Loren J. Field

Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts

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.

Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts.

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 alpha-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 (> 99% pure) were highly differentiated. G418 selected cardiomyocytes were tested for their ability to form grafts in the hearts of adult dystrophic mice. The fate of the engrafted cells was monitored by antidystrophin immunohistology, as well as by PCR analysis with primers specific for the myosin heavy chain-aminoglycoside phosphotransferase transgene. Both analyses revealed the presence of ES-derived cardiomyocyte grafts for as long as 7 wk after implantation, the latest time point analyzed. These studies indicate that a simple genetic manipulation can be used to select essentially pure cultures of cardiomyocytes from differentiating ES cells. Moreover, the resulting cardiomyocytes are suitable for the formation of intracardiac grafts. This selection approach should be applicable to all ES-derived cell lineages.

Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population.

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 KDRlow/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 KDRlow/C-KITneg cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDRlow/C-KITneg 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.

Survey of Studies Examining Mammalian Cardiomyocyte DNA Synthesis

Increases in cardiac mass during fetal life arise predominantly as a consequence of cardiomyocyte proliferation. During neonatal life, there is a transition from hyperplastic to hypertrophic growth such that further increases in myocardial mass are typically not accompanied by cardiomyocyte proliferation. In the adult myocardium, it is generally believed that the vast majority of cardiomyocytes do not proliferate. This view is supported in part by clinical observations: functionally significant myocardial regeneration has not been documented in diseases and/or injuries that result in cardiomyocyte loss. Furthermore, primary myocardial tumors are rarely observed in adults. Although these findings suggest that the proliferative capacity of adult cardiomyocytes is quite low, they do not exclude the existence of a limited degree of hyperplastic growth in either the normal or diseased myocardium. Toward this end, a number of studies examining the proliferative capacity of cardiomyocytes in experimental animals have been reported. Because genome reduplication is a prerequisite for cell proliferation, the majority of these studies have used various methodologies to monitor cardiomyocyte DNA synthesis as a first approximation of cell division. In the present survey, issues that we consider pertinent for accurate assessment of cardiomyocyte DNA synthesis are discussed. The literature examining cardiomyocyte DNA synthesis during normal and pathological myocardial growth is then summarized. Accurate assessment of cardiomyocyte DNA synthesis in vivo is dependent on the selection of an appropriate marker for genome reduplication as well as the criteria used for cardiomyocyte identification. These issues are considered separately below. ### Markers for DNA Synthesis Genome reduplication is accompanied by a variety of cytological, biochemical, and molecular events, many of which can be used as either direct or indirect evidence for DNA synthesis and, by inference, proliferation. These include the presence of cytological landmarks indicative of karyokinesis, the presence of active DNA synthesis, and the expression of genes and/or protein activities …

Increased Vulnerability to Atrial Fibrillation in Transgenic Mice With Selective Atrial Fibrosis Caused by Overexpression of TGF-β1

Studies on patients and large animal models suggest the importance of atrial fibrosis in the development of atrial fibrillation (AF). To investigate whether increased fibrosis is sufficient to produce a substrate for AF, we have studied cardiac electrophysiology (EP) and inducibility of atrial arrhythmias in MHC-TGFcys33ser transgenic mice (Tx), which have increased fibrosis in the atrium but not in the ventricles. In anesthetized mice, wild-type (Wt) and Tx did not show significant differences in surface ECG parameters. With transesophageal atrial pacing, no significant differences were observed in EP parameters, except for a significant decrease in corrected sinus node recovery time in Tx mice. Burst pacing induced AF in 14 of 29 Tx mice, whereas AF was not induced in Wt littermates (P <0.01). In Langendorff perfused hearts, atrial conduction was studied using a 16-electrode array. Epicardial conduction velocity was significantly decreased in the Tx RA compared with the Wt RA. In the Tx LA, conduction velocity was not significantly different from Wt, but conduction was more heterogeneous. Action potential characteristics recorded with intracellular microelectrodes did not reveal differences between Wt and Tx mice in either atrium. Thus, in this transgenic mouse model, selective atrial fibrosis is sufficient to increase AF inducibility.

Cardiomyocyte Cell Cycle Regulation

Although rapid progress is being made in many areas of molecular cardiology, issues pertaining to the origins of heart-forming cells, the mechanisms responsible for cardiogenic induction, and the pathways that regulate cardiomyocyte proliferation during embryonic and adult life remain unanswered. In the present study, we review approaches and studies that have shed some light on cardiomyocyte cell cycle regulation. For reference, an initial description of cardiomyogenic induction and morphogenesis is provided, which is followed by a summary of published cell cycle analyses during these stages of cardiac ontology. A review of studies examining cardiomyocyte cell cycle analysis and de novo cardiomyogenic induction in the adult heart is then presented. Finally, studies in which cardiomyocyte cell cycle activity was experimentally manipulated in vitro and in vivo are reviewed. It is hoped that this compilation will serve to stimulate thought and experimentation in this intriguing area of cardiomyocyte cell biology.

Cell-based cardiac repair: reflections at the 10-year point.

Received February 28, 2005; revision received April 25, 2005; accepted April 29, 2005. It has now been more than a decade since the first experiments were performed using cell transplantation for the prevention and treatment of heart failure.1–3 Although the biomedical community was initially somewhat skeptical of this approach, a large body of experimental evidence was amassed showing that injected cells could create new tissue and improve function of the failing heart. This evidence, coupled with the recognized limitations of heart failure treatments and the intuitively appealing concept of “regenerative medicine,” has contributed to a crescendo of activity in cell-based cardiac repair. Given the flurry of clinical trials that are currently under way, we think it is timely to review progress over the past 10 years and provide a critical assessment of where the field stands and where it appears to be headed. Cell-based cardiac repair began with studies of skeletal myoblasts derived from skeletal muscle satellite cells.1–3 Myoblasts were the initial choice because of their availability from autologous or syngeneic sources, their ability to proliferate, and their ability to withstand ischemia better than many cell types. Although it was originally hoped that these cells would transdifferentiate into cardiomyocytes, it is now clear that myoblasts remain stubbornly committed to form mature skeletal muscle in the heart3–5 (with the exception of rare cell fusion events at the graft–host interface6). Skeletal muscle is one of the few cell types in the body that does not normally express gap junction proteins, and hence, structural and physiological studies indicate that skeletal muscle cells do not form electromechanical junctions with cardiomyocytes when engrafted into the heart.7,8 Despite this, numerous studies have shown beneficial effects of skeletal myoblast grafting into the infarcted heart in rodents and large animals.8–13 Cardiomyocytes would …

Scalable Production of Embryonic Stem Cell-Derived Cardiomyocytes

Cardiomyocyte transplantation could offer a new approach to replace scarred, nonfunctional myocardium in a diseased heart. Clinical application of this approach would require the ability to generate large numbers of donor cells. The purpose of this study was to develop a scalable, robust, and reproducible process to derive purified cardiomyocytes from genetically engineered embryonic stem (ES) cells. ES cells transfected with a fusion gene consisting of the alpha-cardiac myosin heavy chain (MHC) promoter driving the aminoglycoside phosphotransferase (neomycin resistance) gene were used for cardiomyocyte enrichment. The transfected cells were aggregated into embyroid bodies (EBs), inoculated into stirred suspension cultures, and differentiated for 9 days before selection of cardiomyocytes by the addition of G418 with or without retinoic acid (RA). Throughout the culture period, EB and viable cell numbers were measured. In addition, flow cytometric analysis was performed to monitor sarcomeric myosin (a marker for cardiomyocytes) and Oct-4 (a marker for undifferentiated ES cells) expression. Enrichment of cardiomyocytes was achieved in cultures treated with either G418 and retinoic acid (RA) or with G418 alone. Eighteen days after differentiation, G418-selected flasks treated with RA contained approximately twice as many cells as the nontreated flasks, as well as undetectable levels of Oct-4 expression, suggesting that RA may promote cardiac differentiation and/or survival. Immunohistological and electron microscopic analysis showed that the harvested cardiomyocytes displayed many features characteristic of native cardiomyocytes. Our results demonstrate the feasibility of large-scale production of viable, ES cell-derived cardiomyocytes for tissue engineering and/or implantation, an approach that should be transferable to other ES cell derived lineages, as well as to adult stem cells with in vitro cardiomyogenic activity.

Differentiation and long-term survival of C2C12 myoblast grafts in heart.

We have assessed the ability of skeletal myoblasts to form long-term, differentiated grafts in ventricular myocardium. C2C12 myoblasts were grafted directly into the heart of syngeneic mice. Viable grafts were observed as long as 3 mo after implantation. Immunohistological analyses revealed the presence of differentiated myotubes that stably expressed the skeletal myosin heavy chain isoform. Thymidine uptake studies indicated that virtually all of the grafted skeletal myocytes were withdrawn from the cell cycle by 14 d after grafting. Graft myocytes exhibited ultrastructural characteristics typical of differentiated myotubes. Graft formation and the associated myocardial remodeling did not induce overt cardiac arrhythmia. This study indicates that the myocardium can serve as a stable platform for skeletal myoblast grafts. The long-term survival, differentiated phenotype, and absence of sustained proliferative activity observed in myoblast grafts raise the possibility that similar grafting approaches may be used to replace diseased myocardium. Furthermore, the genetic tractability of myoblasts could provide a useful means for the local delivery of recombinant molecules to the heart.

Myocyte and myogenic stem cell transplantation in the heart

Cellular transplantation is emerging as a potential mechanism with which to augment myocyte number in diseased hearts. To date a number of cell types have been shown to successfully engraft into the myocardium, including fetal, neonatal, and embryonic stem cell-derived cardiomyocytes, skeletal myoblasts, and stem cells with apparent cardiomyogenic potential. Here we provide a review of studies wherein myocytes or stem cells with myogenic potential have been transplanted into the heart. In addition, issues pertaining to the tracking and functional consequences of cell transplantation are discussed.