We have isolated a cardiomyogenic cell line (CMG) from murine bone marrow stromal cells. Stromal cells were immortalized, treated with 5-azacytidine, and spontaneously beating cells were repeatedly screened. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine treatment in ∼30% of the cells; they connected with adjoining cells after one week, formed myotube-like structures, began spontaneously beating after two weeks, and beat synchronously after three weeks. They expressed atrial natriuretic peptide and brain natriuretic peptide and were stained with anti-myosin, anti-desmin, and anti-actinin antibodies. Electron microscopy revealed a cardiomyocyte-like ultrastructure, including typical sarcomeres, a centrally positioned nucleus, and atrial granules. These cells had several types of action potentials, such as sinus node-like and ventricular cell-like action potentials. All cells had a long action potential duration or plateau, a relatively shallow resting membrane potential, and a pacemaker-like late diastolic slow depolarization. Analysis of the isoform of contractile protein genes, such as myosin heavy chain, myosin light chain, and α-actin, indicated that their muscle phenotype was similar to that of fetal ventricular cardiomyocytes. These cells expressed Nkx2.5/Csx, GATA4, TEF-1, and MEF-2C mRNA before 5-azacytidine treatment and expressed MEF-2A and MEF-2D after treatment. This new cell line provides a powerful model for the study of cardiomyocyte differentiation.

Heart disease remains a major cause of death despite advances in medical technology. Heart-regenerative therapy that uses pluripotent stem cells (PSCs) is a potentially promising strategy for patients with heart disease, but the inability to generate highly purified cardiomyocytes in sufficient quantities has been a barrier to realizing this potential. Here, we report a nongenetic method for mass-producing cardiomyocytes from mouse and human PSC derivatives that is based on the marked biochemical differences in glucose and lactate metabolism between cardiomyocytes and noncardiomyocytes, including undifferentiated cells. We cultured PSC derivatives with glucose-depleted culture medium containing abundant lactate and found that only cardiomyocytes survived. Using this approach, we obtained cardiomyocytes of up to 99% purity that did not form tumors after transplantation. We believe that our technological method broadens the range of potential applications for purified PSC-derived cardiomyocytes and could facilitate progress toward PSC-based cardiac regenerative therapy.

Maintenance of skeletal muscle mass relies on the dynamic balance between anabolic and catabolic processes and is important for motility, systemic energy homeostasis, and viability. We identified direct target genes of the glucocorticoid receptor (GR) in skeletal muscle, i.e., REDD1 and KLF15. As well as REDD1, KLF15 inhibits mTOR activity, but via a distinct mechanism involving BCAT2 gene activation. Moreover, KLF15 upregulates the expression of the E3 ubiquitin ligases atrogin-1 and MuRF1 genes and negatively modulates myofiber size. Thus, GR is a liaison involving a variety of downstream molecular cascades toward muscle atrophy. Notably, mTOR activation inhibits GR transcription function and efficiently counteracts the catabolic processes provoked by glucocorticoids. This mutually exclusive crosstalk between GR and mTOR, a highly coordinated interaction between the catabolic hormone signal and the anabolic machinery, may be a rational mechanism for fine-tuning of muscle volume and a potential therapeutic target for muscle wasting.

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cells, that expressed EGFP in the cardiomyocyte-specific manner were transplanted directly into BM of lethally irradiated mice, MI was induced, and they were treated with G-CSF. EGFP ؉ actinin ؉ cells were observed in the ischemic myocardium, indicating that CMG cells had been mobilized and differentiated into cardiomyocytes. Together, these results suggest that the origin of the vast majority of BM-derived cardiomyocytes is MSCs.

Although recent reports have described multipotent, self-renewing, neural crest-derived stem cells (NCSCs), the NCSCs in various adult rodent tissues have not been well characterized or compared. Here we identified NCSCs in the bone marrow (BM), dorsal root ganglia, and whisker pad and prospectively isolated them from adult transgenic mice encoding neural crest-specific P0-Cre/Floxed-EGFP and Wnt1-Cre/Floxed-EGFP. Cultured EGFP-positive cells formed neurosphere-like structures that expressed NCSC genes and could differentiate into neurons, glial cells, and myofibroblasts, but the frequency of the cell types was tissue source dependent. Interestingly, we observed NCSCs in the aorta-gonad-mesonephros region, circulating blood, and liver at the embryonic stage, suggesting that NCSCs migrate through the bloodstream to the BM and providing an explanation for how neural cells are generated from the BM. The identification of NCSCs in accessible adult tissue provides a new potential source for autologous cell therapy after nerve injury or disease.

Afferent and efferent cardiac neurotransmission via the cardiac nerves intricately modulates nearly all physiological functions of the heart (chronotropy, dromotropy, lusitropy and inotropy). Afferent information from the heart is transmitted to higher levels of the nervous system for processing (intrinsic cardiac nervous system, extracardiac-intrathoracic ganglia, spinal cord, brain stem and higher centers) which ultimately results in efferent cardiomotor neural impulses (via the sympathetic and parasympathetic nerves). This system forms interacting feedback loops that provide physiological stability for maintaining normal rhythm and life-sustaining circulation. This system also ensures that there is fine-tuned regulation of sympathetic-parasympathetic balance in the heart under normal and stressed states in the short (beat to beat), intermediate (minutes-hours) and long term (days-years). This important neurovisceral /autonomic nervous system also plays a major role in the pathophysiology and progression of heart disease, including heart failure and arrhythmias leading to sudden cardiac death (SCD). Transdifferentiation of neurons in heart failure, functional denervation, cardiac and extra-cardiac neural remodeling have also been identified and characterized during the progression of disease. Recent advances in understanding the cellular and molecular processes governing innervation and the functional control of the myocardium in health and disease provides a rational mechanistic basis for development of neuraxial therapies for preventing SCD and other arrhythmias. Advances in cellular, molecular, and bioengineering realms have underscored the emergence of this area as an important avenue of scientific inquiry and therapeutic intervention.