Year 1

The development of lineage conversion strategies for the generation of vascular progenitor cells (VPCs) and their differentiated derivatives, endothelial and smooth muscle cells, will facilitate the clinical implementation of reprogramming strategies and future personalized medicine. We have previously developed methodologies facilitating the high efficient and rapid generation of human VPCs with potential to give rise to functional human vasculature. Our long-term goals involve the refinement and further development of strategies that not only eliminates the risk of tumor formation due to residual pluripotent stem cell transplantation (a risk inherent to the use of embryonic and induced pluripotent stem cells), but also reduces the time necessary for generation of vessels for the efficient translation and application of these methodologies in acute ischemic situations. Ultimately, we are testing different cell sources as well as refining our reprogramming methodologies to generate: 1) a single and safe methodology for the generation of human vessels; 2) in vivo preclinical data on the functionality and tumor formation potential of the different methodologies used; 3) provide in vivo pre-clinical data on immunocompromised animal models as well as in syngeneic models of cell transplantation to evaluate potential immune rejection of autologous material and the role of inflammation in potential tumor formation. Our in vivo experiments will additionally take place in two different injury models of ischemia: hind limb ischemia and models of cardiac infarction. During the first year of the funded period, we have successfully completed the evaluation of different cell types as a source for the generation of vascular cells and determined that fibroblasts, cells from the skin that can be easily obtained in a patient-specific manner, to be the most reliable source of somatic cells for indirect lineage conversion into vascular progenitor cells. Additionally, we have generated different inducible constructs for the different genes used during reprogramming to a dedifferentiated state, namely Oct4, Sox2, KLF4 and c-Myc. Each gene has been evaluated independently for their potential to generate CD34+ cells, and we have found Sox2 to be indispensable for the process. We have also started the experiments addressing functionality as well as initiated experiments on tumor formation capacity. So far most of the different methodologies tested gave rise to functional cells (albeit with varying efficiencies and timing) and, most importantly, did not result in tumor formation so far. Lastly, we have started to analyze the genetic mutations present in converted endothelial cells and found no significant copy number variations even when integrative approaches were employed.