Directed MSC differentiation via controlled transfection

描述(申请人提供)：干细胞定向分化的能力对于更直接地模拟组织发育过程是至关重要的，尤其是推进基于干细胞的组织再生方法。现有的指导干细胞分化的策略主要集中在用蛋白质生长因子补充培养环境，这些蛋白质生长因子能够诱导特定谱系的分化。再生医学中的一种新兴方法是将编码诱导性生长因子的基因导入细胞，使生长组织中的细胞以转基因方式产生这些因子。以前的研究表明，基因传递可以用来诱导几种组织类型的产生。然而，以前的方法还没有实现对生长因子产生的高水平的空间控制，生长因子的产生可能对复杂的多细胞组织(如血管组织)的再生至关重要。这项建议描述了一项研究计划，旨在通过编码诱导性生长因子的质粒DNA实现对间充质干细胞(MSCs)的高度控制的转基因。特别是，我们计划利用具有良好特性的互补DNA相互作用来可控地将质粒DNA隔离在水凝胶底物上。我们假设，通过互补的DNA相互作用固定在底物上的DNA可以用来转染附着在同一底物上的细胞，并且固定化的DNA的空间图案将使得能够对转染进行空间控制。我们的具体目标是：1)开发和表征同时提供用于细胞黏附的多肽配体和用于定位质粒DNA隔离的寡核苷酸手柄的水凝胶；2)检测载体DNA的可获得性和细胞黏附特性对转染率的依赖；以及3)实现编码两种关键诱导生长因子：血管内皮生长因子和血小板衍生生长因子的基因的hMSCs的空间图案化转染法。这些因子的选择是基于它们分别引导MSCs分化为内皮细胞和平滑肌细胞的能力。我们期望这项拟议的研究最初将产生对底物增强型基因传递的更全面的理解，以及使血管组织再生成为可能的技术。最终，我们定向干细胞分化的基因传递方法原则上可以应用于任何贴壁干细胞类型和任何生长因子。

DESCRIPTION (provided by applicant): The ability to direct differentiation of stem cells is crucial for more direct mimicry of tissue development processes, and it is particularly important to advance stem cell-based approaches to tissue regeneration. Existing strategies to direct stem cell differentiation focus primarily on supplementing culture environments with protein growth factors, which are capable of inducing differentiation down specific lineages. An emerging approach in regenerative medicine involves transfecting cells with genes that encode inductive growth factors, leading to transgenic production of these factors by cells within a growing tissue. Previous studies demonstrate that gene delivery can be used to induce generation of several tissue types. However, previous approaches have not yet achieved a high level of spatial control over growth factor production, which is likely to be vital for regeneration of complex multicellular tissues such as vascular tissue. This proposal describes a research program that aims to achieve highly controlled transfection of mesenchymal stem cells (MSCs) with plasmid DNA encoding inductive growth factors. In particular, we plan to exploit well-characterized complementary DNA interactions to controllably sequester plasmid DNA on hydrogel substrates. We hypothesize that DNA immobilized to a substrate via complementary DNA interactions can be used to transfect cells that are adhered to the same substrate, and that spatial patterning of the immobilized DNA will enable spatial control over transfection. We specifically aim to: 1) develop and characterize hydrogels that simultaneously present peptide ligands for cell adhesion and oligonucleotide handles for localized plasmid DNA sequestering; 2) examine the dependence of transfection efficiency on plasmid DNA availability and cell adhesion characteristics; and 3) achieve spatially patterned transfection of hMSCs with genes encoding two key inductive growth factors: vascular endothelial growth factor and platelet-derived growth factor. These factors are chosen based on their ability direct differentiation of MSCs into endothelial and smooth muscle cells, respectively. We expect that the proposed research will initially produce a more complete understanding of substrate-enhanced gene delivery as well as an enabling technology for vascular tissue regeneration. Ultimately, our gene delivery approach for directed stem cell differentiation can, in principle, be applied to any adherent stem cell type and any growth factor.