In this study, we describe the differentiation, survival, and function of ES cell-derived endothelial cells for cardiovascular applications. The major findings can be summarized as follows: (1) ES cells can be differentiated into endothelial-like cells using a sequential combination of Flk-1+ (early EC marker) and VE-cadherin+ (late EC marker) sorting; (2) these ESC-ECs express typical endothelial markers (Flk-1, eNOS, vWF) and can uptake Dil-ac-LDL as well as participate in vasculogenesis on matrigel assay; (3) transplanted ESC-ECs can improve ventricular function and enhance neoangiogenesis in a mouse model of myocardial infarction; (4) importantly, transplanted cell fate can be monitored noninvasively within the same animals for up to 8 weeks using bioluminescence imaging; (5) however, drastic decrease of cell signal activity was observed within this time period, indicating significant donor cell loss.
In recent years, several animal studies have shown that transplantation of endothelial progenitor cells isolated from the peripheral blood or bone marrow can improve cardiac function 3–6
. But whether these cells will ultimately play a role in human application requires further evaluation. For example, initial results from the TOPCARE-AMI trial showed that patients who received circulating endothelial progenitor cells had improvement in LVEF from 51±10% to 59±10% at 4 months (P
. However, a recent larger study from the same group showed that patients who received circulating endothelial progenitor cells had no improvement in LVEF at 3 months (−0.4±2.2; P
. Although the study design and patient population are different between the two trials, the discrepancy suggests that a detailed mechanistic understanding of how these transplanted cells can improve cardiac function is still lacking. It is possible that patients who have CAD as well as other comorbidities such as diabetes, hypertension, and hypercholesterolemia may posses poorly
functioning endothelial cells to begin with, which may not elicit much long-term functional improvement 8,9
. In this regard, positive beneficial results shown in pre-clinical studies that are based predominantly on transplanting stem cells from young healthy adult animals may not
be representative of the routine clinical scenario involving older patients 6,8,9
. Therefore, therapies utilizing alternative sources of cells possessing limitless potential for self-renewal may be an attractive option.
Compared to adult stem cells, ES cells are unique in their ability to differentiate into virtually all cell types, including neurons, cardiomyocytes, hepatocytes, islet cells, skeletal muscle cells, and endothelial cells 20
. To date, however, the main obstacle remains a lack of reliable methodology to purify cells of interest from other unwanted cell populations. One common approach is to use cell lineage-specific promoters driving GFP or drug-resistant genes. In the case of endothelial cell isolation, most available promoters are still plagued by non-specific expression in other cell types. For instance, the vWF promoter is active in megakaryocytes, the PECAM-1 promoter is active in hematopoietic cells, and the VEGF receptor 2 promoter is active in undifferentiated ES cells 15
. By contrast, the VE-cadherin promoter is known to be constitutively expressed specifically by endothelial cells 21
. Building upon the experience from these studies, we used a combinatorial approach of both early (Flk-1+
and late EC marker (VE-cadherin+
. We were able to differentiate mouse EC cells into endothelial-like cells that express surface markers similar to adult mouse endothelial cells. Upon isolation, these ESC-ECs can form tube-like structures when cultured on matrigel and can uptake Dil-ac-LDL that is a typical phenotype of mature endothelial cells. Our data also concur with other studies describing the functionality of endothelial cells isolated from mouse 12
, monkey 22
, and human ES cells 11
In this study, we determined the longitudinal cell survival kinetics within the same cohort of animals animal using a novel imaging technique, avoiding the sampling biases and errors that may occur when different groups of animals are sacrificed at different time points 23
. Our imaging data suggest that by week 8, <2% of the transplanted ESC-ECs are still alive. This observation conforms with other studies showing poor donor cell survival using serial histology, TUNEL apoptosis assay, or Taqman Sry
PCR techniques 24
. Indeed, our imaging and histologic analysis provide no
definitive proof that donor derived cardiac myocytes or vasculature are being regenerated after transplantation with ESC-ECs. These findings indicate that other mechanisms such as activation of paracrine pathways may play a role 2
, but additional studies will be needed in the future to test this hypothesis. Interestingly, Levenberg et al.
showed that ESC-ECs, when seeded in biodegradable polymer scaffolds, can lead to long-term engraftment and formation of blood-carrying microvessels 11
. Thus, tissue engineering techniques, rather than direct stem cell transplantation, may prove to be a more viable approach in the future 25
In summary, stem cell therapy is an exciting area of investigation. With further validation, the ESC-ECs described here could provide a continual source of endothelial cells for treatment of myocardial ischemia and peripheral vascular disease. Furthermore, we believe molecular imaging will likely play a critical “watchdog” role to monitor the viability of these transplanted cells (or engineered tissues) for cardiovascular diseases and others alike. The in vivo information gathered will provide greater insight into stem cell physiology in living subjects and lay the framework for more complex, refined studies in the future.