Cardioblast-intrinsic Tinman activity controls proper diversification and differentiation of myocardial cells in Drosophila

The NK homeobox gene tinman (tin) is required for the specification of the cardiac, visceral muscle and somatic muscle progenitors in the early dorsal mesoderm of Drosophila. Like its vertebrate counterpart Nkx2.5, the expression of tin is maintained in cardiac cells during cardiac maturation and differentiation; however, owing to the complete lack of a dorsal vessel in tin mutant embryos, the function of tin in these cells has not been defined. Here we show that myocardial cells and dorsal vessels can form even though they lack Tin, and that viable adults can develop, as long as Tin is provided in the embryonic precardiac mesoderm. However, embryos in which tin expression is specifically missing from cardial cells show severe disruptions in the normal diversification of the myocardial cells, and adults exhibit severe defects in cardiac remodeling and function. Our study reveals that the normal expression and activity of Tin in four of the six bilateral cardioblasts within each hemisegment of the heart allows these cells to adopt a cell fate as ;working' myocardium, as opposed to a fate as inflow tract (ostial) cells. This function of tin involves the repression of Dorsocross (Doc) T-box genes and, hence, the restriction of Doc to the Tin-negative cells that will form ostia. We conclude that tin has a crucial role within myocardial cells that is required for the proper diversification, differentiation, and post-embryonic maturation of cardiomyocytes, and we present a pathway involving regulatory interactions among seven-up, midline, tinman and Dorsocross that establishes these developmental events upon myocardial cell specification.     

Hensen's node gives rise to the ventral midline of the foregut: implications for organizing head and heart development

Patterning of the ventral head has been attributed to various cell populations, including endoderm, mesoderm, and neural crest. Here, we provide evidence that head and heart development may be influenced by a ventral midline endodermal cell population. We show that the ventral midline endoderm of the foregut is generated directly from the extreme rostral portion of Hensen's node, the avian equivalent of the Spemann organizer. The endodermal cells extend caudally in the ventral midline from the prechordal plate during development of the foregut pocket. Thus, the prechordal plate appears as a mesendodermal pivot between the notochord and the ventral foregut midline. The elongating ventral midline endoderm delimits the right and left sides of the ventral foregut endoderm. Cells derived from the midline endoderm are incorporated into the endocardium and myocardium during closure of the foregut pocket and fusion of the bilateral heart primordia. Bilateral ablation of the endoderm flanking the midline at the level of the anterior intestinal portal leads to randomization of heart looping, suggesting that this endoderm is partitioned into right and left domains by the midline endoderm, thus performing a function similar to that of the notochord in maintaining left-right asymmetry. Because of its derivation from the dorsal organizer, its extent from the forebrain through the midline of the developing face and pharynx, and its participation in formation of a single midline heart tube, we propose that the ventral midline endoderm is ideally situated to function as a ventral organizer of the head and heart.     

The role of stem cells in cardiac regeneration

After myocardial infarction, injured cardiomyocytes are replaced by fibrotic tissue promoting the development of heart failure. Cell transplantation has emerged as a potential therapy and stem cells may be an important and powerful cellular source. Embryonic stem cells can differentiate into true cardiomyocytes, making them in principle an unlimited source of transplantable cells for cardiac repair, although immunological and ethical constraints exist. Somatic stem cells are an attractive option to explore for transplantation as they are autologous, but their differentiation potential is more restricted than embryonic stem cells. Currently, the major sources of somatic cells used for basic research and in clinical trials originate from the bone marrow. The differentiation capacity of different populations of bone marrow-derived stem cells into cardiomyocytes has been studied intensively. The results are rather confusing and difficult to compare, since different isolation and identification methods have been used to determine the cell population studied. To date, only mesenchymal stem cells seem to form cardiomyocytes, and only a small percentage of this population will do so in vitro or in vivo. A newly identified cell population isolated from cardiac tissue, called cardiac progenitor cells, holds great potential for cardiac regeneration. Here we discuss the potential of the different cell populations and their usefulness in stem cell based therapy to repair the damaged heart.     

Smarcd3b and Gata5 promote a cardiac progenitor fate in the zebrafish embryo

Development of the heart requires recruitment of cardiovascular progenitor cells (CPCs) to the future heart-forming region. CPCs are the building blocks of the heart, and have the potential to form all the major cardiac lineages. However, little is known regarding what regulates CPC fate and behavior. Activity of GATA4, SMARCD3 and TBX5 - the `cardiac BAF' (cBAF) complex, can promote myocardial differentiation in embryonic mouse mesoderm. Here, we exploit the advantages of the zebrafish embryo to gain mechanistic understanding of cBAF activity. Overexpression of smarcd3b and gata5 in zebrafish results in an enlarged heart, whereas combinatorial loss of cBAF components inhibits cardiac differentiation. In transplantation experiments, cBAF acts cell autonomously to promote cardiac fate. Remarkably, cells overexpressing cBAF migrate to the developing heart and differentiate as cardiomyocytes, endocardium and smooth muscle. This is observed even in host embryos that lack endoderm or cardiac mesoderm. Our results reveal an evolutionarily conserved role for cBAF activity in cardiac differentiation. Importantly, they demonstrate that Smarcd3b and Gata5 can induce a primitive, CPC-like state.     

Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart

Arodent cardiac side population cell fraction formed clonal spheroids in serum-free medium, which expressed nestin, Musashi-1, and multi-drug resistance transporter gene 1, markers of undifferentiated neural precursor cells. These markers were lost following differentiation, and were replaced by the expression of neuron-, glial-, smooth muscle cell-, or cardiomyocyte-specific proteins. Cardiosphere-derived cells transplanted into chick embryos migrated to the truncus arteriosus and cardiac outflow tract and contributed to dorsal root ganglia, spinal nerves, and aortic smooth muscle cells. Lineage studies using double transgenic mice encoding protein 0-Cre/Floxed-EGFP revealed undifferentiated and differentiated neural crest-derived cells in the fetal myocardium. Undifferentiated cells expressed GATA-binding protein 4 and nestin, but not actinin, whereas the differentiated cells were identified as cardiomyocytes. These results suggest that cardiac neural crest-derived cells migrate into the heart, remain there as dormant multipotent stem cells-and under the right conditions-differentiate into cardiomyocytes and typical neural crest-derived cells, including neurons, glia, and smooth muscle.     

Persistent expression of the ATP-binding cassette transporter, Abcg2, identifies cardiac SP cells in the developing and adult heart

Stem cells are important in the maintenance and repair of adult tissues. A population of cells, termed side population (SP) cells, has stem cell characteristics as they have been shown to contribute to diverse lineages. In this study, we confirm that Abcg2 is a determinant of the SP cell phenotype. Therefore, we examined Abcg2 expression during murine embryogenesis and observed robust expression in the blood islands of the E8.5 yolk sac and in developing tissues including the heart. During the latter stages of embryogenesis, Abcg2 identifies a rare cell population in the developing organs. We further establish that the adult heart contains an Abcg2 expressing SP cell population and these progenitor cells are capable of proliferation and differentiation. We define the molecular signature of cardiac SP cells and compare it to embryonic stem cells and adult cardiomyocytes using emerging technologies. We propose that the cardiac SP cell population functions as a progenitor cell population for the development, maintenance, and repair of the heart.     

Cardiomyocytes re-enter the cell cycle and contribute to heart development after differentiation from cardiac progenitors expressing Isl1 in chick embryo

Cardiomyocytes are generated from the precardiac mesoderm and the size of the heart increases dramatically during embryogenesis. However, it is unclear how differentiation and proliferation correlate in the cardiac cell line during development. Here, we show that cardiomyocytes re-entered into a proliferative state after differentiation with a concomitant cell cycle arrest in chick embryo. The cells in the course of differentiation from Isl1-positive cardiac precursors to cardiomyocytes did not proliferate, but differentiated cardiomyocytes proliferated even after the acquisition of contractile function. After differentiation, cardiomyocytes developed a proliferative potential to contribute to the increase in cell numbers during heart development. Almost all differentiated cardiomyocytes (82.8%) incorporated bromodeoxyuridine (BrdU) in vitro, indicating the ability of DNA replication. Furthermore, mitotic chromosomes were observed in the cardiomyocytes in which a sarcomeric structure was sustained in the cytoplasm. We conclude that the sequential events of the differentiation to contractile myocytes and the re-entry into the cell cycle are strictly regulated during cardiac cell maturation. These results provide an insight into the maturation mechanism of the cardiac cell line.     

Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart

Despite recent advances in delineating the mechanisms involved in cardiogenesis, cellular lineage specification remains incompletely understood. To explore the relationship between developmental fate and potential, we isolated a cardiac-specific Nkx2.5(+) cell population from the developing mouse embryo. The majority of these cells differentiated into cardiomyocytes and conduction system cells. Some, surprisingly, adopted a smooth muscle fate. To address the clonal origin of these lineages, we isolated Nkx2.5(+) cells from in vitro differentiated murine embryonic stem cells and found approximately 28% of these cells expressed c-kit. These c-kit(+) cells possessed the capacity for long-term in vitro expansion and differentiation into both cardiomyocytes and smooth muscle cells from a single cell. We confirmed these findings by isolating c-kit(+)Nkx2.5(+) cells from mouse embryos and demonstrated their capacity for bipotential differentiation in vivo. Taken together, these results support the existence of a common precursor for cardiovascular lineages in the mammalian heart.     

The dorsal neural tube: A dynamic setting for cell fate decisions

The dorsal neural tube first generates neural crest cells that exit the neural primordium following an epithelial‐to‐mesenchymal conversion to become sympathetic ganglia, Schwann cells, dorsal root sensory ganglia, and melanocytes of the skin. Following the end of crest emigration, the dorsal midline of the neural tube becomes the roof plate, a signaling center for the organization of dorsal neuronal cell types. Recent lineage analysis performed before the onset of crest delamination revealed that the dorsal tube is a highly dynamic region sequentially traversed by fate‐restricted crest progenitors. Furthermore, prospective roof plate cells were shown to originate ventral to presumptive crest and to progressively relocate dorsalward to occupy their definitive midline position following crest delamination. These data raise important questions regarding the mechanisms of cell emigration in relation to fate acquisition, and suggest the possibility that spatial and/or temporal information in the dorsal neural tube determines initial segregation of neural crest cells into their derivatives. In addition, they emphasize the need to address what controls the end of neural crest production and consequent roof plate formation, a fundamental issue for understanding the separation between central and peripheral lineages during development of the nervous system. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 796–812, 2010.     

Vessel and blood specification override cardiac potential in anterior mesoderm

Organ progenitors arise within organ fields, embryonic territories that are larger than the regions required for organ formation. Little is known about the regulatory pathways that define organ field boundaries and thereby limit organ size. Here we identify a mechanism for restricting heart size through confinement of the developmental potential of the heart field. Via fate mapping in zebrafish, we locate cardiac progenitors within hand2-expressing mesoderm and demonstrate that hand2 potentiates cardiac differentiation within this region. Beyond the rostral boundary of hand2 expression, we find progenitors of vessel and blood lineages. In embryos deficient in vessel and blood specification, rostral mesoderm undergoes a fate transformation and generates ectopic cardiomyocytes. Therefore, induction of vessel and blood specification represses cardiac specification and delimits the capacity of the heart field. This regulatory relationship between cardiovascular pathways suggests strategies for directing progenitor cell differentiation to facilitate cardiac regeneration.     

A bipotential precursor population for pancreas and liver within the embryonic endoderm

The pancreas emerges independently from dorsal and ventral domains of embryonic gut endoderm. Gene inactivation experiments in mice have identified factors required for dorsal pancreas development, but factors that initiate the ventral pancreas have remained elusive. In this study, we investigated the hypothesis that the emergence of the ventral pancreas is related to the emergence of the liver. We find that the liver and ventral pancreas are specified at the same time and in the same general domain of cells. Using embryo tissue explantation experiments, we find that the default fate of the ventral foregut endoderm is to activate the pancreas gene program. FGF signalling from the cardiac mesoderm diverts this endoderm to express genes for liver instead of those for pancreas. No evidence was found to indicate that the cell type choice for pancreas or liver involves a selection for growth or viability. Cardiac mesoderm or FGF induces the local expression of sonic hedgehog, which in turn is inhibitory to pancreas but not to liver. The bipotential precursor cell population for pancreas and liver in embryonic development and its fate selection by FGF has features that appear to be recapitulated in the adult pancreas and are reflected in the evolution of these organs.     

A fate map of the murine pancreas buds reveals a multipotent ventral foregut organ progenitor

The definitive endoderm is the embryonic germ layer that gives rise to the budding endodermal organs including the thyroid, lung, liver and pancreas as well as the remainder of the gut tube. DiI fate mapping and whole embryo culture were used to determine the endodermal origin of the 9.5 days post coitum (dpc) dorsal and ventral pancreas buds. Our results demonstrate that the progenitors of each bud occupy distinct endodermal territories. Dorsal bud progenitors are located in the medial endoderm overlying somites 2-4 between the 2 and 11 somite stage (SS). The endoderm forming the ventral pancreas bud is found in 2 distinct regions. One territory originates from the left and right lateral endoderm caudal to the anterior intestinal portal by the 6 SS and the second domain is derived from the ventral midline of the endoderm lip (VMEL). Unlike the laterally located ventral foregut progenitors, the VMEL population harbors a multipotent progenitor that contributes to the thyroid bud, the rostral cap of the liver bud, ventral midline of the liver bud and the midline of the ventral pancreas bud in a temporally restricted manner. This data suggests that the midline of the 9.5 dpc thyroid, liver and ventral pancreas buds originates from the same progenitor population, demonstrating a developmental link between all three ventral foregut buds. Taken together, these data define the location of the dorsal and ventral pancreas progenitors in the prespecified endodermal sheet and should lead to insights into the inductive events required for pancreas specification.     

Fabrication of Mouse Embryonic Stem Cell-Derived Layered Cardiac Cell Sheets Using a Bioreactor Culture System

Bioengineered functional cardiac tissue is expected to contribute to the repair of injured heart tissue. We previously developed cardiac cell sheets using mouse embryonic stem (mES) cell-derived cardiomyocytes, a system to generate an appropriate number of cardiomyocytes derived from ES cells and the underlying mechanisms remain elusive. In the present study, we established a cultivation system with suitable conditions for expansion and cardiac differentiation of mES cells by embryoid body formation using a three-dimensional bioreactor. Daily conventional medium exchanges failed to prevent lactate accumulation and pH decreases in the medium, which led to insufficient cell expansion and cardiac differentiation. Conversely, a continuous perfusion system maintained the lactate concentration and pH stability as well as increased the cell number by up to 300-fold of the seeding cell number and promoted cardiac differentiation after 10 days of differentiation. After a further 8 days of cultivation together with a purification step, around 1×10⁸ cardiomyocytes were collected in a 1-L bioreactor culture, and additional treatment with noggin and granulocyte colony stimulating factor increased the number of cardiomyocytes to around 5.5×10⁸. Co-culture of mES cell-derived cardiomyocytes with an appropriate number of primary cultured fibroblasts on temperature-responsive culture dishes enabled the formation of cardiac cell sheets and created layered-dense cardiac tissue. These findings suggest that this bioreactor system with appropriate medium might be capable of preparing cardiomyocytes for cell sheet-based cardiac tissue.     

Growth and differentiation of human embryonic stem cells for cardiac cell replacement therapy

Due to the limited proliferation capacity of cardiac cells, cell replacement therapy has been proposed to restore cardiac function in patients suffering from ischemic heart disease and congestive heart failure. However, this approach is challenged by an insufficient supply of appropriate cells. Because of their apparent indefinite replicative capacity and their cardiac differentiation potential, human embryonic stem cells (hESCs) are potential candidates as sources of cells for cell replacement therapy. Significant progress has been made in improving culture conditions of undifferentiated hESCs, and using various methods, several laboratories have reported the generation of contracting cardiomyocytes from hESCs in vitro. Application of these cardiomyocytes to the clinic, however, still requires substantial experimentation to show that 1) they are functional in vitro; 2) they are efficacious in animal models of cardiac injury and disease; 3) they are safe and effective in human conditions, and 4) a sufficient amount of cardiomyocytes with expected characteristics can be generated in a reproducible manner. Here we review and discuss current findings on growth and differentiation of hESCs, and on characterization, enrichment and transplantation of hESC-derived cardiomyocytes.