Endothelial progenitor and mesenchymal stem cell-derived cells persist in tissue-engineered patch in vivo:: Application of green and red fluorescent protein-expressing retroviral vector
Endothelial progenitor and mesenchymal stem cell-derived cells persist in tissue-engineered patch in vivo:: Application of green and red fluorescent protein-expressing retroviral vector
复制标题
DOI：
10.1089/ten.2006.0128
复制
发表时间：
2007-03-01
影响因子：
--
通讯作者：
Mayer, John E., Jr.
中科院分区：
文献类型：
--
作者：
Sales, Virna L.;Mettler, Bret A.;Mayer, John E., Jr.
关于组织工程（TE）心脏瓣膜和血管的一个未解决的问题是移植细胞在体内的命运。我们开发了一种策略，使用编码绵羊循环内皮祖细胞（EPC）和骨髓间充质干细胞（BMSC）中绿色和红色荧光蛋白（分别为GFP和RFP）的逆转录病毒载体系统来跟踪TE构建体和邻近组织内种子细胞的解剖位置。我们证明，使用基于高滴度莫洛尼鼠白血病病毒的逆转录病毒载体进行离体稳定转导，可使 GFP(+) EPC-和 RFP+ 间充质干细胞 (MSC) 衍生细胞的转导效率大于 97%。细胞表型和转基因表达在随后的 25 代中也得以维持。使用逆转录病毒载体系统将我们预先接种的细胞与组织驻留祖细胞以及循环内皮和骨髓源性前体细胞区分开来，我们同时将 2x10(6) GFP+ EPC 和 2 x 10(5) RFP+ MSC 共同接种到 TE 贴片上。在一系列绵羊肺动脉补片增强研究中，移植的 GFP(+) EPC - 和 RFP+ MSC 衍生细胞在植入后 7 至 14 天持续存在于 TE 补片内，如使用免疫荧光所鉴定。分析显示，在植入转导细胞之前，TE 贴片的管腔覆盖率为 81%，在植入后第 7 天增加至 96%，并在植入后第 14 天下降至 67%。这表明祖细胞的逆转录病毒表达与这些细胞对 TE 构建体的生理重塑和成熟的介导作用之间存在时间关联。据我们所知，这是第一个心血管组织工程体内研究，使用双标记方法来证明共培养和同时预接种的成体祖细胞的来源、持久性和掺入 TE 血管贴片的直接证据。
An unresolved question regarding tissue-engineered (TE) cardiac valves and vessels is the fate of the transplanted cells in vivo. We have developed a strategy to track the anatomic location of seeded cells within TE constructs and neighboring tissues using a retroviral vector system encoding green and red fluorescent proteins (GFPs and RFPs, respectively) in ovine circulating endothelial progenitor cells (EPCs) and bone marrow-derived mesenchymal stem cells (BMSCs). We demonstrate that stable transduction ex vivo with high-titer Moloney murine leukemia virus-based retroviral vector yields transduction efficiency of greater than 97% GFP(+) EPC- and RFP+ mesenchymal stem cell (MSC)-derived cells. Cellular phenotype and transgene expression were also maintained through 25 subsequent passages. Using a retroviral vector system to distinguish our pre-seeded cells from tissue-resident progenitor cells and circulating endothelial and marrow-derived precursors, we simultaneously co-seeded 2x10(6) GFP+ EPCs and 2 x 10(5) RFP+ MSCs onto the TE patches. In a series of ovine pulmonary artery patch augmentation studies, transplanted GFP(+) EPC - and RFP+ MSC-derived cells persisted within the TE patch 7 to 14 days after implantation, as identified using immunofluorescence. Analysis showed 81% luminal coverage of the TE patches before implantation with transduced cells, increasing to 96% at day 7 and decreasing to 67% at day 14 post-implantation. This suggests a temporal association between retroviral expression of progenitor cells and mediating effects of these cells on the physiological remodeling and maturation of the TE constructs. To our knowledge, this is the first cardiovascular tissue-engineering in vivo study using a double-labeling method to demonstrate a direct evidence of the source, persistence, and incorporation into a TE vascular patch of co-cultured and simultaneously pre-seeded adult progenitor cells.