Characterizing blood progenitor cells differentiated from human iPS and ES cells

描述（由申请人提供）：我们提交本提案是为了响应RFA-OD-09-004“大机会（GO）”和NHLBI申请其九（9）个选定的高优先级主题之一，“表征通过重编程人类胚胎和诱导多能干细胞衍生的分化的心脏，肺和血细胞”。最近在细胞重编程领域取得了相当大的进展，包括从多种成体体细胞类型诱导多能干细胞。为了评估这些独特的细胞来源在模型实验系统开发中的价值，并评估其在潜在治疗中的安全性和有效性，仍有几个迫切的问题需要解决。在这项提案中，我们将解决NHLBI提出的三个问题中的两个：（1）在实验室中通过重新编程干细胞产生的分化状态如何与正常组织和器官对应物的特征和命运进行比较？(2)胚胎干细胞（ES）和诱导多能干细胞（iPS）的分化状态如何相互比较？为此，我们建立了一个来自3个州的5个机构的多学科团队，其中包括来自约翰霍普金斯大学、马里兰州大学、两家生物技术公司（位于CA和MA）和NIH的研究人员。与总是供应短缺的出生后干细胞相比，具有定义的表面标记和功能测定的人类造血系统为我们提供了表征在实验室中大量产生的人类iPS/ES细胞后代的潜力和安全性的最佳选择。我们将首先比较由人iPS细胞产生的造血干祖细胞（HSPC）与iPS细胞所来源的成年供体的造血干祖细胞（Aim 1）。此外，我们将比较iPS细胞及其造血衍生物与NIH批准的人ES细胞系及其造血衍生物。我们将使用尖端技术，如DNA甲基化和染色质相关组蛋白修饰的基因组规模分析，以建立来自不同来源的纯化HSPC的表观遗传和基因表达特征。在目标2中，我们将建立从人iPS细胞大量产生的红细胞（成红细胞）的全基因组分子特征，与在相同培养条件下从原始成人供体的分离的HSPC和人ES细胞离体产生的那些相比。在目标3中，我们将开发一种基于DNA甲基化的检测方法，以评估和监测iPS细胞系及其分化的造血后代的致瘤潜力。GO赠款将支持我们建立临界质量和协同作用，以加速使用最近建立的人类iPS细胞及其造血衍生物的关键突破。这一重点研究将有助于我们阐明和调节基因表达和细胞命运决定的表观遗传决定因素。从这项为期2年的研究中获得的分子水平上的更大理解可以广泛地转化为心脏，血管和肺部疾病的研究，以及我们近期关注的血液疾病。 公共卫生相关性：具有患者特异性、可再生性和多能性的人类iPS细胞为我们提供了前所未有的机会，可以从单个确定的干细胞克隆中产生无限数量的特定细胞类型，例如造血细胞。这项重点研究将有助于我们阐明和调节适用于多能干细胞和出生后干细胞的基因表达和细胞命运规范的表观遗传决定因素。与出生后干细胞相比，具有确定的表面标志物和功能测定的人类造血系统为我们提供了表征人类iPS/ES细胞的潜力和安全性的最佳选择。从这项研究中获得的分子水平上的更大理解可以广泛地转化为心脏，血管和肺部疾病的研究，以及我们近期关注的血液疾病。

DESCRIPTION (provided by applicant):We submit this proposal in response to RFA-OD-09-004 "Grand Opportunities (GO)" and NHLBI requests for applications to one of its nine (9) selected high-priority topics, "Characterizing Differentiated Heart, Lung, and Blood Cells Derived by Reprogramming Human Embryonic and Induced Pluripotent Stem Cells". Considerable progress has recently been made in the field of cellular reprogramming, including the induction of pluripotent stem cells from a diversity of adult somatic cell types. To assess the value of these unique cell sources in the development of model experimental systems, and to evaluate their safety and efficacy in potential therapies, several urgent questions remain to be addressed. In this proposal, we will address two of three questions raised by NHLBI: (1) How do the differentiated states generated by reprogramming stem cells in the laboratory compare with the characteristics and fates of their normal tissue and organ counterparts? (2) How do the differentiated states generated by reprogramming embryonic stem (ES) and induced pluripotent stem (iPS) cells compare with each other? To these ends, we have established a multidisciplinary team from 5 institutions in 3 states that includes investigators from Johns Hopkins University, University of Maryland, two biotechnology companies (in CA and MA) and NIH. The human hematopoietic system with defined surface markers and functional assays provides us the best choice to characterize the potential as well as safety of human iPS/ES cell progeny massively generated in laboratories, as compared to postnatal stem cells that are always in short supplies. We will first compare hematopoietic stem-progenitor cells (HSPCs) generated from human iPS cells with those of an adult donor from whom the iPS cells are derived (Aim 1). In addition, we will compare iPS cells and their hematopoietic derivatives with NIH-approved human ES cell lines and their hematopoietic derivatives. We will use cutting-edge technologies such as genome-scale analyses of DNA methylation and chromatin-associated histone modifications to establish epigenetic and gene expression signatures of purified HSPCs from different sources. In Aim 2, we will establish genome-wide molecular signatures of red blood cells (erythroblasts) massively generated from human iPS cells, as compared to those generated ex vivo under the same culture condition from the isolated HSPCs of the original adult donor and of human ES cells. In Aim 3, we will develop a DNA methylation-based assay to assess and monitor tumorigenic potential of iPS cell lines and their differentiated hematopoietic progeny. A GO grant will support us to build critical mass and synergy, to accelerate critical breakthroughs using recently established human iPS cells and their hematopoietic derivatives. This focused study will help us to elucidate and modulate epigenetic determinants of gene expression and cell fate determination. The greater understanding at the molecular level gained from this 2-year study can be broadly translated to the study of heart, vascular and lung diseases, in addition to the blood disorders that are our near-term focus. PUBLIC HEALTH RELEVANCE: Human iPS cells that are patient-specific, renewable and pluripotent provide unprecedented opportunities for us to generate unlimited numbers of a specific cell type such as hematopoietic cells from a single defined stem cell clone. This focused study will help us to elucidate and modulate epigenetic determinants of gene expression and cell fate specification applicable to both pluripotent and postnatal stem cells. The human hematopoietic system with defined surface markers and functional assays provides us the best choice to characterize the potential as well as safety of human iPS/ES cells, as compared to postnatal stem cells. The greater understanding at a molecular level gained from this study can be broadly translated to the study of heart, vascular and lung diseases in addition to the blood disorders that are our near-term focuses.