PURPOSE:To test the hypothesis that the robust expansion of bovine corneal endothelial cells (BCECs) in vitro is due to the presence of individual endothelial cells with various levels of proliferative potential.METHODS:BCECs and bovine vascular endothelial cells (ECs) derived from aorta, coronary artery, and pulmonary artery were cultivated in optimized medium. These cell populations were confirmed by morphologic features, functional assays, and gene expression profiles. Moreover, ECs were plated in a single-cell clonogenic assay to evaluate colony-forming ability.RESULTS:Both corneal and vascular ECs were confirmed to be pure populations of endothelium uncontaminated with hematopoietic cells. A complete hierarchy of endothelial colony-forming cells (ECFCs) was identified in BCECs by a single-cell clonogenic assay. The distribution of the various types of ECFCs was similar to the control ECs removed from the systemic vessels.CONCLUSIONS:Cultured BCECs display clonal proliferative properties similar to those of vascular ECs.

We have recently identified endothelial colony forming cells (ECFCs) in human blood and blood vessels, and ECFC are elevated in patients with coronary artery disease. Because pigs are a favored model for studying myocardial ischemia, we questioned whether ECFCs also exist in swine and whether myocardial ischemia would alter the number of ECFC in circulation. ECFCs were present in circulating blood and aortic endothelium of healthy pigs. In pigs with an acute myocardial infarction (AMI) (n = 9), the number of circulating ECFC was markedly increased compared to sham control pigs (15 ± 6 vs. 1 ± 1 colonies/100 cc blood, p < 0.05). Moreover, the percentage of circulating high proliferative potential ECFCs (HPP-ECFCs) was significantly increased following AMI induction compared to sham control (38.4 ± 5.8% vs. 0.4 ± 0.4%, p < 0. 05) and to baseline (38.4 ± 5.8% vs. 2.4 ± 2.4%, p < 0. 05) blood samples. This is the first study to report that ECFCs are present in blood and aorta in healthy pigs and that the number and distribution of circulating ECFCs is altered following AMI. Because circulating ECFC are also altered in human subjects with severe coronary artery disease, the pig model of AMI may be an excellent preclinical model to test the role of ECFC in the pathophysiology of AMI.

Murine embryonic stem (mES) cells are pluripotent cells derived from the inner cell mass of the preimplantation blastocyst. These cells are primitive and undifferentiated and have the potential to become a wide variety of specialized cell types. Mouse ES cells can be regarded as a versatile biological tool that has led to major advances in our understanding of cell and developmental biology. To study specific gene function in early developmental events, gene knockout approaches have been traditionally used, however, this is a time-consuming and expensive approach. Recently, we have shown that small interfering RNA is an effective strategy to knockdown target gene expression, during ES cell differentiation, and consequently, one can alter cell fates in ES-derived differentiated cells. This method will be useful to test the function of a wide variety of gene products using the ES cell differentiation system.