NCE-based strategy for nuclear reprogramming and regenerative medicine

基于 NCE 的核重编程和再生医学策略

基本信息

批准号：
7941985
负责人：
JOHN P COOKE
金额：
$ 134.68万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7941985
关键词：
Adult Advisory Committees Animals Applied Genetics Biological Assay Biomedical Engineering Cardiovascular system Cell Extracts Cell Line Cell Survival Cell physiology Cells Chemicals Chemistry Clinical Clinical Trials Colorado Communities Core Facility DNA Integration Development Dose Embryo Endothelial Cells Engraftment Enhancers Epigenetic Process Ethical Issues Fibroblasts Foundations Funding Generations Genetic Human Immune In Vitro Institutes Intellectual Property Karyotype determination procedure Knowledge Lead Methodology Methods Microfluidics Mitochondria Molecular Molecular Abnormality Monitoring Clinical Trials Mus Nuclear Penetration Peptides Phenotype Pluripotent Stem Cells Production Proteins Protocols documentation Reagent Recruitment Activity Regenerative Medicine Reporting Research Research Institute Research Personnel Safety Somatic Cell Source Stem Cell Research Stem cells Stimulus Teratoma Testing Therapeutic Time Transformed Cell Line Translations Universities Valproic Acid Work base biobank c-myc Genes cell bank clinical application comparative genomic hybridization design human embryonic stem cell improved insight novel novel strategies nuclear reprogramming pre-clinical professor public health relevance single cell analysis small molecule small molecule libraries stem cell biology tool translational medicine uptake

项目摘要

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.

描述（由申请人提供）：发现一系列的重编程因子（例如Oct4，Sox2，KLF4和C-Myc）可以诱导成人人类细胞的核重编程以多能元性，这是再生医学的具有里程碑意义的发展。尽管人类诱导的多能干细胞（HIPSC）有望成为治疗细胞的来源，而没有人类胚胎干细胞的伦理问题或免疫屏障（ESC），但使用基于DNA的方法来产生人类诱导的多能细胞（HIPSC）可能会引入遗传或表观遗传错误。为了克服这些安全问题并提高产生安全有效的IPSC的效率，我们将发现并完善诱导重新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的化学文库的小分子，以及设计用于有效核进入的新型细胞 - 佩戴肽。我们预计，基于NCE的核重编程将避免与基于DNA的方法（例如DNA积分）以及缓慢，效率低下和异常重编程相关的关注。产生的知识以及从该知识中得出的工具对于许多干细胞生物学家和再生医学研究人员都会有用。我们的具体目的是：1）开发和完善试剂和协议，以产生基于NCE的HIPSC策略。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细胞的摄取（与基于DNA的策略相比），并更精确地控制重编程刺激的剂量，持续时间和时间。 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由NCE基于NCE的方法产生的IPSC。尤其是，我们将重点关注人IPSC对内皮细胞（EC）的分化，并将相同的严格遗传，表观遗传和线粒体分析以及谱系特异性功能分析施加，以记录HIPSC-EC的保真度对预期的表型。公共卫生相关性：这项工作将提供科学见解，方法和实用性的方法，以有效地将人类体细胞核重编程为整个再生医学界的安全有效的HIPSC。