Functional screen reveals essential roles of miR‐27a/24 in differentiation of embryonic stem cells

MicroRNAs play important roles in controlling the embryonic stem cell (ESC) state. Although much is known about microRNAs maintaining ESC state, microRNAs that are responsible for promoting ESC differentiation are less reported. Here, by screening 40 microRNAs pre-selected by their expression patterns and predicted targets in Dgcr8-null ESCs, we identify 14 novel differentiation-associated microRNAs. Among them, miR-27a and miR-24, restrained by c-Myc in ESC, exert their roles of silencing self-renewal through directly targeting several important pluripotency-associated factors, such as Oct4, Foxo1 and Smads. CRISPR/Cas9-mediated knockout of all miR-27/24 in ESCs leads to serious deficiency in ESC differentiation in vitro and in vivo. Moreover, depleting of them in mouse embryonic fibroblasts can evidently promote somatic cell reprogramming. Altogether, our findings uncover the essential role of miR-27 and miR-24 in ESC differentiation and also demonstrate novel microRNAs responsible for ESC differentiation.     

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.     

Vascular endothelial growth factor promotes cardiomyocyte differentiation of embryonic stem cells

Embryonic stem cells (ESCs) overexpressing the vascular endothelial growth factor (VEGF) improve cardiac function in mouse models of myocardial ischemia and infarction by mechanisms that are poorly understood. Here we studied the effects of VEGF on cardiomyocyte differentiation of mouse ESCs in vitro. We used flow cytometry to determine the expression of alpha-myosin heavy chain (alpha-MHC), cardiac troponin I (cTn-I), and Nkx2.5 in differentiated ESCs. VEGF (20 ng/ml) significantly enhanced alpha-MHC, cTn-I, and Nkx2.5 expression in differentiated ESCs. Western blot analysis confirmed these findings. We found that VEGF receptor FMS-like tyrosine kinase-1 (Flt-1) and fetal liver kinase-1 (Flk-1) expression increased during ESC differentiation. Antibodies against Flk-1 totally blocked and against Flt-1 partially blocked VEGF-induced NKx2.5-positive-stained cells. The ERK inhibitor PD-098059 abolished VEGF-induced cardiomyocyte differentiation of ESCs. Our results suggest that VEGF promotes cardiomyocyte differentiation predominantly by ERK-mediated Flk-1 activation and, to a lesser extent, by Flt-1 activation. These findings may be of significance for stem cell and growth factor therapies to regenerate failing cardiomyocytes.     

Three-dimensional co-culture facilitates the differentiation of embryonic stem cells into mature cardiomyocytes

The cardiomyocyte (CM) differentiation of embryonic stem cells (ESCs) is routinely cultured as two-dimensional (2D) monolayer, which doesn't mimic in vivo physiological environment and may lead to low differentiated level of ESCs. Here, we develop a novel strategy that enhances CM differentiation of ESCs in collagen matrix three-dimensional (3D) culture combined with indirect cardiac fibroblasts co-culture. ESCs were cultured in hanging drops to form embryoid bodies (EBs) and then applied on collagen matrix. The EBs were indirectly co-cultured with cardiac fibroblasts by the hanging cell culture inserts (PET 1 μm). The molecular expressions and ultrastructural characteristics of ESC-derived CMs (ESCMs) were analyzed by real time RT-PCR, immunocytochemistry, and Transmission Electron Microscopy (TEM). We found that the percentage of beating EBs with cardiac fibroblasts co-culture was significantly higher than that without co-culture after differentiation period of 8 days. Type I collagen used as 3D substrates enhanced the late-stage CM differentiation of ESCs and had effect on ultrastructural mature of ESCMs in late-stage development. The combined effects of 3D and co-culture that mimic in vivo physiological environment further improved the efficiency of CM differentiation from ESCs, resulting in fiber-like structures of cardiac cells with organized sarcomeric structure in ESCMs. This novel 3D co-culture system emphasizes the fact that the ESC differentiation is actively responding to cues from their environment and those cues can drive phenotypic control, which provides a useful in vitro model to investigate CM differentiation of stem cells.     

Analysis of Epigenetic Factors in Mouse Embryonic Neural Stem Cells Exposed to Hyperglycemia

Background

Maternal diabetes alters gene expression leading to neural tube defects (NTDs) in the developing brain. The mechanistic pathways that deregulate the gene expression remain unknown. It is hypothesized that exposure of neural stem cells (NSCs) to high glucose/hyperglycemia results in activation of epigenetic mechanisms which alter gene expression and cell fate during brain development.

Methods and Findings

NSCs were isolated from normal pregnancy and streptozotocin induced-diabetic pregnancy and cultured in physiological glucose. In order to examine hyperglycemia induced epigenetic changes in NSCs, chromatin reorganization, global histone status at lysine 9 residue of histone H3 (acetylation and trimethylation) and global DNA methylation were examined and found to be altered by hyperglycemia. In NSCs, hyperglycemia increased the expression of Dcx (Doublecortin) and Pafah1b1 (Platelet activating factor acetyl hydrolase, isoform 1b, subunit 1) proteins concomitant with decreased expression of four microRNAs (mmu-miR-200a, mmu-miR-200b, mmu-miR-466a-3p and mmu-miR-466 d-3p) predicted to target these genes. Knockdown of specific microRNAs in NSCs resulted in increased expression of Dcx and Pafah1b1 proteins confirming target prediction and altered NSC fate by increasing the expression of neuronal and glial lineage markers.

Conclusion/Interpretation

This study revealed that hyperglycemia alters the epigenetic mechanisms in NSCs, resulting in altered expression of some development control genes which may form the basis for the NTDs. Since epigenetic changes are reversible, they may be valuable therapeutic targets in order to improve fetal outcomes in diabetic pregnancy.     

Differential expression of microRNAs in adipose tissue after long-term high-fat diet-induced obesity in mice

Obesity is a major health concern worldwide which is associated with increased risk of chronic diseases such as metabolic syndrome, cardiovascular disease and cancer. The elucidation of the molecular mechanisms involved in adipogenesis and obesogenesis is of essential importance as it could lead to the identification of novel biomarkers and therapeutic targets for the development of anti-obesity drugs. MicroRNAs (miRNAs) have been shown to play regulatory roles in several biological processes. They have become a growing research field and consist of promising pharmaceutical targets in various fields such as cancer, metabolism, etc. The present study investigated the possible implication of miRNAs in adipose tissue during the development of obesity using as a model the C57BLJ6 mice fed a high-fat diet.C57BLJ6 wild type male mice were fed either a standard (SD) or a high-fat diet (HFD) for 5 months. Total RNA was prepared from white adipose tissue and was used for microRNA profiling and qPCR.Twenty-two of the most differentially expressed miRNAs, as identified by the microRNA profiling were validated using qPCR. The results of the present study confirmed previous results. The up-regulation of mmu-miR-222 and the down-regulation of mmu-miR-200b, mmu-miR-200c, mmu-miR-204, mmu-miR-30a*, mmu-miR-193, mmu-miR-378 and mmu-miR-30e* after HFD feeding has also been previously reported. On the other hand, we show for the first time the up-regulation of mmu-miR-342-3p, mmu-miR-142-3p, mmu-miR-142-5p, mmu-miR-21, mmu-miR-146a, mmu-miR-146b, mmu-miR-379 and the down-regulation of mmu-miR-122, mmu-miR-133b, mmu-miR-1, mmu-miR-30a*, mmu-miR-192 and mmu-miR-203 during the development of obesity. However, future studies are warranted in order to understand the exact role that miRNAs play in adipogenesis and obesity.     

p57与同源框基因HOXA10在子宫内膜基质细胞体外蜕膜化过程中的表达

本文旨在从mRNA和蛋白水平研究子宫内膜基质细胞(endometrial stromalcell,ESC)体外蜕膜化过程中p57和同源框基因HOXA10(homeobox A10 gene)的表达变化以及HOXA10的亚细胞定位,从而推测其在蜕膜化过程中的作用。本实验联合使用0.5mmol/L8-溴-cAMP和1×10?6mol/LMPA(medroxyprogesterone acetate)作用1、2、4d(D1、D2、D4)诱导ESC发生蜕膜化,相应时间点提取mRNA和蛋白质行半定量RT-PCR和免疫印迹,同时以2%低血清培养ESC1、4d作为对照(C1、C4)。用间接免疫荧光和基因转染的方法,观察蜕膜化过程中HOXA10的亚细胞定位。结果显示:(1)蜕膜化过程中HOXA10的表达进行性下降,D2开始与对照组(C4)比较具有显著性差异(P<0.05)。(2)相反,蜕膜化过程中p57的表达进行性上升,D2开始与对照组C4比较也有显著性差异(P<0.05)。(3)低血清培养ESC1、4d后,p57和HOXA10的表达没有显著性差异(P>0.05)。(4)蜕膜化过程中HOXA10始终定位于胞核,不发生胞浆胞核穿梭。以上观察结果表明:(1)p57的高表达是ESC脱离细胞周期走向分化的因素之一。(2)HOXA10的低表达可能是p57上调的原因之一。(3)孕激素受体(progesterone receptor,PR)途径参与了促进ESC脱离细胞周期而走向分化的过程。     

Beta-adrenergic signals regulate cardiac differentiation of mouse embryonic stem cells via mitogen-activated protein kinase pathways

As embryonic stem cell-derived cardiomyocytes (ESC-CMs) have the potential to be used in cell replacement therapy, an understanding of the signaling mechanisms that regulate their terminal differentiation is imperative. In previous studies, we discovered the presence of adrenergic and muscarinic receptors in mouse embryonic stem cells (ESCs). However, little is known about the role of these receptors in cardiac differentiation and development, which is critically important in cardiac physiology and pharmacology. Here, we demonstrated that a β-adrenergic receptor (β-AR) agonist significantly enhanced cardiac differentiation as indicated by a higher percentage of beating embryoid bodies and a higher expression level of cardiac markers. Application of β1-AR and β2-AR antagonists partly abolished the effect of the β-AR agonist. In addition, by administering selective inhibitors we found that the effect of β-AR was driven via p38 mitogen-activated protein kinase and extracellular-signal regulated kinase pathway. These findings suggest that ESCs are also a target for β-adrenergic regulation and β-adrenergic signaling plays a role in ESC cardiac differentiation.     

Neuregulin induces GABAA receptor beta2 subunit expression in cultured rat cerebellar granule neurons by activating multiple signaling pathways

The GABAA receptor beta subunit is required to confer sensitivity to gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the CNS. In previous studies we demonstrated that the growth and differentiation factor neuregulin 1 (NRG1) selectively induced expression of the beta2 subunit mRNA and encoded protein in rat cerebellar granule neurons in culture. In the present report we examine the signaling pathways that mediate this effect. These studies demonstrate that the effects of NRG1 on beta2 subunit polypeptide expression require activation of the ErbB4 receptor tyrosine kinase; its effects are inhibited by pharmacological blockade of ErbB4 phosphorylation or reduction of receptor level with an antisense oligodeoxynucleotide. The NRG1-induced activation of ErbB4 stimulates the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) and cyclin-dependent kinase-5 (cdk5) pathways. Pharmacological blockade of any of these pathways inhibits increased beta2 subunit expression, demonstrating that all three pathways are required to mediate the effects of NRG1 on GABAA receptor subunit expression in cerebellar granule neurons. These studies provide novel information concerning the actions of NRG1 on GABAA receptor expression in the CNS.     

The Long-Term Differentiation of Embryonic Stem Cells into Cardiomyocytes: An Indirect Co-Culture Model

Background

Embryonic Stem Cells (ESCs) can differentiate into cardiomyocytes (CMs) in vitro but the differentiation level from ESCs is low. Here we describe a simple co-culture model by commercially available Millicell™ hanging cell culture inserts to control the long-term differentiation of ESCs into CMs.

Methodology/Principal Findings

Mouse ESCs were cultured in hanging drops to form embryoid bodies (EBs) and treated with 0.1 mmol/L ascorbic acid to induce the differentiation of ESCs into CMs. In the indirect co-culture system, EBs were co-cultured with epidermal keratinocytes (EKs) or neonatal CMs (NCMs) by the hanging cell culture inserts (PET membranes with 1 µm pores). The molecular expressions and functional properties of ESC-derived CMs in prolonged culture course were evaluated. During time course of ESC differentiation, the percentages of EBs with contracting areas in NCMs co-culture were significantly higher than that without co-culture or in EKs co-culture. The functional maintenance of ESC-derived CMs were more prominent in NCMs co-culture model.

Conclusions/Significance

These results indicate that NCMs co-culture promote ESC differentiation and has a further effect on cell growth and differentiation. We assume that the improvement of the differentiating efficiency of ESCs into CMs in the co-culture system do not result from the effect of co-culture directly on cell differentiation, but rather by signaling effects that influence the cells in proliferation and long-term function maintenance.     

A p38mapk-p53 cascade regulates mesodermal differentiation and neurogenesis of embryonic stem cells

Embryonic stem cells (ESCs) differentiate in vivo and in vitro into all cell lineages, and they have been proposed as cellular therapy for human diseases. However, the molecular mechanisms controlling ESC commitment toward specific lineages need to be specified. We previously found that the p38 mitogen-activated protein kinase (p38MAPK) pathway inhibits neurogenesis and is necessary to mesodermal formation during the critical first 5 days of mouse ESC commitment. This period corresponds to the expression of specific master genes that direct ESC into each of the three embryonic layers. By both chemical and genetic approaches, we found now that, during this phase, the p38MAPK pathway stabilizes the p53 protein level and that interfering directly with p53 mimics the effects of p38MAPK inhibition on ESC differentiation. Anti-p53 siRNA transient transfections stimulate Bcl2 and Pax6 gene expressions, leading to increased ESC neurogenesis compared with control transfections. Conversely, p53 downregulation leads to a strong inhibition of the mesodermal master genes Brachyury and Mesp1 affecting cardiomyogenesis and skeletal myogenesis of ESCs. Similar results were found with p53^−/− ESCs compared with their wild-type counterparts. In addition, knockout p53 ESCs show impaired smooth muscle cell and adipocyte formation. Use of anti-Nanog siRNAs demonstrates that certain of these regulations result partially to p53-dependent repression of Nanog gene expression. In addition to its well-known role in DNA-damage response, apoptosis, cell cycle control and tumor suppression, p53 has also been involved in vivo in embryonic development; our results show now that p53 mediates, at least for a large part, the p38MAPK control of the early commitment of ESCs toward mesodermal and neural lineages.     

Activation of the neuregulin/ErbB system during physiological ventricular remodeling in pregnancy

The neuregulin-1 (NRG1)/ErbB system has emerged as a paracrine endothelium-controlled system in the heart, which preserves left ventricular (LV) performance in pathophysiological conditions. Here, we analyze the activity and function of this system in pregnancy, which imparts a physiological condition of LV hemodynamic overload. NRG1 expression and ErbB receptor activation were studied by Western blot analyses in rats and mice at different stages of pregnancy. LV performance was evaluated by transthoracic echocardiography, and myocardial performance was assessed from twitches of isolated papillary muscles. NRG1/ErbB signaling was inhibited by oral treatment of animals with the dual ErbB1/ErbB2 tyrosine kinase inhibitor lapatinib. Analyses of LV tissue revealed that protein expression of different NRG1 isoforms and levels of phosphorylated ErbB2 and ErbB4 significantly increased after 1-2 wk of pregnancy. Lapatinib prevented phosphorylation of ErbB2 and ERK1/2, but not of ErbB4 and protein kinase B (Akt), revealing that lapatinib only partially inhibited NRG1/ErbB signaling in the LV. Lapatinib did not prevent pregnancy-induced changes in LV mass and did not cause apoptotic cell death or fibrosis in the LV. Nevertheless, lapatinib led to premature maternal death of ～25% during pregnancy and it accentuated pregnancy-induced LV dilatation, significantly reduced LV fractional shortening, and induced abnormalities of twitch relaxation (but not twitch amplitude) of isolated papillary muscles. This is the first study showing that the NRG1/ErbB system is activated, and plays a modulatory role, during physiological hemodynamic overload associated with pregnancy. Inhibiting this system during physiological overload may cause LV dysfunction in the absence of myocardial cell death.     

The role of neuregulin/ErbB2/ErbB4 signaling in the heart with special focus on effects on cardiomyocyte proliferation

The signaling complex consisting of the growth factor neuregulin-1 (NRG1) and its tyrosine kinase receptors ErbB2 and ErbB4 has a critical role in cardiac development and homeostasis of the structure and function of the adult heart. Recent research results suggest that targeting this signaling complex may provide a viable strategy for treating heart failure. Clinical trials are currently evaluating the effectiveness and safety of intravenous administration of recombinant NRG1 formulations in heart failure patients. Endogenous as well as administered NRG1 has multiple possible activities in the adult heart, but how these are related is unknown. It has recently been demonstrated that NRG1 administration can stimulate proliferation of cardiomyocytes, which may contribute to repair failing hearts. This review summarizes the current knowledge of how NRG1 and its receptors control cardiac physiology and biology, with special emphasis on its role in cardiomyocyte proliferation during myocardial growth and regeneration.     

A New Role of p53 in Maintaining Genetic Stability in Embryonic Stem Cells

Embryonic stem cells (ESCs) are capable of unlimited self-renewal and retain the pluripotency to differentiate into all cell lineages in the body. Since DNA damage occurs during normal cellular proliferation as well as after exposure to DNA damaging agents, it is critical for ESCs to possess stringent mechanisms to maintain genetic stability and prevent the passage of DNA damage to the progeny. Consistent with this notion, the rate of spontaneous mutation in ESCs is several magnitudes lower than that in somatic cells. Our recent findings indicate that tumor suppressor p53 plays an important role in maintaining genetic stability in ESCs by eliminating DNA-damaged ESCs from the replicative ESC pool. In this context, p53 induces the differentiation of DNA-damaged ESCs by directly suppressing the expression of Nanog, which is critical for the self-renewal of ESCs. This newly found role of p53 in cellular differentiation indicates an alternative mechanism for p53 to maintain genetic stability in ESCs and suggests the possibility that p53 might play a similar role in certain tissue stem cells and suppress the development of cancer stem cells.     

Presenilin-1 processing of ErbB4 in fetal type II cells is necessary for control of fetal lung maturation

Maturation of pulmonary fetal type II cells to initiate adequate surfactant production is crucial for postnatal respiratory function. Little is known about specific mechanisms of signal transduction controlling type II cell maturation. The ErbB4 receptor and its ligand neuregulin (NRG) are critical for lung development. ErbB4 is cleaved at the cell membrane by the γ-secretase enzyme complex whose active component is either presenilin-1 (PSEN-1) or presenilin-2. ErbB4 cleavage releases the 80 kDa intracellular domain (4ICD), which associates with chaperone proteins such as YAP (Yes-associated protein) and translocates to the nucleus to regulate gene expression. We hypothesized that PSEN-1 and YAP have a development-specific expression in fetal type II cells and are important for ErbB4 signaling in surfactant production. In primary fetal mouse E16, E17, and E18 type II cells, PSEN-1 and YAP expression increased at E17 and E18 over E16. Subcellular fractionation showed a strong cytosolic and a weaker membrane location of both PSEN-1 and YAP. This was enhanced by NRG stimulation. Co-immunoprecipitations showed ErbB4 associated separately with PSEN-1 and with YAP. Their association, phosphorylation, and co-localization were induced by NRG. Confocal immunofluorescence and nuclear fractionation confirmed these associations in a time-dependent manner after NRG stimulation. Primary ErbB4-deleted E17 type II cells were transfected with a mutant ErbB4 lacking the γ-secretase binding site. When compared to transfection with wild-type ErbB4, the stimulatory effect of NRG on surfactant protein mRNA expression was lost. We conclude that PSEN-1 and YAP have crucial roles in ErbB4 signal transduction during type II cell maturation.     

Engineered heart tissue enables study of residual undifferentiated embryonic stem cell activity in a cardiac environment

Embryonic stem cell (ESC) derivatives are a promising cell source for cardiac cell therapy. Mechanistic studies upon cell injection in conventional animal models are limited by inefficient delivery and poor cell survival. As an alternative, we have used an engineered heart tissue (EHT) based on neonatal rat cardiomyocytes (CMs) cultivated with electrical field stimulation as an in vitro model to study cell injection. We injected (0.001, 0.01, and 0.1 million) and tracked (by qPCR and histology) undifferentiated yellow‐fluorescent protein transgenic mouse ESCs and Flk1 + /PDGFRα+ cardiac progenitor (CPs) cells, to investigate the effect of the cardiac environment on cell differentiation, as well as to test whether our in vitro model system could recapitulate the formation of teratoma‐like structures commonly observed upon in vivo ESC injection. By 8 days post‐injection, ESCs were spatially segregated from the cardiac cell population; however, ESC injection increased survival of CMs. The presence of ESCs blocked electrical conduction through the tissue, resulting in a 46% increase in the excitation threshold. Expression of mouse cardiac troponin I, was markedly increased in CP injected constructs compared to ESC injected constructs at all time points and cell doses tested. As early as 2 weeks, epithelial and ganglion‐like structures were observed in ESC injected constructs. By 4 weeks of ESC injection, teratoma‐like structures containing neural, epithelial, and connective tissue were observed in the constructs. Non‐cardiac structures were observed in the CP injected constructs only after extended culture (4 weeks) and only at high cell doses, suggesting that these cells require further enrichment or differentiation prior to transplantation. Our data indicate that the cardiac environment of host tissue and electrical field stimulation did not preferentially guide the differentiation of ESCs towards the cardiac lineage. In the same environment, injection of CP resulted in a more robust cardiac differentiation than injection of ESC. Our data demonstrate that the model‐system developed herein can be used to study the functional effects of candidate stem cells on the host myocardium, as well as to measure the residual activity of undifferentiated cells present in the mixture. Biotechnol. Bioeng. 2011; 108:704–719. © 2010 Wiley Periodicals, Inc.