Transcription Factor Induced Reprogramming

描述(申请人提供)：本申请旨在研究细胞重新编程的潜在机制，即将成体细胞转化为多能干细胞。我们将利用转录因子介导的重编程转化为诱导多能干细胞(IPSCs)作为实现三个主要目标的工具。在目标1中，我们将(I)测试来自成纤维细胞、造血细胞和肌源性细胞的ipSC是否在转录、表观遗传和功能上是不同的，(Ii)研究提高祖细胞重编程效率的机制，以及(Iii)确定ipSC的形成是否会将基因突变引入细胞。解决IPSCs的表观遗传学和遗传完整性对于这项技术的任何潜在治疗应用都是至关重要的，并可能确定最适合产生患者特定IPSCs的细胞类型。在目标2中，我们将(I)绘制经历重编程的中间细胞群体中的转录和表观遗传学变化，(Ii)测试是否将分化的细胞重编程为多能细胞是否概括了正常发育的阶段，以及(Iii)分别进行获得和丧失功能的筛选，以确定重编程的新调节因子。这一目标将识别在重新编程过程中重要的新分子，其操作可能有助于高效和安全地产生针对患者的IPSCs。在目标3中，我们将使用体外和体内实验来比较IPSCs和ESCs的功能。具体地说，我们将(I)评估IPSC和ESCs来源的神经干细胞和成纤维细胞在体外是否显示出相似的生长和分化特征；(Ii)测试iPSC来源的造血干细胞在连续骨髓移植中是否与ESC来源的造血干细胞一样有效；以及(Iii)完全培育ESC和iPSC来源的小鼠并使其老化，以测试IPSC来源的动物是否过早衰老或患癌症。这一目标将评估体内IPSC来源的成熟细胞的安全性和长期后果，这是将IPSC技术用于人类细胞治疗的先决条件。 公共卫生相关性：我们实验室的目标是通过使用诱导多能干细胞(IPSCs)作为工具来剖析细胞重新编程的机制。在这项建议中，我们将(I)研究体细胞起源在细胞重新编程为IPSCs过程中的作用，(Ii)绘制在经历重新编程的中间细胞中发生的转录和表观遗传事件，以及(Iii)比较IPSCs和胚胎干细胞的发育和分化潜力。总之，这些实验将深入了解转录因子介导的重编程的机制，并为研究和潜在地治疗疾病提供关于IPSC生产的有效性和安全性的关键信息。

DESCRIPTION (provided by applicant): This application seeks to investigate the mechanisms underlying cellular reprogramming, i.e. the conversion of adult cells into pluripotent stem cells. We will utilize transcription factor-mediated reprogramming into induced pluripotent stem cells (iPSCs) as a tool to pursue three major Aims. In Aim 1, we will (i) test whether iPSCs derived from fibroblasts, hematopoietic and myogenic cells are transcriptionally, epigenetically and functionally distinct, (ii) study the mechanism for the increased reprogramming efficiency of progenitors, and (iii) determine if iPSC formation introduces genetic mutations into cells. Addressing the epigenetic and genetic integrity of iPSCs will be crucial for any potential therapeutic applications of this technology and may identify the most suitable cell type for generating patient-specific iPSCs. In Aim 2, we will (i) map the transcriptional and epigenetic changes in intermediate cell populations undergoing reprogramming, (ii) test if reprogramming differentiated cells into pluripotent cells recapitulates stages of normal development, and (iii) perform a gain and loss-of-function screen, respectively, to identify novel regulators of reprogramming. This aim will identify new molecules important during reprogramming, whose manipulation may facilitate the efficient and safe generation of patient-specific iPSCs. In Aim 3, we will investigate the functionality of iPSCs compared with ESCs using in vitro and in vivo assays. Specifically, we will (i) assess whether neural stem cells and fibroblasts derived from iPSCs and ESCs show similar growth and differentiation characteristics in vitro, (ii) test if iPSC-derived hematopoietic stem cells are as potent as ESC-derived hematopoietic stem cells upon serial bone marrow transplantation, and (iii) produce and age entirely ESC and iPSC-derived mice to test if iPSC-derived animals age prematurely or develop cancer. This aim will assess the safety and long-term consequences of iPSCs-derived mature cells in vivo, a prerequisite for using iPSC technology in human cell therapy. PUBLIC HEALTH RELEVANCE: The goal of our lab is to dissect the mechanisms of cellular reprogramming by using induced pluripotent stem cells (iPSCs) as a tool. In this proposal, we will (i) study the role of the somatic cell-of-origin during cellular reprogramming into iPSCs, (ii) map the transcriptional and epigenetic events that occur in intermediate cells undergoing reprogramming, and (iii) compare the developmental and differentiation potentials of iPSCs and embryonic stem cells. Collectively, these experiments will give insight into the mechanisms of transcription-factor-mediated reprogramming and provide crucial information on the efficacy and safety of iPSC production for studying and potentially treating diseases.