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

描述（由申请人提供）：该申请涉及广泛的挑战领域（11）再生医学- 11- hl -101*开发基于干细胞的心血管，肺部和血液疾病疗法。造血干细胞（HSC）的移植来源于组织匹配的高质量骨髓、动员的外周血或脐带血，这是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，而脐带血虽然可供储备，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）定向分化为造血干细胞提供了一个潜在的有吸引力的替代这些传统来源。最近关于体细胞重编程的报道通过诱导多能性使这种方法成为未来基于细胞的治疗更令人兴奋的前景。人类胚胎干细胞（hESCs）衍生的造血干细胞作为上述传统细胞重建来源的治疗替代品的使用受到迄今为止所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生的特点是，原始造血干细胞从早期胚胎中出现的部位，经过胎儿肝脏的一段时间扩张，暂时迁移到骨髓，而骨髓是整个成年生命的造血部位。因此，随着原始中胚层的形成和出现，造血干细胞的增殖和维持与一系列离散的微环境生态位有关，这些微环境生态位的细胞组成和性质随居住器官的不同而变化。该提案将验证这样一个假设，即从多能干细胞来源（ES和iPS）中高效地体外衍生具有长期再生潜力的可移植HSC，需要将分化的多能细胞暴露于这些不同微环境的元素中，以一种概括造血系统的时间和器官特异性个体发生的方式。本提案的具体目标是：1)在化学定义的培养基中使用一种新的依赖于WNT的方法来培养hES细胞的中胚层。为了实现对中胚层规格的严格外源控制，我们将利用由Wnt驱动的最近优化的系统。power方法将通过使用(i)通过hMIXL启动子驱动表达GFP的人类ES系进一步增强，从而鉴定Flk-1+造血中胚层；（ii） PI实验室鉴定的其他HSC细胞表面标记物（包括ACE/CD143和PODXL）。2)。为了在造血个体发育过程中，在基质细胞群的存在下培养hES细胞的中胚层命运后代，这些基质细胞群暂时再现了干细胞龛，es衍生的感兴趣群体（依赖于Aim 1）将在各种条件下与PI实验室先前从AGM、胎儿肝脏和成人骨髓（成骨细胞和血管龛）中产生的有条件永生化基质细胞系（来自永生化小鼠）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，将通过向免疫缺陷的NOD/LtSz-Scid IL2R缺失小鼠经股内注射，检测其HSC含量。HSC含量将通过有限稀释和连续移植来定量。该提案的优势在于研究人员联盟，他们在人类HSC生物标志物的鉴定和造血基质细胞的调节（Simmons； PI）， hES细胞的中胚层规范（Nakayama）以及基质细胞系的衍生和分子分析（Brouard）方面共同带来了相当多的专业知识。

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.