Double-Gated Selection of Ligands that Target Surface Markers of Differentiation

描述（由申请人提供）：人胚胎干（hES）细胞具有无限自我更新的能力，并分化为体内几乎任何细胞类型。它们为开发主要退行性疾病的新疗法提供了巨大的潜力，例如心脏病、阿尔茨海默病、帕金森病、多发性硬化症、皮肤老化和黄斑变性等。最近在将皮肤细胞重编程为具有与胚胎干细胞相同特性的多能细胞方面的进展增加了多能干细胞用于研究的可用性，并且如果它们能够被证明是安全的，则可能最终为临床应用提供细胞。然而，无论多能细胞的来源如何，理解多能细胞如何在体外分化以促进开发将多能细胞分化为用于细胞替代疗法以及用于药物开发的期望的成熟细胞类型的有效方法是关键的未满足的需求。随着多能干细胞向各种谱系的特化和定型进展，细胞表面分子发生的变化知之甚少。使用传统的噬菌体展示选择方法绘制分化干细胞的表面在异质细胞的混合物上是有问题的，因为更丰富的细胞和受体可能掩盖不太常见的细胞和受体。我们在这里提出应用一种新的靶向基因递送技术与展示文库选择相结合，以直接选择靶向来自hES细胞（WA09）的谱系特异性祖细胞的配体。我们独特的选择策略是基于使用2个门来选择配体。为了被选择，配体靶向载体必须首先通过表面标记物相互作用内化到祖细胞中。第二个门是在分化细胞中载体报告基因的调节表达，所述分化细胞是配体靶向的祖细胞的后代。以这种方式，将仅选择靶向特定分化细胞类型（如本文提出的心肌细胞）的祖细胞的配体。我们将首先通过使用EGF靶向颗粒靶向神经祖细胞和从星形胶质细胞中遗传选择靶向颗粒来测试这个概念。在利用神经祖细胞通路对载体进行优化后，我们将构建一个大的随机肽库，并从中筛选心肌细胞靶向肽。所选择的肽将表征其靶向和分离心肌细胞祖细胞的能力。我们的长期目标是开发和商业化靶向配体和试剂盒，用于干细胞祖细胞靶向、分离和分化为治疗上有用的细胞类型和用于药物筛选的细胞。公共卫生相关性：胚胎干细胞和其他多能干细胞，如最近发现的重编程皮肤细胞，具有无限自我更新的能力，并分化成体内几乎任何类型的细胞。因此，它们有可能通过提供用于治疗几乎任何退行性疾病或损伤（例如心脏病、糖尿病、MS、AD和帕金森病）的细胞来深刻地有益于公共健康。了解多能干细胞如何在实验室中发育，以便为安全有效的再生治疗准备正确类型的细胞，这一点至关重要。拟议的项目将使用创新方法开发试剂，以促进干细胞研究，并实现用于治疗和药物开发的细胞的分离，扩大和生产。

DESCRIPTION (provided by applicant): Human embryonic stem (hES) cells have the ability to self renew indefinitely and differentiate into virtually any cell type in the body. They offer tremendous potential for the development of new treatments for major degenerative diseases such as heart disease, Alzheimer's disease, Parkinson's, multiple sclerosis, skin aging and macular degeneration, to name a few. Recent developments in reprogramming skin cells to pluripotent cells with the same properties as embryonic stem cells have increased the availability of pluripotent stem cells for research and may ultimately provide cells for clinical applications is they can be proven safe. However, regardless of the source of pluripotent cells, there is a critical unmet need to understand how pluripotent cells differentiate in vitro to facilitate development of efficient methods of directing differentiating of pluripotent cells to desirable mature cell types for cell replacement therapy as well as for drug development. The changes in cell surface molecules that occur as pluripotent stem cells progress toward specification and commitment to various lineages are poorly understood. Mapping the surface of differentiating stem cells using traditional phage display selection methods is problematic on a mixture of heterogenous cells because the more abundant cells and receptors may mask the less common ones. We propose here to apply a novel targeted gene delivery technology combined with display library selection to directly select ligands that target lineage specific progenitors derived from hES cells (WA09). Our unique selection strategy is based on selection of the ligands using 2 gates. To be selected, the ligand-targeted vector must first internalize into the progenitor cells through a surface marker interaction. The second gate is regulated expression of a vector reporter gene in the differentiated cells that are descended from the ligand targeted progenitors. In this manner, only ligands that target progenitors of a specific differentiated cell type like cardiomyocytes as proposed here would be selected. We will first test the concept by using an EGF targeted particle to target neural progenitor cells and genetically selecting targeted particles from astrocytes. After optimizing the vector using the neural progenitor pathway, we will construct a large random peptide library and select it for cardiomyocyte targeting peptides. The selected peptides will be characterized for their ability to target and isolate cardiomyocyte progenitors. Our long term goal is to develop and commercialize targeting ligands and kits for stem cell progenitor targeting, isolation and differentiation into therapeutically useful cell-types and cells for drug screening. PUBLIC HEALTH RELEVANCE: Embryonic and other pluripotent stem cells such as recently discovered reprogrammed skin cells have the ability to self renew indefinitely and differentiate into virtually any cell type in the body. They therefore have the potential to profoundly benefit public health by providing cells for treating virtually any degenerative disease or injury such as heart disease, diabetes, MS, AD, and Parkinson's disease to name a few. It is critical to understand how pluripotent stem cells develop in the laboratory so that the right types of cells can be prepared for safe and effective regenerative treatments. The proposed project will use innovative methods to develop reagents that will facilitate stem cell research and enable the isolation, scale up and production of cells for therapy and drug development.