Directed MSC differentiation via controlled transfection

通过受控转染定向 MSC 分化

• 批准号: 7078204
• 负责人: WILLIAM L. MURPHY
• 金额: $ 18.19万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7078204
• 关键词: bioengineering /biomedical engineering; biomaterials; biotechnology; cell adhesion; cell differentiation; gene delivery system; intermolecular interaction; mesenchyme; oligonucleotides; plasmids; platelet derived growth factor; polyethylene glycols; regeneration; reporter genes; stem cells; tissue /cell culture; tissue engineering; transfection /expression vector; vascular endothelial growth factors; vascular endothelium; vascular smooth muscle

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.

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是至关重要的，并且推进基于干细胞的组织再生方法是特别重要的。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，其能够诱导特定谱系的分化。再生医学中的一种新兴方法涉及用编码诱导生长因子的基因转染细胞，导致生长组织中的细胞转基因产生这些因子。先前的研究表明，基因递送可用于诱导几种组织类型的产生。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，这可能对复杂多细胞组织如血管组织的再生至关重要。该提案描述了一个研究计划，旨在实现高度控制转染间充质干细胞（MSC）与质粒DNA编码诱导生长因子。特别是，我们计划利用充分表征的互补DNA相互作用，以可控地隔离水凝胶基质上的质粒DNA。我们假设，通过互补DNA相互作用固定到基底上的DNA可用于转染粘附在同一基底上的细胞，并且固定化DNA的空间图案化将使得能够对转染进行空间控制。我们的具体目标是：1）开发和表征同时呈现用于细胞粘附的肽配体和用于局部质粒DNA螯合的寡核苷酸柄的水凝胶; 2）检查转染效率对质粒DNA可用性和细胞粘附特征的依赖性;和3）实现用编码两种关键诱导生长因子的基因对hMSC的空间模式化转染：血管内皮生长因子和血小板衍生生长因子。这些因子的选择是基于它们分别将MSC直接分化为内皮细胞和平滑肌细胞的能力。我们预计，拟议中的研究将首先产生一个更完整的理解基板增强基因传递以及使血管组织再生的技术。最终，我们用于定向干细胞分化的基因递送方法原则上可以应用于任何粘附干细胞类型和任何生长因子。