NCE-based strategy for nuclear reprogramming and regenerative medicine

描述（由申请人提供）：一小组重编程因子（例如Oct 4、Sox 2、Klf 4和c-Myc）可诱导成人细胞核重编程为多能性的发现是再生医学的里程碑式发展。虽然人类诱导多能干细胞（hiPSC）有望成为治疗性细胞的来源，但在没有人类胚胎干细胞（ESC）的伦理问题或免疫障碍的情况下，使用基于DNA的方法产生人类诱导多能干细胞（hiPSC）可能在核重编程期间引入遗传或表观遗传错误。为了克服这些安全性问题并提高产生安全有效的iPSC的效率，我们将发现和改进诱导重编程或提高其效率的新化学实体（NCE）。这些新的化学实体包括源自独特化学文库的小分子以及设计用于有效进入核的新型细胞渗透肽。我们预计，基于NCE的核重编程将避免与基于DNA的方法，如DNA整合，以及缓慢，低效和异常的重编程相关的问题。所产生的知识，以及从这些知识中获得的工具，将对许多干细胞生物学家和再生医学研究人员有用。我们的具体目标是：1）开发和改进用于产生hiPSC的有效的基于NCE的策略的试剂和方案。我们将使用小分子和肽来表征重编程效率。这种方法将提供无偏的细胞摄取（与基于DNA的策略相比），以及对重编程刺激的剂量、持续时间和时机的更精确控制。2)表征这些hiPSC的安全性和功能。我们将应用遗传、表观遗传和线粒体分析来表征通过基于NCE的方法产生的iPSC。特别是，我们将专注于人iPSC向内皮细胞（EC）的分化，并应用同样严格的遗传，表观遗传和线粒体分析，以及谱系特异性功能测定，以记录hiPSC-EC对预期表型的保真度。 公共卫生相关性：这项工作将为整个再生医学界提供科学见解，方法和试剂，用于将人体细胞有效地核重编程为安全有效的hiPSC。

DESCRIPTION (provided by applicant): The discovery that a small set of reprogramming factors (e.g. Oct4, Sox2, Klf4 and c-Myc) can induce the nuclear reprogramming of adult human cells to pluripotentiality, is a landmark development in regenerative medicine. Although human induced pluripotential stem cells (hiPSCs) promise a source of therapeutic cells, without the ethical issues or immune barriers of human embryonic stem cells (ESCs), the use of DNA-based methods for generating human induced pluripotential cells (hiPSCs) may introduce genetic or epigenetic errors during nuclear reprogramming. To overcome these safety concerns and to increase the efficiency of generating safe and effective iPSC, we will discover and refine new chemical entities (NCE) that induce reprogramming or enhance its efficiency. These new chemical entities include small molecules derived from a unique chemical library together with novel cell-permeant peptides designed for effective nuclear entry. We anticipate that NCE-based nuclear reprogramming will avoid concerns associated with DNA-based approaches such as DNA integration, as well as slow, inefficient and aberrant reprogramming. The knowledge that is generated, and the tools that are derived from that knowledge, will be useful for many stem cell biologists and investigators in regenerative medicine. Our specific aims are: 1) To develop and refine reagents and protocols for an efficient NCE-based strategy of generating hiPSCs. We will characterize the reprogramming efficiency using small molecules and peptides. This approach will provide for unbiased cell uptake (by comparison to DNA-based strategies), and more precise control over the dose, duration and timing of the reprogramming stimuli. 2) To characterize the safety and function of these hiPSCs. We will apply the genetic, epigenetic and mitochondrial analyses to characterize iPSCs generated by the NCE-based approach. Especially, we will focus on the differentiation of human iPSC to endothelial cells (EC) and apply the same rigorous genetic, epigenetic and mitochondrial analyses, as well as lineage-specific functional assays, to document the fidelity of hiPSC-EC to the expected phenotype. PUBLIC HEALTH RELEVANCE: This work will provide scientific insights, methodologies and reagents of utility for efficient nuclear reprogramming of human somatic cells into safe and effective hiPSCs for the entire regenerative medicine community.