Purpose-Human embryonic stem cell (hESC)-derived cardiomyocytes are a promising cell source for cardiac repair. Whether these cells can be transported long distance, survive, and mature in hearts subjected to ischemia/reperfusion with minimal infarction is unknown. Taking advantage of a constitutively GFP-expressing hESC line we investigated whether hESC-derived cardiomyocytes could be shipped and subsequently form grafts when transplanted into the left ventricular wall of athymic nude rats subjected to ischemia/reperfusion with minimal infarction. Co-localization of GFP-epifluorescence and cardiomyocyte specific marker staining was utilized to analyze hESCderived cardiomyocyte fate in a rat ischemia/reperfused myocardium.Methods-Differentiated, constitutively green fluorescent protein (GFP) expressing hESCs (HES3-GFP; Envy) containing about 13% cardiomyocytes were differentiated in Singapore, and shipped in culture medium at 4°C to Los Angeles (shipping time ~3 days). The cells were dissociated and a cell suspension (2×10 6 cells for each rat, n=10) or medium (n=10) was injected directly into the myocardium within the ischemic risk area 5 minutes after left coronary artery occlusion in athymic nude rats. After 15 minutes of ischemia the coronary artery was reperfused. The hearts were harvested at various time points later and processed for histology, immunohistochemical staining, and fluorescence microscopy. In order to assess whether the hESC-derived cardiomyocytes might evade immune surveillance, 2×10 6 cells were injected into immune competent Sprague-Dawley rat hearts (n=2), and the hearts were harvested at 4 weeks after cell injection and examined as in the previous procedures.Results-Even following 3 days of shipping, the hESC-derived cardiomyocytes within embryoid bodies (EBs) showed active and rhythmic contraction after incubation in the presence of 5% CO 2 at 37°C. In the nude rats, following cell implantation, H&E, immunohistochemical staining and GFP epifluorescence demonstrated grafts in 9 out of 10 hearts. Cells that demonstrated GFP epifluorescence also stained positive (co-localized) for the muscle marker alpha-actinin and exhibited cross striations (sarcomeres). Furthermore cells that stained positive for the antibody to GFP (immunohistochemistry) also stained positive for the muscle marker sarcomeric actin and Conclusions-hESCs-derived cardiomyocytes can survive several days of shipping. Grafted cells survived up to 4 weeks after transplantation in hearts of nude rats subjected to ischemia/reperfusion with minimal infarction. They continued to express cardiac muscle markers, exhibit sarcomeric structure, and were well interspersed with the endogenous myocardium. However, hESC-derived cells did not escape immune surveillance in the xenograft setting in that they elicited a rejection phenomenon in immune competent rats.

Aim A limitation of cell therapy for heart disease is the fact that stem cells injected directly into the myocardium are capable of entering the vasculature and migrating to remote organs. We determined whether retention of mesenchymal stem cells (MSCs) in the infarcted myocardium could be improved by implanting the cells in a collagen matrix. Methods A myocardial infarction was induced by ligation of the left anterior descending coronary artery in Fischer rats. A total of 7 days after myocardial infarction, saline (n = 12), saline plus 2 million bone marrow-derived rat MSCs labeled with isotopic colloidal nanoparticles containing europium (n = 13), collagen (n = 13) or collagen plus 2 million labeled MSCs (n = 13) were directly injected into the infarcted myocardium. Tissues from the infarcted myocardium, noninfarcted myocardium, lung, liver, spleen and kidney were sampled 4 weeks later. Distribution of grafted MSCs was quantitatively analyzed by measuring the nanoparticle radioactivity in these tissues. Cardiac function was assessed by left ventriculography. Results There were zero nanoparticles detected in the tissues that received saline or collagen alone into the heart. Nanoparticles were detected in the heart and remote organs in the saline plus MSC group. Labeled cells (expressed as cell number/g tissue weight) were present in three out of 13 lungs (mean of 12,724 ± 7060 cells/g), four out of 13 livers (12,301 ± 5924 cells/g), 11 out of 13 spleens (57,228 ± 11,483 cells/g), zero out of 13 kidneys, 13 out of 13 infarcted myocardium (8,006,835 ± 1,846,462 cells/g) and nine out of 13 noninfarcted myocardium (167,331 ± 47,007 cells/g). However, compared with the saline plus MSC group, nanoparticles were detected to a lesser extent in remote organs in collagen plus MSC group. Nanoparticles were detected in two out of 13 lungs (4631 ± 3176 cells/g; p = NS), zero out of 13 livers (0 cells/g; p <0.05 vs saline plus MSC), four out of 13 spleens (24,060 ± 17,373 cells/g; p <0.05), zero out of 13 kidneys (p = NS) and five out of 13 noninfarcted myocardium (51,522 ± 21,548 cells/g; p <0.05). In the collagen plus MSC group, nanoparticles were detected in 12 out of 13 infarcted myocardium (4,830,050 ± 592,215 cells/g), which did not significantly differ from that in the saline plus MSC group (p = NS). Both saline plus MSCs and collagen alone improved left ventricular ejection fraction compared with saline treatment. However, collagen plus MSCs failed to improve cardiac function. Conclusions Collagen matrix as a delivery vehicle significantly reduced the relocation of transplanted MSCs to remote organs and noninfarcted myocardium.

Mild increase in s-crea is a marker for patients with increased cardiac risk factors and the risk for poor outcomes. Both ES definitions are highly predictive of the outcomes.