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.
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. The factor required for blood vessel development is only required for a short window of time and then must be removed from the system for the cells to progress to mature blood vessels. Using a viral vector to introduce the modified genes to cells, we are taking advantage of a system that allows us to regulate the expression of an endothelial gene by the addition of a common drug to the cells. Once the drug is removed from the system, gene expression is ended. This allows us to mimic the pattern of the factor seen in normal development of blood vessel cells.
We have established a method in the laboratory to reliably generate endothelial cells from unmodified hESCs based on methods from previously published studies. These laboratory generated cells mimic human endothelial cells in many tests including gene expression and surface protein expression. In addition, we have shown that expression of the transcription factor required for endothelial cell development in hESCs induces the cells to express other genes associated with blood vessel endothelium. We are in the process of introducing the viral system to the hESCs so that we can temporally induce the endothelial gene and increase the numbers of endothelial cells that we generate using our differentiation method.
To test the ability of the cells that we have generated in the laboratory to aid in human condition, we have been testing models of cardiovascular blockage in a mouse. We have thus far tested models that mimic a complete coronary artery blockage in the heart, a complete blockage in a leg artery and a model which tests how well the introduced cells are able to integrate into the mouse circulatory system. All of these models will be further tested to determine which is most effective for the endothelial cells we have generated.