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: 8581567
• 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/8581567
• 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（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（包括所有骨髓和淋巴谱系）的祖细胞。与许多其他成体干细胞不同，可以明确评估细胞群中 HSC 的存在。这是通过移植实验来实现的，其中将供体细胞静脉注射到受到致命辐射的免疫缺陷受体小鼠中。单个 HSC 可以在长达六个月的时间内重建完整的造血系统。事实上，造血干细胞负责骨髓移植患者造血室的重新增殖。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) 的功能定义为其分化为生物体所有细胞的能力及其无限的自我更新能力，是体外衍生 HSC 的一个有吸引力的起点。 PSC 衍生的 HSC (PSC-HSC) 作为对控制 HSC 和造血室生成以及血液疾病的遗传程序进行建模的平台将具有无价的价值。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（如果可能）用同种异体或自体骨髓移植治疗的多种疾病，特别是血液恶性肿瘤。 Daley 实验室首先通过 ESC 的定向分化、同源盒转录因子 HoxB4 的异位表达以及与 OP9 基质细胞共培养来生产可移植的小鼠 PSC-HSC（Kyba 等，2002）。虽然这些 PSC-HSC 对宿主造血室做出多谱系贡献，但这些细胞的淋巴潜能和长期植入受到限制。由于更好地理解体内协调和指定 HSC 的发育程序将指导更忠实地衍生 PSC-HSC，我们对小鼠胚胎关键发育阶段的纯化 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，与早期胚胎中更原始的 HSC 相比，Hoxb4 诱导的 PSC-HSC 在整体上与成人的最终 HSC 更相似，这表明只有一组有限的程序可以区分 PSC-HSC 和 HSC。这里提出的研究将确定两个候选遗传程序对当前 PSC-HSC 有限的自我更新和淋巴潜力的贡献。目标 1 将确定 Hoxb4 诱导的 PSC-HSC 中后部 HoxA 表达的功能后果。目标 2 将评估 Notch 信号传导对 Hoxb4 诱导的 PSC-HSC 淋巴细胞生成缺陷的影响。 这项为期五年的培训计划旨在使帕特里克·卡汉博士成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/肿瘤科乔治·戴利博士的实验室进行，该实验室也是乔治·戴利博士的实验室所在地。 Orkin、Williams 和 Zon 是国际知名的研究人员，他们在干细胞生物学、发育、造血、化学生物学、转录、细胞周期和癌症遗传学领域不断进行突破性的研究。该候选人在圣路易斯华盛顿大学研究生院接受了计算生物学家培训，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 Daley 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和 PSC 血液定向分化之间的差异，长期目标是提高体外衍生群体模仿体内对应群体的保真度。要成为一名跨学科的独立研究者，并完成拟议的研究，候选人需要巩固其发育生物学基础，扩展其实验专业知识（例如 FACS 分析、骨髓移植和体外造血测定），并提高成为竞争性研究者所需的领导和管理技能，包括资助能力。广泛的正式培训、课程作业、与咨询委员会的会议以及戴利博士的专门监督和指导将确保这些目标的实现。