Year 3

Cardiovascular disease is a major concern for medicine and is caused by damage to blood vessels. We have begun a project to generate endothelial cells, the cells which line the blood vessels, from human embryonic stem cells (hESCs) using gene transfer technology and regulated gene expression.

Little is known about the early stages of blood vessel endothelial differentiation of the human embryo. It is imperative that we understand normal development in order to mimic it in the laboratory. We have used hESCs to model embryonic development and determine the pattern of gene expression in the early stages of differentiation. Using what is known about mouse embryonic development as a model, we have determined that gene expression in differentiating human cells closely follows that of differentiating mouse cells. In particular, we have determined the timing of the expression pattern of a gene that is required for the generation of endothelial cells. This knowledge will allow us to induce expression of this gene at the proper time during differentiation in the cells in the laboratory to increase the number of blood vessel cells we can generate.

We have established a method in the laboratory to reliably generate endothelial cells from unmodified hESCs. Timing of gene expression during development is extremely important and improper timing can result in cells being unable to respond to the signal generated by the gene or unable to progress further in development. We have found that introduction of a single factor into the differentiating hESCs results in either as little as two or as much as 20 times more endothelial cells depending upon the time of administration than in cells without this added factor. In live animal studies by other groups, it was shown that expression of this gene after the time of normal expression has little effect on the animal . In human embryonic stem cells, we found that the same effect is seen when the gene is administered at the peak of expression or up to 7 days after the peak of expression.

All of our methods thus far have used a technique that alters the genetic makeup of the cells we are testing. We are now exploring methods that do not alter the genes of the cells that could be used to generate cells that could be administered to patients.