Pluripotent stem cell-derived HSCs: improvements and molecular mechanisms

多能干细胞衍生的 HSC：改进和分子机制

• 批准号: 9176876
• 负责人: Patrick Cahan
• 金额: $ 12.1万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9176876
• 关键词: Achievement; Adult; Advisory Committees; Affect; Allogenic; Area; Autologous Bone Marrow Transplantation; Bioinformatics; Biological Assay; Biology; Blood; Blood Cells; Bone Marrow; Bone Marrow Transplantation; Boston; Cell Culture Techniques; Cell Cycle; Cells; Chemicals; Chimerism; Coculture Techniques; Copy Number Polymorphism; DNA copy number; Derivation procedure; Development; Developmental Biology; Disease; Disease model; Dissection; Ectopic Expression; Embryo; Engraftment; Ensure; Exhibits; Foundations; Gene Expression Profile; Gene Expression Profiling; Gene Targeting; Generations; Genes; Genetic Programming; Genetic Transcription; Goals; Health; Hematologic Neoplasms; Hematological Disease; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic System; Hematopoietic stem cells; Hereditary Disease; Home environment; Homeobox; Human; Immune; In Vitro; Laboratories; Leadership; Learning; Lymphoid; Lymphoid Cell; Lymphopoiesis; Mammals; Mentorship; Methods; Modeling; Molecular; Mus; Myelogenous; Oncology; Organism; Pathway interactions; Patients; Pediatric Hospitals; Pluripotent Stem Cells; Population; Postdoctoral Fellow; Production; Regulator Genes; Relative (related person); Research; Research Personnel; Research Training; Role; Schools; Signal Transduction; Specific qualifier value; Staging; Stem Cell Development; Stem Cell Research; Stromal Cells; Supervision; T-Lymphocyte; Testing; Training; Training Programs; Transplantation; Universities; Washington; Work; adult stem cell; cancer genetics; cancer stem cell; clinical application; clinically relevant; design; embryonic stem cell; gene therapy; improved; in vitro Model; in vivo; induced pluripotent stem cell; insight; knock-down; meetings; notch protein; novel; progenitor; programs; receptor; reconstitution; research study; self-renewal; skills; stem; stem cell biology; stem cells; therapy development; transcription factor

DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSC), largely residing in the bone marrow of adult mammals, are the progenitors of all blood cells, including all myeloid and lymphoid lineages. Unlike many other adult stem cells, the presence of an HSC within a population of cells can be assessed definitively. This is accomplished by transplantation experiments, where donor cells are injected intravenously into lethally irradiated, immunodeficient recipient mice. A single HSC can reconstitute the complete hematopoietic system for up six months. Indeed, HSCs are responsible for the repopulation of the hematopoietic compartment of bone marrow transplantation patients. While HSC transplantation remains the most clinically successful stem cell treatment, the availability of HSCs suitable for therapy is limited. Pluripotent stem cells (PSC), functionally defined by their ability to differentiate into all cells of an organism and their unlimited self-renewal, are an appealing starting point for the in vitro derivation of HSCs. PSC-derived HSCs (PSC-HSC) would be invaluable as a platform to model the genetic programs that govern the HSC and production of the hematopoietic compartment, as well as hematological diseases. Furthermore, with the advent of induced pluripotent stem cells, patient-specific PSC- HSC ultimately may be used to treat a variety of diseases currently treated (when possible) with allogeneic or autologous bone marrow transplants, especially hematological malignancies. The Daley lab first produced transplantable mouse PSC-HSCs by directed differentiation of ESCs, ectopic expression of the homeobox transcription factor HoxB4, and co-culture with OP9 stromal cells (Kyba et al 2002). While these PSC-HSCs make multilineage contributions to the host hematopoietic compartment, the lymphoid potential and long-term engraftment of these cells were limited. Reasoning that a better understanding of the developmental programs that orchestrate and specify the HSC in vivo would guide a more faithful derivation of PSC-HSCs, we performed gene expression profiling of purified HSCs and their immediate precursors at key developmental stages from the mouse embryo (McKinney-Freeman et al 2012). From this study, we learned that the Hoxb4- induced PSC-HSCs were globally more similar to the definitive HSCs of the adult than the more primitive HSCs of the early embryo, suggesting that only a limited set of programs distinguish PSC-HSC from HSC. The studies proposed here will determine the contribution of two candidate genetic programs to the limited self- renewal and lymphoid potential of current PSC-HSC. Aim 1 will determine the functional consequences of posterior HoxA expression in Hoxb4-induced PSC-HSCs. Aim 2 will assess the contribution of Notch signaling to deficient lymphopoiesis in Hoxb4-induced PSC-HSCs. The five-year training program has been designed to establish Dr. Patrick Cahan as an independent investigator in Stem Cell Biology. The proposed research and training program will be carried out in Dr. George Daley's lab at Boston Children's Hospital in the Division of Hematology/Oncology, which is also home to the laboratories of Drs. Orkin, Williams, and Zon, internationally known investigators who have consistently produced ground-breaking research in the areas of stem cell biology, development, hematopoiesis, chemical biology, transcription, cell cycle, and cancer genetics. The candidate was trained as a Computational Biologist in graduate school at Washington University in St. Louis, where he investigated the functional consequences of DNA copy number variation in hematopoietic stem and progenitors. As a postdoctoral fellow in the Daley Lab at Boston Children's Hospital, Dr. Cahan's central area of research is elucidating the differences between in vivo blood development and directed differentiation of blood from PSCs with the long-term goal of improving the fidelity with which in vitro derived populations mimic their in vivo counterparts. To become an interdisciplinary and independent investigator, and to complete the proposed research, the candidate needs to solidify his foundation of Developmental Biology, to expand his experimental expertise (e.g. FACS analysis, bone marrow transplant, and in vitro hematopoietic assays), and improve the leadership and administrative skills required to become a competitive investigator, including grantsmanship. An extensive program of formal training, course-work, meetings with the advisory committee, and dedicated supervision and mentorship by Dr. Daley will ensure that these goals are achieved.

描述（由申请人提供）：造血干细胞（HSC）主要存在于成年哺乳动物的骨髓中，是所有血细胞（包括所有髓系和淋巴系）的祖细胞。与许多其他成体干细胞不同，HSC在细胞群中的存在可以明确评估。这是通过移植实验来实现的，其中供体细胞被静脉注射到致死辐射的免疫缺陷受体小鼠中。单个HSC可以重建完整的造血系统长达六个月。事实上，HSC负责骨髓移植患者造血区室的再增殖。虽然HSC移植仍然是临床上最成功的干细胞治疗方法，但适合治疗的HSC的可用性有限。多能干细胞（PSC），功能上定义为它们分化成生物体的所有细胞的能力和它们的无限自我更新，是HSC体外衍生的一个有吸引力的起点。PSC衍生的HSC（PSC-HSC）将作为对控制HSC和造血区室的产生以及血液学疾病的遗传程序进行建模的平台是非常宝贵的。此外，随着诱导多能干细胞的出现，患者特异性PSC-HSC最终可用于治疗目前用同种异体或自体骨髓移植治疗的多种疾病（当可能时），特别是血液恶性肿瘤。Daley实验室首先通过ESC的定向分化、同源框转录因子HoxB 4的异位表达以及与0 P9基质细胞的共培养来产生可移植的小鼠PSC-HSC（Kyba等2002）。虽然这些PSC-HSC对宿主造血区室做出多谱系贡献，但这些细胞的淋巴潜能和长期植入是有限的。由于更好地理解体内协调和指定HSC的发育程序将指导PSC-HSC的更忠实的衍生，我们在小鼠胚胎的关键发育阶段对纯化的HSC及其直接前体进行了基因表达谱分析（McKinney-Freeman et al 2012）。从这项研究中，我们了解到，Hoxb 4诱导的PSC-HSC在总体上比早期胚胎的更原始的HSC更类似于成年人的定形HSC，这表明只有有限的一组程序将PSC-HSC与HSC区分开来。本文提出的研究将确定两个候选遗传程序对当前PSC-HSC的有限自我更新和淋巴潜能的贡献。目的1将确定Hoxb 4诱导的PSC-HSC中后部HoxA表达的功能后果。目的2将评估Notch信号传导对Hoxb 4诱导的PSC-HSC中淋巴细胞生成缺陷的贡献。 为期五年的培训计划旨在将帕特里克卡汉博士确立为干细胞生物学的独立研究者。拟议的研究和培训计划将在波士顿儿童医院血液学/肿瘤学部的乔治戴利博士实验室进行，该实验室也是Orkin博士、威廉姆斯博士和Zon博士实验室的所在地，他们是国际知名的研究人员，在干细胞生物学、发育、造血、化学生物学、转录、细胞周期、和癌症遗传学这位候选人在圣路易斯的华盛顿大学研究生院接受了计算生物学家的培训，在那里他研究了造血干细胞和祖细胞中DNA拷贝数变异的功能后果。作为波士顿儿童医院戴利实验室的博士后研究员，Cahan博士的中心研究领域是阐明体内血液发育与PSC血液定向分化之间的差异，其长期目标是提高体外衍生群体模仿体内对应物的保真度。要成为一名跨学科和独立的研究者，并完成拟议的研究，候选人需要巩固他的发育生物学基础，扩大他的实验专业知识（例如，流式细胞仪分析，骨髓移植和体外造血测定），并提高成为一名有竞争力的研究者所需的领导和管理技能，包括granectin。戴利博士的广泛正式培训、课程作业、与咨询委员会的会议以及专门的监督和指导计划将确保实现这些目标。