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

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*，以开发基于干细胞的治疗心血管、肺部和血液疾病。将会有血：干细胞位驱动的干细胞从ES细胞衍生干细胞移植来自高质量来源的组织匹配的骨髓、动员的外周血或脐带血的造血干细胞(HSCs)移植代表着治疗恶性血液病和越来越多的非恶性血液病的治疗选择。合适的骨髓通常供不应求，而脐带血虽然可以储存，但含有的造血干细胞数量要少得多，这使得它不太适合成人移植。人类胚胎干细胞(HES)定向分化为HSCs提供了一个潜在的有吸引力的替代这些传统来源。最近通过诱导多能性方法进行体细胞重编程的报道使这一方法成为未来基于细胞的治疗的一个更令人兴奋的前景。使用来自人类胚胎干细胞(HESCs)的HSC作为上述传统重建细胞来源的治疗替代方案，受到迄今已描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育的特点是原始的HSC从它们在早期胚胎中的出现部位经过胎儿肝脏的扩张期迁移到骨髓，这是成年生命中的造血部位。因此，在它们被具体化并从原始中胚层出现后，随后的HSC的增殖和维持伴随着一系列离散的微环境生态位，这些微环境的细胞组成和特性随着居住器官的不同而不同。这项建议将检验这样一种假设，即从多能干细胞来源(ES和iPS)高效地体外获得具有长期再填充潜力的可移植HSC将需要将分化的多能细胞暴露在这些不同的微环境中，以一种概括造血系统的时间和器官特异性个体发育的方式。这项建议的具体目的是：1.)在化学定义的介质中使用一种新的依赖WNT的协议来指定HES细胞的中胚层。为了实现对中胚层规格的严格外源控制，我们将使用由WNT驱动的最近优化的系统。POWER方法将通过使用(I)由hMIXL启动子驱动的表达GFP的人类ES系来鉴定Flk-1造血中胚层；(Ii)在PI的实验室中发现的HSC的其他细胞表面标记(包括ACE/CD143和PODXL)的使用将进一步增强。2.)为了培养中胚层命中注定的HES细胞的后代，在基质细胞群体的存在下，这些间质细胞群体暂时概括了造血个体发育过程中存在的干细胞龛。在不同的条件下，ES来源的感兴趣群体(依赖于AIM 1)将与PI的实验室先前从AGM、胎肝和成人骨髓(成骨细胞和血管壁龛)产生的条件永生化的基质细胞系(来自永生化的小鼠)按顺序共培养。在每种条件下产生的原始造血细胞将被检测其免疫表型特性和克隆性造血祖细胞的含量。在最能促进原始造血细胞出现的条件下产生的细胞将通过向免疫缺陷NOD/LtSz-SCID IL2R缺失小鼠股内注射来检测它们的HSC含量。HSC的含量将通过有限稀释和连续移植来定量。这一建议的优势在于研究人员的联合，他们共同带来了在人类造血干细胞生物标记物的识别和基质细胞对造血的调控(Simmons；PI)、HES细胞的中胚层规范(Nakayama)以及基质细胞系的派生和分子分析(Brouard)方面的相当专业知识。 公共卫生相关性：移植来自高质量来源的组织匹配的骨髓、动员的外周血或脐带血的造血干细胞(HSCs)目前是治疗血液系统恶性肿瘤和越来越多的非恶性血液疾病的首选治疗选择。来自这些来源的适当匹配的HSC的有限供应是对血液学移植的安全性和更广泛的实用性的重大限制，促使人们寻找替代的HSC来源。这项建议旨在开发显着改进的策略，从多能干细胞中提取HSC，作为克服这一限制的一种手段，并进一步开发治疗血液疾病的新型细胞疗法。

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.