Establishment of a cardiovascular stem cell culture system and its application for gene therapy

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Masuda demonstrated that a neuropathogenic murine retrovirus PVC-211 transduced exogenous genes into rat endothelial cells more efficiently than other viruses and that treatment of the target endothelial cells with 2-deoxyglucose and vector inoculation in the absence of heparin further increased the transduction efficiency. In order to solve the problem that retrovirus-mediated gene transfer is irreversible, Masuda also developed two novel retroviral vectors, exploiting the Cre loxP homologous recombination system ; one is a "Flip-Flop" vector which enables reversible regulation of transduced genes by Cre-mediated DNA inversion, and the other is an excisable vector, which allows excision of transduced genes by Cre-mediated DNA deletion. The latter is useful for analyzing the host genome at proviral integration sites and mutagenic effects of proviral insertion.Kurihara et al. established a system with which differentiation of primary neural crest cells derived from mouse embryos into vascular smooth muscle cells can be induced. Using this system, Kurihara et al. Demonstrated that colony formation of the smooth muscle cells require endothelin-1(ET-1)and that unlike BMP-2 and BMP-4, FGF-2 was inhibitory to differentiation of neural crest cells into smooth muscle cells. In addition, studies on ET-1 knockout mice suggested that ET- 1 might regulate differentiation of the vascular system through its effects on expression of a bHLH type DNA-binding protein, dHAND.With this 2-year project, we were able to [1] establish and analyze the system for cardiovascular stem cell primary culture and differentiation induction and [2] develop vector systems for reversible gene transduction into cardiovascular cells. Based on these achievements, further studies will be carried out to establish cell lines, which retain the ability to differentiate into vascular smooth muscle cells, and generate animal models for gene therapy of cardiovascular diseases.