Generation of vascular progenitor cells from ES cells and iPS cells for medical applications

Objective and Aim: Establishing of induced pluripotency in the cells derived from mice and humans to generate endothelial cells for cell based therapies of vascular or parenchymal diseases of the lung. Endothelium is of crucial importance for the maintenance of vessel function, thus endothelial dysfunction is seen in a variety of pathological conditions. Human embryonic stem (hES) cells – derived differentiated cells hold broad treatment applications. Generation of these cells is ethically debatable. Development of pluripotent stem cells – from somatic cells – holds promise for regenerative medicine and biomedical research. This enables the generation of patient- and disease-specific pluripotent stem cells, which may produce therapeutic cell populations without immune rejection and ethical controversy.

Methods and Results: Generation of murine (miPS) and human induced pluripotent stem cells (hiPS) by reprogramming of somatic cells to pluripotent ES cell-like cells, named induced pluripotent stem cells. The preliminary data, concerning production of integrase deficient construct by means of site-directed mutagenesis strategy (SDM). Throughout cloning procedure integrase deficient lentiviral (IDLV) particles were obtained. The integrase deficiency was confirmed by transduction of HEK cells followed by FACS analysis of GFP expression upon several passages. Furthermore, induction of pluripotency in mouse embryonic fibroblast (MEF's) was achieved. A method for generation of new cell lines from murine ES cells using lentiviral transduction for monitoring of endothelial differentiation and selection by means of antibiotic resistance was successfully set up. Tests carried out to validate different promoter-resistance-gene combinations. Set of vectors had been created, containing promoters: mouse VE-Cadherin; mouse VEGFR2, human VE-Cadherin and human VEGFR2 in connection with resistance gene against three antibiotics: puromycin, hygromycin and neomycin.

Conclusion and Outlook: The experiments have proved that for a successful antibiotic selection of desired cell type further investigations are required. Current work is focused on promoter/resistance gene combinations and optimization of endothelial differentiation and accumulation of generated pure endothelial cell subsets.