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: 8706144
• 负责人: Patrick Cahan
• 金额: $ 15.63万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 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/8706144
• 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; 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; 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; Stem cells; 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; oncology; progenitor; programs; public health relevance; receptor; reconstitution; research study; self-renewal; skills; stem; stem cell biology; 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(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，是所有血细胞的祖细胞，包括所有髓系和淋巴系。与许多其他成体干细胞不同的是，一组细胞中是否存在HSC可以得到明确的评估。这是通过移植实验实现的，在移植实验中，捐赠者细胞被静脉注射到受到致命性照射的免疫缺陷受体小鼠体内。单个HSC可以重建完整的造血系统长达六个月。事实上，造血干细胞对骨髓移植患者的造血室的再生负有责任。虽然造血干细胞移植仍然是临床上最成功的干细胞治疗，但适合治疗的造血干细胞的可用性是有限的。多能干细胞(PSC)具有分化为生物体所有细胞的能力和无限制的自我更新能力，是体外诱导HSCs的良好起点。PSC来源的HSC(PSC-HSC)将是一个非常有价值的平台，可以用来模拟控制HSC和造血室的产生以及血液病的遗传程序。此外，随着诱导多能干细胞的出现，患者特异性的PSC-HSC最终可能被用于治疗目前(如果可能的话)使用同种异体或自体骨髓移植治疗的各种疾病，特别是血液系统恶性肿瘤。Daley实验室首次通过ESCs的定向分化、异位表达同源盒转录因子HOXB4以及与OP9基质细胞共培养来培育出可移植的小鼠PSC-HSCs(Kyba等人，2002年)。虽然这些PSC-HSC对宿主的造血室做出了多系贡献，但这些细胞的淋巴潜能和长期植入是有限的。由于更好地理解在体内协调和指定HSC的发育程序将指导更忠实地派生PSC-HSCs，我们对小鼠胚胎中纯化的HSCs及其直接前体进行了基因表达谱分析(McKinney-Freeman等人，2012)。从这项研究中，我们了解到HOXB4诱导的PSC-HSCs与成人的最终HSCs比更原始的早期胚胎HSCs更相似，这表明只有有限的一组程序将PSC-HSC与HSC区分开来。这里提出的研究将确定两种候选遗传方案对当前PSC-HSC有限的自我更新和淋巴潜能的贡献。目的1确定HOXB4诱导的PSC-HSCs后部HoxA表达的功能后果。目的2将评估Notch信号在HOXB4诱导的PSC-HSCs淋巴生成缺陷中的作用。这项为期五年的培训计划旨在确立帕特里克·卡汉博士作为干细胞生物学独立研究员的地位。拟议的研究和培训计划将在乔治·戴利博士在波士顿儿童医院血液/肿瘤科的实验室进行，这里也是Orkin、Williams和Zon博士的实验室，他们是国际知名的研究人员，一直在干细胞生物学、发育、造血、化学生物学、转录、细胞周期和癌症遗传学领域进行开创性研究。这位候选人在圣路易斯的华盛顿大学研究生院接受过计算生物学家的培训，在那里他研究了DNA拷贝数变化在造血干细胞和祖细胞中的功能后果。作为波士顿儿童医院戴利实验室的博士后研究员，卡恩博士的主要研究领域是阐明体内血液发育和定向从PSCs分化血液之间的差异，长期目标是提高体外来源的群体模拟体内群体的保真度。要成为一名跨学科和独立的研究人员，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，FACS分析、骨髓移植和体外造血分析)，并提高成为一名有竞争力的研究人员所需的领导和管理技能，包括资质。广泛的正规培训、课程作业、与咨询委员会的会议以及戴利博士的专职监督和指导将确保这些目标的实现。