Hematopoietic stem cells from human pluripotent stem cells and modeling of blood disorders

描述(申请人提供)：血液系统是由稀有的造血干细胞(HSCs)维持的发育等级，能够延长自我更新和多谱系分化。由于其在移植后完全重建血液系统的能力，造血干细胞是一种非常有价值的治疗细胞类型。在我与John Dick博士一起接受博士培训期间，我获得了专业知识，并对了解脐带血中的人类造血干细胞做出了贡献(NAT免疫学2010；科学2011)。在乔治·戴利博士的博士后培训期间，我将所有的努力和专业知识都用于从患者诱导的多能干细胞(IPSCs)中培养出造血干细胞和其他有价值的人类血细胞，如可输血的红细胞。我的工作证明了这一重要目标的可行性，证明了人类IPSC来源的血液前体可以使用转录因子HOXA9、ERG和RORA(Cell Stem Cell 2013)转换(或重新指定)为可移植的多系祖细胞。将祖细胞重新定向为干细胞的想法是一种很有前途的方法，但我们仍然缺乏产生真正的造血干细胞。在我的研究计划的指导部分，我将专注于识别转录因子的组合，这些转录因子可以重新指定 将IPSCs转化为具有长期多系移植潜能的HSCs。在独立阶段，我将研究这些调控网络在正常造血中的作用。在体外和体内，重新指定的IPSC祖细胞是一种特别有效的红细胞来源。人类红细胞在体内经历珠蛋白转换以表达成人血红蛋白，类似于出生后发生的胎儿到成人的珠蛋白转换。出于这个原因，我已经开始使用来自钻石黑扇贫血和镰状细胞贫血患者的IPSC系对两种先天性贫血进行研究。通过使用转录因子将iPSC衍生的祖细胞植入小鼠体内，我将 创建第一个人体血液疾病的活体模型，目的是询问潜在的疾病机制，并作为药物测试的平台(目标2a)。在独立阶段，我将更深入地研究，使用IPSC因子系统研究染色体5q缺失的骨髓增生异常综合征，剖析缺失区间内的单个基因对疾病病理生物学的贡献(目标2b)，这将确定靶向治疗的潜在途径。戴利博士是国际上受人尊敬的干细胞生物学研究员，塞缪尔·E·勒克斯四世血液学教授，波士顿儿童医院(BCH)干细胞移植项目主任。戴利博士曾指导过36名行业主要研究人员和小组领导人，并获得了哈佛医学院颁发的A·克利福德·巴格卓越导师奖。BCH是享有盛誉的研究机构和儿科医学中心。BCH是大哈佛研究和医学界医疗和学术机构网络的一部分，其中包括哈佛医学院、Dana-Farber癌症研究所、布里格姆妇女医院、贝丝以色列女执事医疗中心、哈佛干细胞研究所等。这种丰富的环境为年轻科学家提供了培训、开展高影响力研究和促进专业互动的绝佳机会。戴利博士和我已经制定了一份详细的职业发展计划，这将使我能够获得必要的技术、指导和领导技能。我将进一步得到一个干细胞生物学高级领导人委员会的指导：伦纳德·宗博士、斯图尔特·奥尔金博士和戈登·凯勒博士。K99/R00奖项的支持将使我能够全力以赴地执行这一雄心勃勃的项目，目标是在指导阶段结束时发表几篇影响深远的论文。利用我在造血、多能干细胞和原生人体系统方面的专业知识，我将申请R00基金，在一家在干细胞生物学方面享有领先声誉的顶级研究所建立一个独立研究项目。我相信，这项提案中概述的Sklls和创新研究计划的组合将使我能够确立自己作为独立调查员的地位。我的职业目标是成为干细胞生物学领域的领导者，开展多样化和协作性的研究项目，提供对基础生物学的基本见解，同时为翻译、药物设计和基于细胞的疗法创造真正的机会。

DESCRIPTION (provided by applicant): The blood system is a developmental hierarchy maintained by rare hematopoietic stem cells (HSCs) capable of extended self-renewal and multilineage differentiation. Because of their ability to fully reconstitute the blood system upon transplantation, HSCs are a highly valued therapeutic cell type. During my Ph.D. training with Dr. John Dick, I have gained expertise and contributed to understanding of human HSCs from umbilical cord blood (Nat Immunol 2010; Science 2011). In my postdoctoral training with Dr. George Daley, I have directed all my efforts and expertise to the generation of HSCs and other valuable human blood cells, such as transfusable red blood cells, from patient induced-pluripotent stem cells (iPSCs). My work has demonstrated the feasibility of this important goal by showing that human iPSC-derived blood precursors can be converted (or respecified) into transplantable multilineage progenitors using transcription factors: HOXA9, ERG, and RORA (Cell Stem Cell 2013). The idea of respecifying progenitors into stem cells is a promising approach, however we are still short of generating true HSCs. In the mentored part of my research proposal, I will focus on identifying combinations of transcription factors that respecify iPSCs into HSCs with long-term multilineage transplantation potential. In the independent phase, I will study the role of these regulatory networks in normal hematopoiesis. Respecified iPSC progenitors are a particularly potent source of red blood cells in vitro and in vivo. Human erythrocytes undergo globin switching in vivo to express adult hemoglobin mimicking the fetal-to-adult globin switch that occurs after birth. For this reason, I have initiated the study of two congenital anemias using iPSC lines from patients with Diamond Blackfan anemia and sickle cell anemia. By using the transcription factors to engraft iPSC-derived progenitors in mice, I will create the first in vivo models of human blood disorders, with the goal of interrogating the underlying disease mechanisms and as a platform for drug testing (Aim 2a). In the independent phase, I will delve deeper, using the iPSC factor system to study myelodysplastic syndromes with a chromosome 5q deletion to dissect the contribution of individual genes within the deletion interval to disease pathobiology (Aim 2b), which will identify potential avenues for targeted therapies. Dr. Daley is an internationally respected investigator in stem cell biology, the Samuel E. Lux IV Professor of Hematology, and serves as the Director of the Stem Cell Transplantation Program at Boston Children's Hospital (BCH). Dr. Daley has mentored 36 principal investigators and group leaders in industry, and has received the A. Clifford Barger Excellence in Mentoring Award from Harvard Medical School. BCH is a prestigious research institute and a pediatric medical center. BCH is part of a network of medical and academic institutions within the greater Harvard research and medical community, that includes Harvard Medical School, Dana-Farber Cancer Institute, Brigham and Women's Hospital, Beth Israel Deaconess Medical Center, Harvard Stem Cell Institute, and others. This rich environment provides a superb opportunity for a young scientists to train, carry out high impact research, and foster professional interactions. Dr. Daley and I have developed a detailed career development plan that will allow me to acquire the needed technical, mentorship, and leadership skills. I will be further guided by a committee of senior leaders in stem cell biology: Drs. Leonard Zon, Stuart Orkin, and Gordon Keller. The support of this K99/R00 award will allow me to dedicate my full energies to carry out this ambitious project with the goal of producing several high-impact papers by the end of mentored phase. Using my expertise in hematopoiesis, pluripotent stem cells, and primary human systems, I will apply for R00 funds to establish an independent research program at a top institute with a reputation as a leader in stem cell biology. I believe that this combination of sklls and an innovative research plan outlined in this proposal will allow me to establish myself as an independent investigator. My career goal is to become a leader in the field of stem cell biology, to carry out a diverse and collaborative research program that provides fundamental insights into basic biology, while creating real opportunities for translation, drug design, and cell-based therapies.