM3DH), respectively (P < 0.05). Therefore, cECM3DH supports the generation of primary CMs and ESC-derived CMs, APcECM3DH was more conducive to promoting CM maturation, which contributes to building 3D model for pathogenesis exploration, drug screening, and regenerative medicine of heart diseases.

The chromatin, topological and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo.

Poised enhancers (PEs) represent a genetically distinct set of distal regulatory elements that control the expression of major developmental genes. Before becoming activated in differentiating cells, PEs are already bookmarked in pluripotent cells with unique chromatin and topological features that could contribute to their privileged regulatory properties. However, since PEs were originally characterized in embryonic stem cells (ESC), it is currently unknown whether PEs are functionally conserved in vivo. Here, we show that the chromatin and 3D structural features of PEs are conserved among mouse pluripotent cells both in vitro and in vivo. We also uncovered that the interactions between PEs and their target genes are globally controlled by the combined action of Polycomb, Trithorax and architectural proteins. Moreover, distal regulatory sequences located close to developmental genes and displaying the typical genetic (i.e. CpG islands) and chromatin (i.e. high accessibility and H3K27me3 levels) features of PEs are commonly found across vertebrates. These putative PEs show high sequence conservation within specific vertebrate clades, with only a few being evolutionary conserved across all vertebrates. Lastly, by genetically disrupting PEs in mouse and chicken embryos, we demonstrate that these regulatory elements play essential roles during the induction of major developmental genes in vivo.

Effect of the phenylpyrrole fungicide fludioxonil on cell proliferation and cardiac differentiation in mouse embryonic stem cells.

Fludioxnil is extensively used as a fungicide in agricultural application, but its possible impact on embryonic development is not yet well understood. In this study, the potential effect of fludioxonil on cardiac differentiation was evaluated in mouse embryonic stem cells (mESCs). The water-soluble tetrazolium (WST) and colony formation assays were conducted to confirm the effect of fludioxonil on proliferation of mESCs. The effect of fludioxonil on the ability of mESCs to form mouse embryoid bodies (mEBs) was determined by the hanging drop assay, whereas the ability of cardiomyocyte differentiation in the early stage was evaluated by determining the beating ratio (ratio of the number of contracting cells to the number of attached EBs) of cardiomyocytes. The viability of mESCs was significantly decreased (less than 50 %) at 10-5 M fludioxonil. Results of the colony formation assay revealed suppressed colony formation at 10-5 M fludioxonil (about 50 % at 5 days). Furthermore, the expressions of cell-cycle related proteins, i.e., cyclin D1, cyclin E, p21 and p27, were altered and trending towards inhibiting cell growth. Exposure to fludioxonil also resulted in reduced size of the mEB and induced increasing expression levels of the pluripotency markers Oct4, Sox2 and Nanog. Development of the beating ratio in the process of differentiation to cardiomyocytes derived from mESCs was completely inhibited after exposure to 10-5 M fludioxonil during the early stage of differentiation (day 5), whereas the beating ratio gradually increased after 5-day treatment. Simultaneously, expressions of the cardiomyocyte-related proteins, Gata4, Hand1 and cTnI, were inhibited after exposure to 10-5 M fludioxonil. Taken together, these results imply that fludioxonil may impact on the developmental process of mESCs, particularly the cardiac lineage.

Generation of mouse-human chimeric embryos.

Naive human pluripotent stem cells (hPSCs) can be used to generate mature human cells of all three germ layers in mouse-human chimeric embryos. Here, we describe a protocol for generating mouse-human chimeric embryos by injecting naive hPSCs converted from the primed state. Primed hPSCs are treated with a mammalian target of rapamycin inhibitor (Torin1) for 3 h and dissociated to single cells, which are plated on mouse embryonic fibroblasts in 2iLI medium, a condition essentially the same for culturing mouse embryonic stem cells. After 3-4 d, bright, dome-shaped colonies with mouse embryonic stem cell morphology are passaged in 2iLI medium. Established naive hPSCs are injected into mouse blastocysts, which produce E17.5 mouse embryos containing 0.1-4.0% human cells as quantified by next-generation sequencing of 18S ribosomal DNA amplicons. The protocol is suitable for studying the development of hPSCs in mouse embryos and may facilitate the generation of human cells, tissues and organs in animals.

Differentiation of embryonic stem cells into a putative hair cell-progenitor cells via co-culture with HEI-OC1 cells.

Several studies have shown how different cell lines can influence the differentiation of stem cells through co-culture systems. The House Ear Institute-Organ of Corti 1 (HEI-OC1) is considered an important cell line for in vitro auditory research. However, it is unknown if HEI-OC1 cells can promote the differentiation of embryonic stem cells (ESCs). In this study, we investigated whether co-culture of ESCs with HEI-OC1 cells promotes differentiation. To this end, we developed a co-culture system of mouse ESCs with HEI-OC1 cells. Dissociated or embryonic bodies (EBs) of ESCs were introduced to a conditioned and inactivated confluent layer of HEI-OC1 cells for 14 days. The dissociated ESCs coalesced into an EB-like form that was smaller than the co-cultured EBs. Contact co-culture generated cells expressing several otic progenitor markers as well as hair cell specific markers. ESCs and EBs were also cultured in non-contact setup but using conditioned medium from HEI-OC1 cells, indicating that soluble factors alone could have a similar effect. The ESCs did not form into aggregates but were still Myo7a-positive, while the EBs degenerated. However, in the fully differentiated EBs, evidence to prove mature differentiation of inner ear hair cell was still rudimentary. Nevertheless, these results suggest that cellular interactions between ESCs and HEI-OC1 cells may both stimulate ESC differentiation.

Melatonin mitigates the adverse effect of hypoxia during myocardial differentiation in mouse embryonic stem cells.

BACKGROUND: Hypoxia causes oxidative stress and affects cardiovascular function and the programming of cardiovascular disease. Melatonin promotes antioxidant enzymes such as superoxide dismutase, glutathione reductase, glutathione peroxidase, and catalase. OBJECTIVES: This study aims to investigate the correlation between melatonin and hypoxia induction in cardiomyocytes differentiation. METHODS: Mouse embryonic stem cells (mESCs) were induced to myocardial differentiation. To demonstrate the influence of melatonin under hypoxia, mESC was pretreated with melatonin and then cultured in hypoxic condition. The cardiac beating ratio of the mESC-derived cardiomyocytes, mRNA and protein expression levels were investigated. RESULTS: Under hypoxic condition, the mRNA expression of cardiac-lineage markers (Brachyury, Tbx20, and cTn1) and melatonin receptor (Mtnr1a) was reduced. The mRNA expression of cTn1 and the beating ratio of mESCs increased when melatonin was treated simultaneously with hypoxia, compared to when only exposed to hypoxia. Hypoxia-inducible factor (HIF)-1α protein decreased with melatonin treatment under hypoxia, and Mtnr1a mRNA expression increased. When the cells were exposed to hypoxia with melatonin treatment, the protein expressions of phospho-extracellular signal-related kinase (p-ERK) and Bcl-2-associated X proteins (Bax) decreased, however, the levels of phospho-protein kinase B (p-Akt), phosphatidylinositol 3-kinase (PI3K), B-cell lymphoma 2 (Bcl-2) proteins, and antioxidant enzymes including Cu/Zn-SOD, Mn-SOD, and catalase were increased. Competitive melatonin receptor antagonist luzindole blocked the melatonin-induced effects. CONCLUSIONS: This study demonstrates that hypoxia inhibits cardiomyocytes differentiation and melatonin partially mitigates the adverse effect of hypoxia in myocardial differentiation by regulating apoptosis and oxidative stress through the p-AKT and PI3K pathway.

Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells.

Ammonia has a cytotoxic effect and can therefore be used as a selection agent for enrichment of zone I hepatocytes. However, it has not yet been determined whether ammonia-treated hepatocyte-like cells are able to proliferate in vitro. In this study, we employed an ammonia selection strategy to purify hepatocyte-like cells that were differentiated from human embryonic stem cells (ESCs) and from induced pluripotent stem cells (iPSCs). The resistance to cytotoxicity or cell death by ammonia is likely attributable to the metabolism of ammonia in the cells. In addition to ammonia metabolism-related genes, ammonia-selected hepatocytes showed increased expression of the cytochrome P450 genes. Additionally, the ammonia-selected cells achieved immortality or at least an equivalent life span to human pluripotent stem cells without affecting expression of the liver-associated genes. Ammonia treatment in combination with in vitro propagation is useful for obtaining large quantities of hepatocytes.

Individual and combined effects of BPA, BPS and BPAF on the cardiomyocyte differentiation of embryonic stem cells.

Exposure to many kinds of bisphenols (BPs) is common, and the effects of BP mixtures may differ from those of individual BPs. Therefore, evaluating combined exposure effects is necessary. Our study evaluated the individual and combined exposure effects of bisphenol A (BPA), bisphenol S (BPS) and bisphenol AF (BPAF) on embryonic development using an embryonic stem cell test (EST) and a concentration additive (CA) model at relatively high doses to uncover the interaction model of the three BPs. Environmentally relevant concentrations were then used to evaluate the possible effects of the individual and combined BPs at actual human exposure levels. Exposure to relatively high-dose BPA, BPS and BPAF inhibited embryonic stem cell differentiation into cardiomyocytes and exhibited weak embryo toxicity. Individually, BPA, BPS and BPAF inhibited endoderm, mesoderm and ectoderm marker expression but enhanced pluripotency marker expression. Combined exposure to BPs had an additive effect on cardiomyocyte differentiation and embryonic stem cell proliferation based on the CA model. Environmentally relevant individual or combined BP doses (10 ng/ml individual BPA, BPS and BPAF doses or 1 ng/ml and 10 ng/ml BP mixture doses) failed to cause oxidative stress, DNA damage or apoptosis changes in stem cell differentiation. The cardiomyocyte differentiation ratio also did not change significantly. Individual and combined exposure to environmentally relevant BP doses led to a significant increase in collagen expression. BPAF and the combination of BPs increased the type 1 collagen level, while the combination also increased the type 3 collagen level, which may be related to p38 pathway activation. The p38 pathway inhibitor SB203580 inhibited the increase in collagen during cardiomyocyte differentiation caused by low-dose BPs. These results suggest that relatively high-dose BPs in combination have an additive effect on cardiomyocyte differentiation. Low-dose BPs individually and in combination may affect cardiomyocyte collagen through the p38 pathway.

Retinoic acid signaling is critical during the totipotency window in early mammalian development.

Totipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such '2-cell-like-cells' (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.