The functional heart is comprised of distinct mesoderm-derived lineages including cardiomyocytes, endothelial cells and vascular smooth muscle cells. Studies in the mouse embryo and the mouse embryonic stem cell differentiation model have provided evidence indicating that these three lineages develop from a common Flk-1(+) (kinase insert domain protein receptor, also known as Kdr) cardiovascular progenitor that represents one of the earliest stages in mesoderm specification to the cardiovascular lineages. To determine whether a comparable progenitor is present during human cardiogenesis, we analysed the development of the cardiovascular lineages in human embryonic stem cell differentiation cultures. Here we show that after induction with combinations of activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF, also known as FGF2), vascular endothelial growth factor (VEGF, also known as VEGFA) and dickkopf homolog 1 (DKK1) in serum-free media, human embryonic-stem-cell-derived embryoid bodies generate a KDR(low)/C-KIT(CD117)(neg) population that displays cardiac, endothelial and vascular smooth muscle potential in vitro and, after transplantation, in vivo. When plated in monolayer cultures, these KDR(low)/C-KIT(neg) cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDR(low)/C-KIT(neg) fraction give rise to colonies that contain all three lineages when plated in methylcellulose cultures. Results from limiting dilution studies and cell-mixing experiments support the interpretation that these colonies are clones, indicating that they develop from a cardiovascular colony-forming cell. Together, these findings identify a human cardiovascular progenitor that defines one of the earliest stages of human cardiac development.

The mammalian heart has a very limited regenerative capacity and, hence, heals by scar formation. Recent reports suggest that haematopoietic stem cells can transdifferentiate into unexpected phenotypes such as skeletal muscle, hepatocytes, epithelial cells, neurons, endothelial cells and cardiomyocytes, in response to tissue injury or placement in a new environment. Furthermore, transplanted human hearts contain myocytes derived from extra-cardiac progenitor cells, which may have originated from bone marrow. Although most studies suggest that transdifferentiation is extremely rare under physiological conditions, extensive regeneration of myocardial infarcts was reported recently after direct stem cell injection, prompting several clinical trials. Here, we used both cardiomyocyte-restricted and ubiquitously expressed reporter transgenes to track the fate of haematopoietic stem cells after 145 transplants into normal and injured adult mouse hearts. No transdifferentiation into cardiomyocytes was detectable when using these genetic techniques to follow cell fate, and stem-cell-engrafted hearts showed no overt increase in cardiomyocytes compared to sham-engrafted hearts. These results indicate that haematopoietic stem cells do not readily acquire a cardiac phenotype, and raise a cautionary note for clinical studies of infarct repair.

Cardiomyocyte DNA synthesis and binucleation indexes were determined during murine development. Cardiomyocyte DNA synthesis occurred in two temporally distinct phases. The first phase occurred during fetal life and was associated exclusively with cardiomyocyte proliferation. The second phase occurred during early neonatal life and was associated with binucleation. Collectively, these results suggest that cardiomyocyte reduplication ceases during late fetal life. Northern and Western blot analyses identified several candidate genes that were differentially expressed during the reduplicative and binucleation phases of cardiomyocyte growth.

This study describes a simple approach to generate relatively pure cultures of cardiomyocytes from differentiating murine embryonic stem (ES) cells. A fusion gene consisting of the ␣ -cardiac myosin heavy chain promoter and a cDNA encoding aminoglycoside phosphotransferase was stably transfected into pluripotent ES cells. The resulting cell lines were differentiated in vitro and subjected to G418 selection. Immunocytological and ultrastructural analyses demonstrated that the selected cardiomyocyte cultures ( Ͼ 99% pure) were highly differentiated. G418 selected cardiomyocytes were tested for their ability to form grafts in the hearts of adult dystrophic mice.

To study the mechanisms that activate expression of the atrial natriuretic factor (ANF) gene during pressure-induced hypertrophy, we have developed and characterized an in vivo murine model of myocardial cell hypertrophy. We employed microsurgical techniques to produce a stable 35-to 45-mmHg pressure gradient across the thoracic aorta of the mouse that is associated with rapid and transient expression of an immediate-early gene program (c-fos/cjun/junB/Egr-1/nur-77), an increase in heart weight/body weight ratio, and up-regulation of the endogenous ANF gene. These responses that are identical to those in cultured cell and other in vivo models of hypertrophy. To determine whether tissue-specific and inducible expression of the ANF gene can be segregated, we used a transgenic mouse line in which 500 base pairs of the human ANF promoter region directs atrial-specific expression of the simian virus 40 large tumor antigen (T antigen), with no detectable expression in the ventricles. Thoracic aortic banding of these mice led to a 20-fold increase in the endogenous ANF mRNA in the ventricle but no detectable expression of the T-antigen marker gene. This result provides evidence that atrial-specific and inducible expression of the ANF gene can be segregated, suggesting that a distinct set of regulatory cis sequences may mediate the up-regulation of the ANF gene during in vivo pressure overload hypertrophy. This murine model demonstrates the utility of microsurgical techniques to study in vivo cardiac physiology in transgenic mice and should allow the application of genetic approaches to identify the mechanisms that activate ventricular expression of the ANF gene during in vivo hypertrophy.In response to diverse stimuli, such as hypertension, valvular heart disease, and endocrine disorders, the myocardium adapts to increased workloads through the hypertrophy of individual muscle cells (for a review, see refs. 1 and 2). Although the signaling mechanisms that mediate the hypertrophic response of cardiac muscle cells remain unclear, transcriptional activation of cardiac target genes, including contractile proteins and embryonic markers, appears to play a pivotal role in this adaptive response (3, 4). In this regard, the reactivation of atrial natriuretic factor (ANF) gene expression in ventricular cells occurs in response to diverse hypertrophic stimuli (genetic, hormonal, volume overload, pressure overload, hypertension, etc.) in multiple species (5-11), including humans, and could be considered one of the conserved features of ventricular cell hypertrophy.To study the transcriptional regulation of cardiac genes, workers in our laboratory (3, 12, 13) and others (14-16) have extensively characterized cultured myocardial cell models in which several features of hypertrophy can be induced after stimulation with defined agents, such as a-adrenergic agonists (3, 12, 14-17) or endothelin 1 (13). In this model, the inducibility of a constitutively expressed contractile protein gene, myosin light chain 2 (MLC-2), is mediat...