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

Masuda证明了一种神经致病性小鼠逆转录病毒PVC-211比其他病毒更有效地将外源基因转导到大鼠内皮细胞中，并且在没有肝素的情况下，用2-脱氧葡萄糖和载体接种处理靶内皮细胞进一步提高了转导效率。为了解决逆转录病毒介导的基因转移不可逆的问题，Masuda还开发了两种新型逆转录病毒载体，利用Cre loxP同源重组系统；一种是“Flip-Flop”载体，可以通过cre介导的DNA反转对转导基因进行可逆调节，另一种是可切除载体，可以通过cre介导的DNA缺失来切除转导基因。后者可用于分析前病毒整合位点的宿主基因组和前病毒插入的诱变效应。Kurihara等人建立了一个系统，该系统可以诱导小鼠胚胎原代神经嵴细胞向血管平滑肌细胞分化。Kurihara等人利用这个系统。证实平滑肌细胞的集落形成需要内皮素-1(ET-1)，与BMP-2和BMP-4不同，FGF-2抑制神经嵴细胞向平滑肌细胞的分化。此外，对ET-1敲除小鼠的研究表明，ET-1可能通过影响bHLH型dna结合蛋白dHAND的表达来调节血管系统的分化。在这个为期2年的项目中，我们能够建立和分析心血管干细胞原代培养和分化诱导系统，[2]能够开发可逆基因转导到心血管细胞的载体系统。在这些成果的基础上，将开展进一步的研究，建立保留向血管平滑肌细胞分化能力的细胞系，并为心血管疾病的基因治疗建立动物模型。

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.