Enhancement of cardiomyocyte differentiation from human embryonic stem cells

Several approaches have been used to encourage the differentiation of cardiomyocytes from human embryonic stem cells.However,the differentiation efficiency is low,and appropriate culture protocols are needed to produce adequate numbers of cardiomyocytes for therapeutic cell transplantation.This study investigated the effects of serum on cardiomyocyte differentiation in suspension culture medium during embryoid body(EB) formation by human embryonic stem cells.The addition of ascorbic acid,dimethylsulfoxide and 5-aza-2'-deoxycytidine during days 5-7 at the EB-forming stage resulted in an increase in the numbers of rhythmically contracting clusters of derived cardiomyocytes.Treatment with 0.1 mmol L-1 ascorbic acid alone,or more notably in combination with 10 μmol L-1 5-aza-2'-deoxycytidine,induced the formation of beating cells within EBs.Most of the beating clusters had spontaneous contraction rates similar to those found in human adults,and their contractile ac-tivity lasted for up to 194 days.     

Effects of electrical fields on cardiomyocyte differentiation of embryonic stem cells

The effects of electromagnetic fields (EMFs) on the differentiation of cardiomyocytes in embryoid bodies derived from pluripotent embryonic stem (ES) cells were investigated. A single direct current (DC) field pulse was applied to 4-day-old embryoid bodies. The electrical field induced a hyperpolarization of the anode-facing side of embryoid bodies and a depolarization at the cathode-facing side. Significant effects of a single electrical field pulse applied for 90 s on cardiomyocyte differentiation were achieved with field strengths of 250 and 500 V/m, which increased both the number of embryoid bodies differentiating beating foci of cardiomyocytes and the size of the beating foci. The 500-V/m electrical field increased intracellular reactive oxygen species (ROS), but not [Ca(2+)](i) and activated nuclear factor kappa B (NF-kappaB). A comparable increase in the number of beating embryoid bodies was achieved by an incubation for 1 h with H(2)O(2) (1-10 nM), indicating that the electrical field effect was transduced via the intracellular generation of ROS. Because the radical scavengers dehydroascorbate and pyrrolidinedithiocarbamate (APDC) and the NF-kappaB antagonist N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) inhibited cardiac differentiation, we assume that ROS and NF-kappaB may play a role in early cardiac development.     

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.     

Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells

Many applications of human embryonic stem cells (hESCs) will require fully defined growth and differentiation conditions including media devoid of fetal calf serum. To identify factors that control lineage differentiation we have analyzed a serum-free (SF) medium conditioned by the cell line END2, which efficiently induces hESCs to form cardiomyocytes. Firstly, we noted that insulin, a commonly used medium supplement, acted as a potent inhibitor of cardiomyogenesis in multiple hESC lines and was rapidly cleared by medium conditioning. In the presence of insulin or IGF-1, which also suppressed cardiomyocyte differentiation, the PI3/Akt pathway was activated in undifferentiated hESC, suggesting that insulin/IGF-1 effects were mediated by this signaling cascade. Time course analysis and quantitative RT-PCR revealed impaired expression of endoderm and mesoderm markers in the presence of insulin, particularly if added during early stages of hESC differentiation. Relatively high levels of the neural ectoderm marker Sox1 were expressed under these conditions. Secondly, comparative gene expression showed that two key enzymes in the prostaglandin I2 (PGI2) synthesis pathway were highly up-regulated in END2 cells compared with a related, but non-cardiogenic, cell line. Biochemical analysis confirmed 6-10-fold higher PGI2 levels in END2 cell-conditioned medium (END2-CM) vs. controls. Optimized concentrations of PGI2 in a fully synthetic, insulin-free medium resulted in a cardiogenic activity equivalent to END2-CM. Addition of the p38 mitogen-activated protein kinase-inhibitor SB203580, which we have shown previously to enhance hESC cardiomyogenesis, to these insulin-free and serum-free conditions resulted in a cardiomyocyte content of >10% in differentiated cultures without any preselection. This study represents a significant step toward developing scalable production for cardiomyocytes from hESC using clinically compliant reagents compatible with Good Manufacturing Practice.     

Chromatin organization and differentiation in embryonic stem cell models

Embryonic stem cells derived from mammalian embryos represent indispensable tools for mammalian genetics. Their key features--self-renewal and pluripotency--enable them, on the one hand, to be propagated in culture almost indefinitely and, on the other, to be used to study the molecular details of cell commitment and differentiation. In the past few years, it has become clear that chromatin and epigenetic modifications have a central role in maintaining the gene expression programs that are important for both self-renewal and cell commitment. Therefore, studies focused on the chromatin profiles of embryonic stem cells are likely to be very informative for understanding pluripotency and the process of differentiation, and ultimately for using embryonic stem cells as a tool for cell replacement therapy or as models for the study of genetic diseases, cancer progression or drug testing.     

胚胎干细胞来源的拟胚体分化早期血管平滑肌标志物的表达时相

目的:研究胚胎血管发育早期SMα-actin、SM22α、myocardin、平滑肌肌球蛋白重链(SMMHC)的表达规律,并初步探讨在此阶段血小板源性生长因子-BB(PDGF-BB)对血管平滑肌细胞(VSMCs)分化的影响。方法:采用转染平滑肌特异性蛋白SM22α启动子控制下表达增强型绿色荧光蛋白(GFP)报告基因载体的胚胎干细胞制备拟胚体(EBs),用免疫荧光染色、RT-PCR、Western blot分析SMα-actin、SM22α、myocardin、SMMHC的表达时相;然后分别用0μmol/L(对照组)、10μmol/L、50μmol/L AG1296(血小板源性生长因子受体抑制剂)处理EBs,观察三组SMα-actin、SM22α、myocardin、SMMHC在基因及蛋白水平上的表达变化。结果:胚胎血管发育早期SMα-actin、myocardin、SM22α、SMMHC分别在EBs第0(胚胎干细胞)、8、11、13d开始有表达。AG1296三种浓度处理后SMα-actin、myocardin、SM22α、SMMHC蛋白表达及myocardin、SM22α和SMMHC mRNA表达均无明显差异。结论:EBs发育过程中存在着自发的VSMCs分化,SMα-actin表达最早,依次为myocardin、SM22α、SMMHC;PDGF-BB对EBs分化早期VSMCs标志物表达的调控可能不是必要的。     

Electrophysiological properties of embryonic stem cell-derived neurons

In vitro generation of functional neurons from embryonic stem (ES) cells and induced pluripotent stem cells offers exciting opportunities for dissecting gene function, disease modelling, and therapeutic drug screening. To realize the potential of stem cells in these biomedical applications, a complete understanding of the cell models of interest is required. While rapid advances have been made in developing the technologies for directed induction of defined neuronal subtypes, most published works focus on the molecular characterization of the derived neural cultures. To characterize the functional properties of these neural cultures, we utilized an ES cell model that gave rise to neurons expressing the green fluorescent protein (GFP) and conducted targeted whole-cell electrophysiological recordings from ES cell-derived neurons. Current-clamp recordings revealed that most neurons could fire single overshooting action potentials; in some cases multiple action potentials could be evoked by depolarization, or occurred spontaneously. Voltage-clamp recordings revealed that neurons exhibited neuronal-like currents, including an outward current typical of a delayed rectifier potassium conductance and a fast-activating, fast-inactivating inward current, typical of a sodium conductance. Taken together, these results indicate that ES cell-derived GFP(+) neurons in culture display functional neuronal properties even at early stages of differentiation.     

Paracrine factors of vascular endothelial cells facilitate cardiomyocyte differentiation of mouse embryonic stem cells

For myocardial regeneration therapy, the low differentiation capability of functional cardiomyocytes sufficient to replace the damaged myocardial tissue is one of the major difficulties. Using Nkx2.5-GFP knock-in ES cells, we show a new efficient method to obtain cardiomyocytes from embryonic stem (ES) cells. The proportion of GFP-positive cells was significantly increased when ES cells were cultured with a conditioned medium from aortic endothelial cells (ECs), accompanied by upregulation of cardiac-specific genes as well as other mesodermal genes. The promotion was more prominent when EC-conditioned medium was added at an early stage of ES cell differentiation culture (Day 0-3). Inhibitors of bone morphogenic protein (BMP), cyclooxygenase (COX), and nitric oxide synthetase (NO) prevented the promotion of cardiomyogenesis by EC-conditioned medium. These results suggest that supplementation of EC-conditioned medium enables cardiomyocytes to be obtained efficiently through promotion of mesoderm induction, which is regulated by BMP, COX, and NOS.     

Fibroblast growth factor-10 promotes cardiomyocyte differentiation from embryonic and induced pluripotent stem cells

Background

The fibroblast growth factor (FGF) family is essential to normal heart development. Yet, its contribution to cardiomyocyte differentiation from stem cells has not been systemically studied. In this study, we examined the mechanisms and characters of cardiomyocyte differentiation from FGF family protein treated embryonic stem (ES) cells and induced pluripotent stem (iPS) cells.

Methodology/Principal Findings

We used mouse ES cells stably transfected with a cardiac-specific α-myosin heavy chain (αMHC) promoter-driven enhanced green fluorescent protein (EGFP) and mouse iPS cells to investigate cardiomyocyte differentiation. During cardiomyocyte differentiation from mouse ES cells, FGF-3, -8, -10, -11, -13 and -15 showed an expression pattern similar to the mesodermal marker Brachyury and the cardiovascular progenitor marker Flk-1. Among them, FGF-10 induced cardiomyocyte differentiation in a time- and concentration-dependent manner. FGF-10 neutralizing antibody, small molecule FGF receptor antagonist PD173074 and FGF-10 and FGF receptor-2 short hairpin RNAs inhibited cardiomyocyte differentiation. FGF-10 also increased mouse iPS cell differentiation into cardiomyocyte lineage, and this effect was abolished by FGF-10 neutralizing antibody or PD173074. Following Gene Ontology analysis, microarray data indicated that genes involved in cardiac development were upregulated after FGF-10 treatment. In vivo, intramyocardial co-administration of FGF-10 and ES cells demonstrated that FGF-10 also promoted cardiomyocyte differentiation.

Conclusion/Significance

FGF-10 induced cardiomyocyte differentiation from ES cells and iPS cells, which may have potential for translation into clinical applications.     

Transient inhibition of BMP signaling by Noggin induces cardiomyocyte differentiation of mouse embryonic stem cells

Embryonic stem (ES) cells are a promising source of cardiomyocytes, but clinical application of ES cells has been hindered by the lack of reliable selective differentiation methods. Differentiation into any lineage is partly dependent on the regulatory mechanisms of normal early development. Although several signals, including bone morphogenetic protein (BMP), Wnt and FGF, are involved in heart development, scarce evidence is available about the exact signals that mediate cardiomyocyte differentiation. While investigating the involvement of BMP signaling in early heart formation in the mouse, we found that the BMP antagonist Noggin is transiently but strongly expressed in the heart-forming region during gastrulation and acts at the level of induction of mesendoderm to establish conditions conducive to cardiogenesis. We applied this finding to develop an effective protocol for obtaining cardiomyocytes from mouse ES cells by inhibition of BMP signaling.     

小鼠胚胎干细胞移植入成体大鼠脑内的区域特异性存活与分化

全能区域非特异性的胚胎干细胞是研究成体不同脑区控制干细胞分化能力的十分有力的工具。胚胎干细胞源性神经前体细胞移植入成体脑后可分化为功能性神经元,但是未分化的胚胎干细胞在成体脑内各个部位的存活、生长与分化的潜能差异尚不清楚。本文旨在探讨成体脑组织对胚胎干细胞的影响及胚胎干细胞在成体脑内的一系列行为。将少量转绿色荧光蛋白未分化的小鼠胚胎干细胞移植入成体大鼠脑内不同部位,分别于移植5、14和28d后处死大鼠,进行形态学观察及免疫组化定性,以了解未分化的小鼠胚胎干细胞在大鼠脑内不同区域的存活、生长与分化。结果发现未分化的小鼠胚胎干细胞可逐步整合入受体组织并向nestin阳性神经前体细胞分化。移植细胞及其后裔在海马生长最为旺盛,而在隔区最差（P〈0．01）;移植细胞分化为神经干细胞的效率也是在海马最高,而在隔区最低（P〈0．01）。提示只有部分脑区适合胚胎干细胞及其后裔生存,并提供促进其分化的有益环境。因此,由于位置特异的微环境因子及环境因素的存在,宿主组织特性对决定中枢神经系统疾病的细胞替代疗法策略是相当重要的。     

Modulation of the pentose phosphate pathway induces endodermal differentiation in embryonic stem cells

Embryonic stem (ES) cells can differentiate in vitro into a variety of cell types. Efforts to produce endodermal cell derivatives, including lung, liver and pancreas, have been met with modest success. Understanding how the endoderm originates from ES cells is the first step to generate specific cell types for therapeutic purposes. Recently, it has been demonstrated that inhibition of Myc or mTOR induces endodermal differentiation. Both Myc and mTOR are known to be activators of the Pentose Phosphate Pathway (PPP). We found that, differentely from wild type (wt), ES cells unable to produce pentose sugars through PPP differentiate into endodermal precursors in cell culture conditions generally non-permissive to generate them. The same effect was observed when wt ES cells were differentiated in presence of chemical inhibitors of the PPP. These data highlight a new role for metabolism. Indeed, to our knowledge, it is the first time that modulation of a metabolic pathway is described to be crucial in determining ES cell fate.