Self-Organization Phenomena in Embryonic Stem Cell-Derived Embryoid Bodies: Axis Formation and Breaking of Symmetry during Cardiomyogenesis (original) (raw)
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Development
The cardiogenic potency of cells in the epiblast of the early primitive-streak stage (early PS) embryo was tested by heterotopic transplantation. The results of this study show that cells in the anterior and posterior epiblast of the early PS-stage embryos have similar cardiogenic potency, and that they differentiated to heart cells after they were transplanted directly to the heart field of the late PS embryo. That the epiblast cells can acquire a cardiac fate without any prior act of ingression through the primitive streak or movement within the mesoderm suggests that neither morphogenetic event is critical for the specification of the cardiogenic fate. The mesodermal cells that have recently ingressed through the primitive streak can express a broad cell fate that is characteristic of the pre-ingressed cells in the host when they were returned to the epiblast. However, mesoderm cells that have ingressed through the primitive streak did not contribute to the lateral plate mesoderm...
Cell Biology International, 2006
Embryonic stem cells (ESCs) are expected to become a powerful tool for future regenerative medicine and developmental biology due to their capacity for self-renewal and pluripotency. The present study involves characterization and particularly, the ultrastructure of ESC-derived cardiomyocytes (ESC-CMs). Spontaneously differentiated murine (C57BL/6) ESC-CMs were cultured for 21 days. At different stages, growth characteristics of the CMs were assessed by immunocytochemistry, RT-PCR, transmission electron microscopy, and by addition of chronotropic drugs. EB-derived spontaneously beating cells expressed markers characteristic of CMs including a-actinin, desmin, troponin I, sarcomeric myosin heavy chain (MHC), pan-cadherin, connexin 43, cardiac a-MHC, cardiac b-MHC, atrial natriuretic factor (ANF), and myosin light chain isoform-2V (MLC-2V) and responded to drugs in a maturation-and dose-dependent manner. At the ultrasructural level, maturation proceeded with increasing time in culture. In 7 þ 21 days CMs, all sarcomeric components, such as Z-discs, A-, I-and H-bands as well as M-lines, T-tubules, intercalated discs, and the sarcoplasmic reticulum were present. Our data suggest that ESCs can differentiate into functional mature CMs in vitro. Furthermore, ESC-CMs may provide an ideal model for the study of cardiomyocytic development and may be useful for cell therapy of various cardiac diseases. Ó
Electron microscopic study of mouse embryonic stem cell-derived cardiomyocytes
Cytotechnology, 2012
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 a-actinin. Transmission and scanning electron microscopy (TEM & SEM) were used to study the structural characteristics of ESCderived 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.
Establishment of cardiac cytoarchitecture in the developing mouse heart
Developmental Biology, 2006
Cardiomyocytes are characterized by an extremely well-organized cytoarchitecture. We investigated its establishment in the developing mouse heart with particular reference to the myofibrils and the specialized types of cell-cell contacts, the intercalated discs (ICD). Early embryonic cardiomyocytes have a polygonal shape with cell-cell contacts distributed circumferentially at the peripheral membrane and myofibrils running in a random orientation in the sparse cytoplasm between the nucleus and the plasma membrane. During fetal development, the cardiomyocytes elongate, and the myofibrils become aligned. The restriction of the ICD components to the bipolar ends of the cells is a much slower process and is achieved for adherens junctions and desmosomes only after birth, for gap junctions even later. By quantifying the specific growth parameters of prenatal cardiomyocytes, we were able to identify a previously unknown fetal phase of physiological hypertrophy. Our results suggest (1) that myofibril alignment, bipolarization and ICD restriction happen sequentially in cardiomyocytes, and (2) that increase of heart mass in the embryo is not only achieved by hyperplasia alone but also by volume increase of the individual cardiomyocytes (hypertrophy). These observations help to understand the mechanisms that lead to the formation of a functional heart during development at a cellular level.
Mechanisms of Development, 1993
Pluripotent embryonic stem cells (ESC, ES ceils) of line D3 were differentiated in vitro via embryo-like aggregates (embryoid bodies) of defined cell number into spontaneously beating cardiomyocytes. By using RT-PCR technique, a-and /3-cardiac myosin heavy chain (MHC) genes were found to be expressed in embryoid bodies of early to terminal differentiation stages. The exclusive expression of the/3-cardiac MHC gene detected in very early differentiated embryoid bodies proved to be dependent on the number of ES cells developing in the embryoid body. Cardiomyocytes enzymatically isolated from embryoid body outgrowths at different stages of development were further characterized by immunocytological and electrophysiological techniques. All cardiomyocytes appeared to be positive in immunofluorescence assays with monoclonal antibodies against cardiac-specific a-cardiac MHC, as well as muscle-specific sarcomeric myosin heavy chain and desmin. The patch-clamp technique allowed a more detailed characterization of the in vitro differentiated cardiomyocytes which were found to represent phenotypes corresponding to sinusnode, atrium or ventricle of the heart. The cardiac cells of early differentiated stage expressed pacemaker-like action potentials similar to those described for embryonic cardiomyocytes. The action potentials of terminally differentiated cells revealed shapes, pharmacological characteristics and hormonal regulation inherent to adult sinusnodal, atrial or ventricular cells. In cardiomyocytes of intermediate differentiation state, action potentials of very long duration (0.3-1 s) were found, which may represent developmentally controlled transitions between different types of action potentials. Therefore, the presented ES cell differentiation system permits the investigation of commitment and differentiation of embryonic cells into the cardiomyogenic lineage in vitro.
European Journal of Cell Biology, 1999
Mouse embryonic stem (ES) cells, when cultivated as embryoid bodies, differentiate in vitro into cardiomyocytes of ventricle·, atrium· and pacemaker· like cell types characterized by develop· mentally controlled expression of cardiac·specific genes, struc· tural proteins and ion channels. Using this model system, we show here, (1) that during cardiac myofibrillogenesis sarco· meric proteins are organized in a developmentally regulated manner following the order: titin (Z·disk), a·actinin, myome· sin, titin (M.band), myosin heavy chain, a'actin, cardiac tropo· nin T and M'protein, recapitulating the sarcomeric organization in the chicken embryonal heart in vivo. Our data support the view that the formation of I·Z·J complexes is developmentally delayed with respect to A·band assembly. We show (2) that the process of cardiogenic differentiation in vitro is influenced by medium components: Using a culture medium supplemented with glucose, amino acids, vitamins and selenium ions, we were able to increase the efficiency of cardiac differentiation of wild· type, as well as of Ih integrin·deficient Oh -/-) ES cells, and to improve the degree of organization of sarcomeric structures in wild· type and in III -/cardiac cells.