N-cadherin is essential for retinoic acid-mediated cardiomyogenic differentiation in mouse embryonic stem cells

Contraction forces developed by cardiomyocytes are transmitted across the plasma membrane through end-to-end connections between the myocytes, called intercalated disks, which enable the coordinated contraction of heart muscle. A component of the intercalated disk, the adherens junction, consists of the cell adhesion molecule, N-cadherin. Embryos lacking N-cadherin die at mid-gestation from cardiovascular abnormalities. We have evaluated the role of N-cadherin in cardiomyogenesis using N-cadherin-null mouse embryonic stem (ES) cells grown as embryoid bodies (EBs) in vitro. Myofibrillogenesis, the spatial orientation of myofibers, and intercellular contacts including desmosomes were normal in N-cadherin-null ES cell-derived cardiomyocytes. The effect of retinoic acid (RA), a stage and dose-dependent cardiogenic factor, was assessed in differentiating ES cells. all-trans (at) RA increased the number of ES cell-derived cardiomyocytes by approximately 3-fold (at 3 x 10(-9) M) in wt EBs. However, this effect was lost in N-cadherin-null EBs. In the presence of supplemented at-RA, the emergence of spontaneously beating cardiomyocytes appeared to be delayed and slightly less efficient in N-cadherin-null compared with wt and heterozygous EBs (frequencies of EBs with beating activity at 5 days: 54+/-18% vs. 96+/-0.5%, and 93+/-7%, respectively; peak frequencies of EBs with beating activity: 83+/-8% vs. 96+/-0.5% and 100%, respectively). In conclusion, cardiomyoyctes differentiating from N-cadherin-null ES cells in vitro show normal myofibrillogenesis and intercellular contacts, but impaired responses to early cardiogenic effects mediated by at-RA. These results suggest that N-cadherin may be essential for RA-induced cardiomyogenesis in mouse ES cells in vitro.     

Electron microscopic study of mouse embryonic stem cell-derived cardiomyocytes

Differentiation of embryonic stem cell (ESC)-derived embryoid bodies (EBs) is a heterogeneous process. ESCs can differentiate in vitro into different cell types including beating cardiomyocytes. The main aim of the present study was to develop an improved preparation method for scanning electron microscopic study of ESC-derived cardiac bundles and to investigate the fine structural characteristics of mouse ESCs-derived cardiomyocytes using electron microscopy. The mouse ESCs differentiation was induced by EBs’ development through hanging drop, suspension and plating stages. Cardiomyocytes appeared in the EBs’ outgrowth as beating clusters that grew in size and formed thick branching bundles gradually. Cardiac bundles showed cross striation even when they were observed under an inverted microscope. They showed a positive immunostaining for cardiac troponin I and α-actinin. Transmission and scanning electron microscopy (TEM & SEM) were used to study the structural characteristics of ESC-derived cardiomyocytes. Three weeks after plating, differentiated EBs showed a superficial layer of compact fibrous ECM that made detailed observation of cardiac bundles impossible. We tried several preparation methods to remove unwanted cells and fibers, and finally we revealed the branching bundles of cardiomyocytes. In TEM study, most cardiomyocytes showed parallel arrays of myofibrils with a mature sarcomeric organization marked by H-bands, M-lines and numerous T-tubules. Cardiomyocytes were connected to each other by intercalated discs composed of numerous gap junctions and fascia adherences.     

Molecular and pharmacological properties of human embryonic stem cell-derived cardiomyocytes

Human embryonic stem cells (hESCs) can be coaxed to differentiate into specific cell types, including cardiomyocyte-like cells. These cells express cardiac-specific markers and display functional similarities to their adult counterparts. Based on these properties, hESC-derived cardiomyocytes have the potential to be extremely useful in various in vitro applications and to provide the opportunity for cardiac cell replacement therapies. However, before this can become a reality, the molecular and functional characteristics of these cells need to be investigated in more detail. In the present study we differentiate hESCs into cardiomyocyte-like cells via embryoid bodies (EBs). The fraction of spontaneously beating clusters obtained from the EBs averaged approximately 30% of the total number of EBs used. These cell clusters were isolated, dissociated into single-cell suspensions, and frozen for long-term storage. The cryopreserved cells could be successfully thawed and subcultured. Using electron microscopy, we observed Z discs and tight junctions in the hESC-derived cardiomyocytes, and by immunohistochemical analysis we detected expression of cardiac-specific markers (cTnI and cMHC). Notably, using BrdU labeling we also could demonstrate that some of the hESC-derived cardiomyocytes retain a proliferative capacity. Furthermore, pharmacological stimulation of the cells resulted in responses indicative of functional adrenergic and muscarinic receptor coupling systems. Taken together, these results lend support to the notion that hESCs can be used as a source for the procurement of cardiomyocytes for in vitro and in vivo applications.     

Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A

Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1(+) common progenitor cells, and identified highly cardiogenic progenitors as Flk1(+)/CXCR4(+)/VE-cadherin(-) (FCV) cells. We have also reported that cyclosporin-A (CSA) drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1(+) cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1(+) cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs.     

Transforming growth factor-beta2 enhances differentiation of cardiac myocytes from embryonic stem cells

Stem cell therapy holds great promise for the treatment of injured myocardium, but is challenged by a limited supply of appropriate cells. Three different isoforms of transforming growth factor-beta (TGF-beta) -beta1, -beta2, and -beta3 exhibit distinct regulatory effects on cell growth, differentiation, and migration during embryonic development. We compared the effects of these three different isoforms on cardiomyocyte differentiation from embryonic stem (ES) cells. In contrast to TGF-beta1, or -beta3, treatment of mouse ES cells with TGF-beta2 isoform significantly increased embryoid body (EB) proliferation as well as the extent of the EB outgrowth that beat rhythmically. At 17 days, 49% of the EBs treated with TGF-beta2 exhibited spontaneous beating compared with 15% in controls. Cardiac myocyte specific protein markers sarcomeric myosin and alpha-actin were demonstrated in beating EBs and cells isolated from EBs. In conclusion, TGF-beta2 but not TGF-beta1, or -beta3 promotes cardiac myocyte differentiation from ES cells.     

Establishment of a human embryonic germ cell line and comparison with mouse and human embryonic stem cells

Human embryonic stem (ES) cells and embryonic germ (EG) cells are pluripotent and are invaluable material for in vitro studies of human embryogenesis and cell therapy. So far, only two groups have reported the establishment of human EG cell lines, whereas at least five human ES cell lines have been established. To see if human EG cell lines can be reproducibly established, we isolated primordial germ cells (PGCs) from gonadal ridges and mesenteries (9 weeks post-fertilization) and cultured them on mouse STO cells. As with mouse ES colonies, the PGC-derived cells have given rise to multilayered colonies without any differentiation over a year of continuous culture. They are karyotypically normal and express high levels of alkaline phosphatase, Oct-4, and several cell-surface markers. Histological and immunocytochemical analysis of embryoid bodies (EBs) formed from floating cultures of the PGC-derived cell colonies revealed ectodermal, endodermal, and mesodermal tissues. When the EBs were cultured in the presence of insulin, transferrin, sodium selenite, and fibronectin for 1 week, markers of primitive neuroectoderm were expressed in cells within the EBs as well as in cells growing out from the EBs. These observations indicate that our PGC-derived cells satisfy the criteria for pluripotent stem cells and hence may be EG cells.     

Highly efficient derivation of ventricular cardiomyocytes from induced pluripotent stem cells with a distinct epigenetic signature

Cardiomyocytes derived from pluripotent stem cells can be applied in drug testing, disease modeling and cell-based therapy. However, without procardiogenic growth factors, the efficiency of cardiomyogenesis from pluripotent stem cells is usually low and the resulting cardiomyocyte population is heterogeneous. Here, we demonstrate that induced pluripotent stem cells (iPSCs) can be derived from murine ventricular myocytes (VMs), and consistent with other reports of iPSCs derived from various somatic cell types, VM-derived iPSCs (ViPSCs) exhibit a markedly higher propensity to spontaneously differentiate into beating cardiomyocytes as compared to genetically matched embryonic stem cells (ESCs) or iPSCs derived from tail-tip fibroblasts. Strikingly, the majority of ViPSC-derived cardiomyocytes display a ventricular phenotype. The enhanced ventricular myogenesis in ViPSCs is mediated via increased numbers of cardiovascular progenitors at early stages of differentiation. In order to investigate the mechanism of enhanced ventricular myogenesis from ViPSCs, we performed global gene expression and DNA methylation analysis, which revealed a distinct epigenetic signature that may be involved in specifying the VM fate in pluripotent stem cells.     

长期培养小鼠胚胎干细胞拟胚体(EB)的观察

胚胎干细胞在体外培养条件下能够维持自我更新,并具有向多种细胞类型分化的能力,因此被广泛用于研究细胞分化的分子机理以及药物筛选.形成拟胚体(Embryoid body,EB)是胚胎干细胞分化常用的技术手段.为了便于今后利用EB做进一步的药物筛选及分化研究,严格规范了形成EB的条件,得到了分化状态均一性很高的EB.利用这一条件,观察到在分化条件下长期培养(长达60 d)的EB中仍有表达各项多能性指标的细胞集落.有关这一现象的进一步分析工作正在进行中.     

Evaluation of hollow fiber culture for large-scale production of mouse embryonic stem cell-derived hematopoietic stem cells

Hematopoietic stem cells (HSCs) have the ability to differentiate into all types of blood cells and can be transplanted to treat blood disorders. However, it is difficult to obtain HSCs in large quantities because of the shortage of donors. Recent efforts have focused on acquiring HSCs by differentiation of pluripotent stem cells. As a conventional differentiation method of pluripotent stem cells, the formation of embryoid bodies (EBs) is often employed. However, the size of EBs is limited by depletion of oxygen and nutrients, which prevents them from being efficient for the production of HSCs. In this study, we developed a large-scale hematopoietic differentiation approach for mouse embryonic stem (ES) cells by applying a hollow fiber (HF)/organoid culture method. Cylindrical organoids, which had the potential for further spontaneous differentiation, were established inside of hollow fibers. Using this method, we improved the proliferation rate of mouse ES cells to produce an increased HSC population and achieved around a 40-fold higher production volume of HSCs in HF culture than in conventional EB culture. Therefore, the HF/organoid culture method may be a new mass culture method to acquire pluripotent stem cell-derived HSCs.     

In vitro differentiation of mouse embryonic stem cells after activation by retinoic acid

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of blastocysts. ES cells are able to differentiate into the three primitive layers (endoderm, mesoderm, and ectoderm) of the organism, including the germline. In recent reports mouse ES cells have been successfully applied in the treatment of spinal cord injury, hereditary myelin disorder of the central nervous system, and diabetes mellitus. In this study, we investigated the induction of mouse ES cell differentiation, using culture of embryoid bodies (EBs) into the diverse tissues. EBs were formed by culturing ES cells (129/SV strain) in DMEM supplemented with 10% FBS, in the absence of feeder cells and leukemia inhibitory factor (LF). EBs were induced to differentiate by treatment with retinoic acid (RA). In control medium (non-RA medium) beating muscles, blood vessels, hemocytes, and cartilages were frequently observed in EBs. Moreover, when EBs were cultured in medium including RA (5 x 10(-8) M, and 5 x 10(-9) M), differentiation of the optic vesicle, lens, retina, and neural groove was observed. In this study we demonstrated that an efficient system for inducing the differentiation of ES cells using EBs.     

PPARgamma-dependent and PPARgamma-independent effects on the development of adipose cells from embryonic stem cells.

Peroxisome proliferator-activated receptor (PPAR) gamma was shown to be required for adipocyte formation both in vivo and in vitro. However, the role of PPARgamma in the initial steps of adipose cell development was not distinguished from its role in the terminal steps. We now show that PPARgamma is expressed early in embryoid bodies (EBs) derived from embryonic stem cells and in E.8.5 mouse embryos. Addition of a specific ligand for PPARgamma in developing EBs over-expressing PPARgamma did not commit stem cells towards the adipose lineage. In differentiated PPARgamma(-/-) EBs, only markers characteristic of preadipocytes were found to be expressed. PPARdelta is present in EBs but did not compensate for the lack of PPARgamma in terminal differentiation. Taken together, these results favor a critical PPARgamma-independent phase culminating in preadipocyte formation that precedes a PPARgamma-dependent phase in the development of adipose cells from pluripotent stem cells.     

In vitro differentiation of rat embryonic stem cells into functional cardiomyocytes

The recent breakthrough in the generation of rat embryonic stem cells (rESCs) opens the door to application of gene targeting to create models for the study of human diseases. In addition, the in vitro differentiation system from rESCs into derivatives of three germ layers will serve as a powerful tool and resource for the investigation of mammalian development, cell function, tissue repair, and drug discovery. However, these uses have been limited by the difficulty of in vitro differentiation. The aims of this study were to establish an in vitro differentiation system from rESCs and to investigate whether rESCs are capable of forming terminal-differentiated cardiomyocytes. Using newly established rESCs, we found that embryoid body (EB)-based method used in mouse ESC (mESC) differentiation failed to work for the serum-free cultivated rESCs. We then developed a protocol by combination of three chemical inhibitors and feeder-conditioned medium. Under this condition, rESCs formed EBs, propagated and differentiated into three embryonic germ layers. Moreover, rESC-formed EBs could differentiate into spontaneously beating cardiomyocytes after plating. Analyses of molecular, structural, and functional properties revealed that rESC-derived cardiomyocytes were similar to those derived from fetal rat hearts and mESCs. In conclusion, we successfully developed an in vitro differentiation system for rESCs through which functional myocytes were generated and displayed phenotypes of rat fetal cardiomyocytes. This unique cellular system will provide a new approach to study the early development and cardiac function, and serve as an important tool in pharmacological testing and cell therapy.     

Efficient generation of transgene- and feeder-free induced pluripotent stem cells from human dental mesenchymal stem cells and their chemically defined differentiation into cardiomyocytes

Advance in stem cell research resulted in several processes to generate induced pluripotent stem cells (iPSCs) from adult somatic cells. In our previous study, the reprogramming of iPSCs from human dental mesenchymal stem cells (MSCs) including SCAP and DPSCs, has been reported. Herein, safe iPSCs were reprogrammed from SCAP and DPSCs using non-integrating RNA virus vector, which is an RNA virus carrying no risk of altering host genome. DPSCs- and SCAP-derived iPSCs exhibited the characteristics of the classical morphology with human embryonic stem cells (hESCs) without integration of foreign genes, indicating the potential of their clinical application. Moreover, induced PSCs showed the capacity of self-renewal and differentiation into cardiac myocytes. We have achieved the differentiation of hiPSCs to cardiomyocytes lineage under serum and feeder-free conditions, using a chemically defined medium CDM3. In CDM3, hiPSCs differentiation is highly generating cardiomyocytes. The results showed this protocol produced contractile sheets of up to 97.2% TNNT2 cardiomyocytes after purification. Furthermore, derived hiPSCs differentiated to mature cells of the three embryonic germ layers in vivo and in vitro of beating cardiomyocytes. The above whole protocol enables the generation of large scale of highly pure cardiomyocytes as needed for cellular therapy.     

MiR-218 regulated cardiomyocyte differentiation and migration in mouse embryonic stem cells by targeting PDGFRα

MicroRNAs (miRNAs) have been identified as key players in cardiogenesis and heart pathophysiological processes. However, many miRNAs are still not recognized for their roles in cardiomyocytes differentiation. In this study, we evaluated the effects of microRNA-218 (miR-218) in cardiomyocyte differentiation of the mouse embryonic stem cells (ESCs) in vitro. The percentage of the beating embryoid bodies (EBs) in miR-218 mimic-treated cells was reduced to 32% compared with miR-218 mimic negative control (56%) on day 5 + 3. The amplitude of the intracellular Ca²⁺ transients in the cardiomyocytes derived from ESCs was reduced upon miR-218 overexpression, followed by the decreased calcium-related proteins and cell junction proteins expressions. Besides, miR-218 expression in ESCs was related to the directional spreading ability of EBs during differentiation. The increased expression of miR-218 could promote the migration of ESCs in vitro, while the decreased expression of miR-218 could inhibit the migration by the transwell experiment. Meanwhile, miR-218 could regulate cell migration–related proteins Cdc42 and Rac1. Platelet-derived growth factor receptor α (PDGFRα) was further confirmed to be a direct target of miR-218 both physically and functionally by dual-luciferase reporter assay. Our data further described that overexpression of PDGFRα rescued the miR-218-mediated inhibition of cardiomyocyte differentiation and restored the miR-218-mediated promotion of cell migration. In conclusion, miR-218 was demonstrated to exert an inhibitory function and promoted cell migration via targeting PDGFRα during cardiomyocyte differentiation from ESCs. The current study revealed the role of miR-218 and may provide an important hint for cardiomyocyte differentiation of ESCs and induced pluripotent stem cells.