Cell Transplantation Work

When heart muscle is irreversibly injured, as by a heart attack, it heals by scar formation. The ensuing loss of contractile function can initiate heart failure, a common disease process with high morbidity and mortality. Although cardiac transplantation is a successful therapeutic option, the limited supply of donor hearts has produced much interest in cell-based strategies to achieve cardiac repair (i.e. "cell transplantation"). Human embryonic stem cells (hereafter, "hESCs") are among the most attractive cell types for this application. hESCs are a unique cell type derived from residual pre-implantation stage embryos that result from in vitro fertilization and are then donated for research purposes. These cells have the unique ability to give rise to all of the cell types of the body, including cardiomyocytes. hESCs have a number of potential advantages over some of the other cell types alluded to for application in cardiac transplantation: this unquestioned capacity to generate cardiomyocytes (a property which remains controversial for other candidate cell types), well-defined protocols for their isolation and maintenance, and a tremendous capacity for expansion in vitro.

In preliminary experiments, we explored the ability of hESC-derived cardiomyocytes to produce human heart muscle in vivo by directly grafting them into the uninjured hearts of immunodeficient rats. hESCs were differentiated as embryoid bodies, enriched for cardiomyocytes (to ~15% purity), heat-shocked to improve survival, and injected into the uninjured left ventricular wall of athymic rats (0.5-10.0 x 10⁶ cells). Hearts were studied from 1 day to 4 weeks after transplantation, using human-specific probes to follow graft lineage. The grafts initially consisted largely of cytokeratin-positive epithelial cells, with minority components of sarcomeric myosin-positive cardiomyocytes and α-fetoprotein-positive endoderm. Surprisingly, the endodermal and epithelial elements were lost over time, while cardiomyocytes appeared to expand. By 4 weeks, the grafts consisted entirely of cardiomyocytes. Importantly, no heterologous elements or teratomas were observed. Engrafted human cardiomyocytes were glycogen-rich and expressed multiple cardiac markers, including the human-specific β-myosin heavy chain, myosin light chain 2v, and atrial natriuretic factor. These human cardiomyocytes proliferated much more than previously seen with rodent-derived cardiomyocytes, as demonstrated by 24.5% and 14.4% of graft cardiomyocytes expressing the proliferation marker Ki-67, as well as 6.4% and 2.7% of these cells incorporating the thymidine analogue BrdU at 1 and 4 weeks following transplantation, respectively. Thus, hESCs can be used to create human myocardium in the rat heart. This system permits studies of human myocardial development and physiology, and provides evidence for the feasibility of using hESCs in myocardial repair. Studies are currently underway to examine whether similar implants of hESC-derived myocardium can be successfully formed within the experimentally-infarcted heart and whether such an intervention ameliorates cardiac dysfunction.

PICTURES FOLLOWING:

Above is a phase-contrast photomicrograph depicting the typical growth pattern and morphology of an undifferentiated hESC culture (here from the H7 line). The undifferentiated cells reside within tightly packed colonies (left half of the field) and are bounded by a looser "mat" of differentiated cells with a stromal morphology.

By contrast, the above phase-contrast photomicrograph demonstrates the morphology of the differentiated progeny derived from hESC cultures. Depicted here are embryoid body outgrowths derived from the previously shown H7 undifferentiated cultures, photographed after growth for approximately 3 weeks under conditions to promote differentiation.

Above is a pair of adjacent histologic sections taken from the heart of a nude rat that had been engrafted 4 weeks earlier with hESC-derived cardiomyocytes. The upper panel depicts the typical, peculiar vacuolated appearance of these cells on a densely fibrous background by routine H&E stain. The lower panel shows the corresponding field, here immunostained with an antibody recognizing sarcomeric myosin heavy chain, a marker for striated muscle. Note that both the host (upper left) and graft (lower right) cardiomyocytes are strongly immunoreactive for this marker. Not shown are additional sections subjected to in situ hybridization for unambiguous human DNA markers (ALU and Y chromosome repetitive elements), which identified the nuclei of graft but not host myocardium.