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There Will Be Blood: Stem Cell Niche Driven Derivation of HSC from ES Cells

将会有血液：干细胞利基驱动从 ES 细胞衍生 HSC

基本信息

批准号：
7810978
负责人：
Naoki Nakayama
金额：
$ 100万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-07 至 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7810978
关键词：
Address Adult Area BMP4 Biological Assay Biological Markers Blood Blood Vessels Bone Marrow Cardiovascular system Cell Line Cell Therapy Cell Transplantation Cell surface Cells Coculture Techniques Derivation procedure Development Elements Embryo Engineering Fetal Liver Future Growth Hematologic Neoplasms Hematological Disease Hematopoiesis Hematopoietic Hematopoietic Stem Cell Transplantation Hematopoietic System Hematopoietic stem cells Human IL2RA gene Immunosuppression In Vitro Incentives Infection Injection of therapeutic agent Knockout Mice Life Lung Maintenance Mesoderm Methodology Molecular Analysis Morbidity - disease rate Non-Malignant Organ Osteoblasts Patients Pluripotent Stem Cells Population Property Protocols documentation Regenerative Medicine Regulation Reporting Research Personnel Risk Safety Second Primary Cancers Series Site Somatic Cell Source Stem cells Stromal Cells System Testing Therapeutic Tissues Transplantation Umbilical Cord Blood activin A embryonic stem cell graft vs host disease human embryonic stem cell improved induced pluripotent stem cell interest migration mortality novel peripheral blood pluripotency podocalyxin promoter public health relevance reconstitution residence stem stem cell niche success

项目摘要

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (11) Regenerative Medicine - 11-HL-101* To develop stem cell based therapies for cardiovascular, lung and blood diseases. There Will be Blood: Stem cell niche driven derivation of HSC from ES cells Transplantation of hematopoietic stem cells (HSCs) derived from high-quality sources of tissue-matched bone marrow, mobilized peripheral blood or umbilical cord blood represents the therapeutic option of choice for the treatment of hematological malignancies and an increasing array of non-malignant blood disorders. Suitable bone marrow is often in short supply, and cord blood, although bankable, contains much lower numbers of HSCs, which makes it less suitable for adult transplantation. Directed differentiation of human embryonic stem (hES) cells toward HSCs offers a potentially attractive alternative to these conventional sources. The recent reports of somatic cell reprogramming by means of induced pluripotency makes this approach an even more exciting prospect for future cell-based therapies. The use of HSC derived from human embryonic stem cells (hESCs) as a therapeutic alternative to the traditional sources of reconstituting cells listed above is limited by the low efficiency of derivation protocols that have been described to date. The ontogeny of the mammalian hematopoietic system is marked by a temporally regulated migration of primitive HSC from their site of emergence in the early embryo via a period of expansion in the fetal liver to the bone marrow, the site of hematopoiesis throughout adult life. Thus following their specification and emergence from primitive mesoderm, the subsequent proliferation and maintenance of HSC occurs in association with a series of discrete microenvironmental niches whose cellular composition and properties vary with the organ of residence. This proposal will test the hypothesis that efficient in vitro derivation of transplantable HSC with long term repopulating potential from pluripotent stem cell sources (ES and iPS) will require exposure of the differentiating pluripotent cells to elements of these distinct microenvironmental niches in a manner that recapitulates both the temporal and organ-specific ontogeny of the hematopoietic system. Specific aims of this proposal are: 1.) To use a novel WNT- dependent protocol in chemically defined medium for the mesoderm specification of hES cells. To achieve stringent exogenous control over mesoderm specification we will utilize a recently optimized system driven by Wnt. The power approach will be further enhanced by the use of (i) a human ES line engineered to express GFP driven by the hMIXL promoter to allow identification of Flk-1+ hematopoietic mesoderm; (ii) additional cell surface markers of HSC identified in the PI's lab (including ACE/CD143 and PODXL). 2.) To culture the mesoderm-fated progeny of hES cells in the presence of stromal cell populations which temporally recapitulate stem cell niches present during hematopoietic ontogeny ES-derived populations of interest (dependent on Aim 1) will be co-cultured in sequence under various conditions with conditionally immortalised stromal cell lines (derived from the Immortomouse) previously generated in the PI's lab from the AGM, fetal liver and adult bone marrow (osteoblast and vascular niches). Primitive hematopoietic cells generated under each condition will be assayed for their immunophenotypic properties and content of clonogenic hematopoietic progenitor cells. Cells generated under conditions that best promote the emergence of primitive hematopoietic cells will be assayed for their content of HSC by intrafemoral injection into immunodeficient NOD/LtSz-Scid IL2R null mice. HSC content will be quantitated by limitdilution and serial transplants. The strength of this proposal lies in the consortium of investigators who collectively bring considerable expertise in the identification of biomarkers of human HSC and the stromal cell regulation of hematopoiesis (Simmons; PI), the mesodermal specification of hES cells (Nakayama) and in the derivation and molecular analysis of stromal cell lines (Brouard). PUBLIC HEALTH RELEVANCE: Transplantation of hematopoietic stem cells (HSCs) derived from high-quality sources of tissue-matched bone marrow, mobilized peripheral blood or umbilical cord blood currently represents the therapeutic option of choice for the treatment of hematological malignancies and an increasing array of non-malignant blood disorders. The limited supply of suitably matched HSC from these sources is a significant limitation to the safety and broader utility of haematological transplantation, prompting the search for alternative sources of HSC. This proposal seeks to develop markedly improved strategies to derive HSC from pluripotent stem cells as a means to overcome this limitation and to further the development of novel cellular therapies for treating blood diseases.

描述（由申请人提供）：本申请涉及广泛的挑战领域（11）再生医学-11-HL-101 *，以开发用于心血管、肺和血液疾病的基于干细胞的疗法。会有血：干细胞小生境驱动的造血干细胞（HSC）从ES细胞的衍生移植源自组织匹配的骨髓、动员的外周血或脐带血的高质量来源的造血干细胞（HSC）代表了用于治疗恶性血液病和越来越多的非恶性血液疾病的治疗选择。合适的骨髓通常供应不足，而脐带血虽然可以储存，但含有的HSC数量要少得多，这使得它不太适合成人移植。人胚胎干细胞（hES）向HSC的定向分化为这些常规来源提供了一个潜在的有吸引力的替代方案。最近的报道，体细胞重编程的手段诱导多能性，使这种方法更令人兴奋的前景为未来的细胞为基础的治疗。使用衍生自人胚胎干细胞（hESC）的HSC作为上述重建细胞的传统来源的治疗替代方案受到迄今为止所述的衍生方案的低效率的限制。哺乳动物造血系统的个体发生的标志是原始HSC从其在早期胚胎中出现的部位经在胎儿肝脏中的扩增期到骨髓（整个成人生命中的造血部位）的时间调节的迁移。因此，在它们从原始中胚层的特化和出现之后，HSC的随后增殖和维持与一系列离散的微环境龛相关联地发生，所述微环境龛的细胞组成和性质随居住器官而变化。该提议将测试以下假设：从多能干细胞来源（ES和iPS）有效地体外衍生具有长期再增殖潜力的可移植HSC将需要以概括造血系统的时间和器官特异性个体发生的方式将分化的多能细胞暴露于这些不同微环境小生境的元素。本提案的具体目标是：1）采用一种新的WNT依赖的化学成分确定的培养基中的hES细胞的中胚层特化方案。为了实现对中胚层规格的严格外源控制，我们将利用最近优化的由Wnt驱动的系统。通过使用（i）经工程改造以表达由hMIXL启动子驱动的GFP以允许鉴定Flk-1+造血中胚层的人ES系;（ii）在PI实验室中鉴定的HSC的其他细胞表面标志物（包括ACE/CD143和PODXL），将进一步增强功效方法。2.）的情况。在存在基质细胞群的情况下培养hES细胞的中胚层命运的后代，所述基质细胞群暂时重现在造血个体发育ES衍生的感兴趣的群体期间存在的干细胞小生境。（取决于Aim 1）将在各种条件下与条件永生化基质细胞系按顺序共培养（来源于Immortomouse），先前在PI实验室中从AGM、胎儿肝脏和成人骨髓（成骨细胞和血管龛）中产生。将测定在每种条件下生成的原始造血细胞的免疫表型特性和克隆生成造血祖细胞的含量。将在最佳促进原始造血细胞出现的条件下产生的细胞通过股内注射到免疫缺陷型NOD/LtSz-Scid IL2R缺失小鼠中来测定其HSC含量。HSC含量将通过有限稀释和连续移植进行定量。该提议的优势在于研究者联盟，他们共同带来了相当多的专业知识，用于鉴定人HSC的生物标志物和造血的基质细胞调节（Simmons; PI）、hES细胞的中胚层特化（Nakayama）以及基质细胞系的衍生和分子分析（Brouard）。公共卫生关系：来源于组织匹配的骨髓、动员的外周血或脐带血的高质量来源的造血干细胞（HSC）的移植目前代表了用于治疗血液恶性肿瘤和越来越多的非恶性血液疾病的治疗选择。这些来源的合适匹配的HSC的有限供应是对血液移植的安全性和更广泛的效用的显著限制，促使寻找HSC的替代来源。该提案旨在开发显著改进的策略，以从多能干细胞中获得HSC，作为克服这种限制的手段，并进一步开发用于治疗血液疾病的新型细胞疗法。