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A c-Kit (CD117) is a well-known cell surface marker for adult somatic stem cells. We harvested c-Kit-positive cardiac stem cells (CSCs) from adult rat hearts by performing magnetic-activated cell sorting (MACS) and subjected them to long-term bulk culture more than 40 times. We made 11 attempts to obtain c-Kit-positive cells from adult (6–8-month-old) rats. Our initial expectation was of obtaining cells with homogenous cardiac phenotypes. However, each CSC bulk culture expressed varying degrees of the genes and cell surface markers belonging to cardiac and other mesenchymal lineages. The results suggested that these CSCs retained multiple developmental potential to some extent. Consequently, we investigated these CSCs in detail, hoping to establish the regeneration method by using c-Kit-positive cardiac cells [1–12].

Heart disease is a leading cause of death in adults and children. We, and others, have described complex signaling, transcriptional, and translational networks that guide early differentiation of cardiac progenitors and later morphogenetic events during cardiogenesis. We found that networks of transcription factors and miRNAs function through intersecting positive and negative feedback loops to reinforce differentiation and proliferation decisions. We have utilized a combination of major cardiac regulatory factors to induce direct reprogramming of cardiac fibroblasts into cardiomyocyte-like cells with global gene expression and electrical activity similar to cardiomyocytes. The in vivo efficiency of reprogramming into cells that are more fully reprogrammed was greater than in vitro and resulted in improved cardiac function after injury. We have also identified a unique cocktail of transcription factors and small molecules that reprogram human fibroblasts into cardiomyocyte-like cells and are testing these in large animals. Knowledge regarding the early steps of cardiac differentiation in vivo has led to effective strategies to generate necessary cardiac cell types for regenerative approaches and may lead to new strategies for human heart disease.

The significance of the epicardium that covers the heart and the roots of the great arteries should not be underestimated as it is a major component with impact on development, disease, and repair. The epicardium differentiates from the proepicardial organ located at the venous pole (vPEO). The differentiation capacities of the vPEO into epicardium-derived cells (EPDCs) have been extensively described. A hitherto escaped part of the epicardium derives from a second proepicardial organ located at the arterial pole (aPEO) and covers the intrapericardial part of the aorta and pulmonary trunk. In avian and mouse embryos, disturbance of epicardium differentiation causes a spectrum of cardiac anomalies including coronary artery abnormalities, deficient annulus fibrosis with rhythm disturbances, valve malformations, and non-compaction cardiomyopathies. Late in prenatal life the epicardium becomes dormant, losing the activity of many genes.

In human cardiac diseases, both arterial and venous epicardium can be activated again into EPDCs. The epicardial reactivation observed after experimental myocardial infarction and during aneurysm formation of the ascending aorta provides clinical relevance. EPDCs applied for cell therapy demonstrate repair processes synergistic with the resident cardiac progenitor stem cells that probably share an embryonic origin with EPDCs. Future therapeutic strategies might be possible addressing cell autonomous-based and signaling capacities of the adult epicardium.

In recent years, regenerative medicine using cells for treating tissue defects has been in the spotlight as a new treatment for severe heart failure. Direct injection of bone marrow-derived cells and isolated skeletal myoblasts has already been used clinically as a method to improve cardiac function by regenerating cardiac muscle cells and blood vessels. The research on reconstructing functional three-dimensional (3D) cardiac grafts using tissue engineering methods has also now been addressed as a treatment for the next generation. Our laboratory has proposed an original tissue engineering technology called “cell sheet engineering” that stacks cell sheets to reconstruct functional 3D tissues. Transplantation of cell sheets has already shown it can cure damaged hearts, and it seems clear that the field of cell sheet tissue engineering can offer realistic treatment for patients with severe cardiac disorders.

Heart transplantation can drastically improve survival in patients with a failing heart; however, the shortage of donor hearts remains a serious problem with this treatment strategy and the successful clinical application of regenerative medicine is eagerly awaited. To this end, we developed a novel method to generate human induced pluripotent stem cells (iPSCs) from circulating human T lymphocytes using Sendai virus containing Yamanaka factors. To establish an efficient cardiac differentiation protocol, we then screened factors expressed in the future heart site of early mouse embryos and identified several growth factors and cytokines that can induce cardiomyocyte differentiation and proliferation. Subsequent transcriptome and metabolome analysis on undifferentiated stem cells and cardiomyocytes to devise a specific metabolic environment for cardiomyocyte selection revealed completely different mechanisms of glucose and lactate metabolism. Based on these findings, we succeeded in metabolically selecting cardiomyocytes using glucose-free and lactate-supplemented medium, with up to 99 % purity and no teratoma formation. Using our aggregation technique, we also showed that >90 % of the transplanted cardiomyocytes survived in the heart and showed physiological growth after transplantation. We expect that combining these techniques will achieve future heart regeneration.

In searching for remedial etiologies for congenital heart disease (CHD), we have focused on identifying interactive signaling pathways or “hubs” in which mutations disrupt fundamental cell biological functions in cardiac progenitor cells in a lineage-specific manner. Based on the frequency of heart defects seen in a clinical setting, we emphasize two signaling hubs – (e.g., ) , (). We discuss them in the context of valve and septal development and the lineages which give origin to their progenitor cells. We also explore developmental windows that are potentially amenable to remedial therapy using homeostatic mechanisms like those revealed by a chimeric mice model, i.e., + , that shows bone marrow-derived cells track to the heart, engraft, and give rise to bona fide fibroblasts. We propose to use this model to deliver genetic payloads or protein cargos during the neonatal period to override biochemical or structural deficits of CHD associated with valve and septal signaling hubs or fibroblast/myocyte interactions. Preliminary tests of the model indicate remedial potential for cardiac injuries.

Heterotaxy syndrome is characterized by a wide variety of cardiac and extra-cardiac congenital malformations that are primarily induced by disorders of the left-right axis determination during early embryonic development. Prognosis of the disease remains unsatisfactory because the syndrome is often associated with complicated congenital heart diseases. Long-term follow-up of heterotaxy patients, particularly those who underwent Fontan procedure, is now one of the most important issues in pediatric and adult congenital heart disease clinics. Collaborative studies between pediatric cardiologists and basic scientists are essential for improving the prognosis of heterotaxy syndrome.

Our body possesses three body axes, anteroposterior, dorsoventral, and left-right (L-R) axes. L-R asymmetry is achieved by three consecutive steps: symmetry breaking at the node, differential patterning of the lateral plate by a signaling molecule Nodal, and finally situs-specific organogenesis. Breaking of L-R symmetry in the mouse embryo takes place in the ventral node, where two types of cilia are found. Whereas centrally located motile cilia generate a leftward fluid flow, peripherally located immotile cilia sense a flow-dependent signal. Although Ca signaling is implicated in flow sensing, it is still not clear what triggers Ca signaling, a determinant molecule transported by the flow or mechanical force induced by the flow.

A central role for cilia in the pathogenesis of congenital heart disease was uncovered by our large-scale mouse mutagenesis screen for mutations causing congenital heart disease. This is supported by human clinical studies, which showed a high prevalence of ciliary dysfunction and respiratory symptoms and disease in patients with congenital heart disease. Our mouse studies indicate this involves essential roles for both primary and motile cilia in the pathogenesis of congenital heart disease. As laterality defects were also observed with high prevalence among the congenital heart disease mutants, this further suggested an important role for left-right patterning in the pathogenesis of congenital heart disease. This finding is reminiscent of the high prevalence of heterotaxy among human fetuses with congenital heart disease, indicating the fetal mouse screen may provide a window into the unborn human fetal population. Clinically, congenital heart disease patients with ciliary dysfunction were found to have more respiratory symptoms and disease, a finding with significant clinical implications, as congenital heart disease patients undergoing surgical palliation often have respiratory complications with high morbidity. While this is usually attributed to the heart disease, we propose this may involve intrinsic airway clearance deficits from ciliary dysfunction. Thus the presurgical screening of congenital heart disease patients for respiratory ciliary dysfunction may provide opportunities to provide perioperative pulmonary therapy to enhance airway clearance for at-risk patients. Such change in the standard of care may improve outcome, especially for those congenital heart disease patients who must endure multiple rounds of cardiac surgeries.

Early progressive pulmonary arterial hypertension (PAH) is often observed in patients with heterotaxy/polysplenia especially who have an intracardiac systemic-to-pulmonary shunt. However, its etiology is uncertain and its management is not well established. There was only a Japanese report about PAH in consecutive patients with heterotaxy/polysplenia syndrome [1]. They seemed to develop pulmonary vascular obstructive disease earlier and more severe than expected, even in cases with only pre-tricuspid systemic-to-pulmonary shunt although more detailed analysis is required.