Directed MSC differentiation via controlled transfection

描述（由申请人提供）：指导干细胞分化的能力对于更直接地模拟组织发育过程至关重要，并且对于推进基于干细胞的组织再生方法尤其重要。现有的指导干细胞分化的策略主要集中在补充蛋白质生长因子的培养环境，这些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用编码诱导生长因子的基因转染细胞，从而使生长组织中的细胞通过转基因方式产生这些因子。先前的研究表明，基因传递可用于诱导多种组织类型的产生。然而，以前的方法尚未实现对生长因子产生的高水平空间控制，而生长因子的产生可能对复杂的多细胞组织（如维管组织）的再生至关重要。本提案描述了一项研究计划，旨在实现转染间充质干细胞（MSCs）与编码诱导生长因子的质粒DNA的高度控制。特别是，我们计划利用特性良好的互补DNA相互作用来控制地将质粒DNA隔离在水凝胶底物上。我们假设，通过互补DNA相互作用固定到底物的DNA可用于转染粘附在相同底物上的细胞，并且固定DNA的空间模式将使转染的空间控制成为可能。我们的具体目标是：1)开发和表征水凝胶，同时提供用于细胞粘附的肽配体和用于局部质粒DNA隔离的寡核苷酸处理；2)检测转染效率与质粒DNA可用性和细胞粘附特性的关系；3)用编码血管内皮生长因子和血小板源性生长因子两种关键诱导生长因子的基因对hMSCs进行空间模式转染。这些因子的选择是基于它们分别将MSCs直接分化为内皮细胞和平滑肌细胞的能力。我们期望所提出的研究将最初产生对底物增强基因传递的更完整的理解，以及血管组织再生的使能技术。最终，我们的定向干细胞分化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.