Growth factors and engineered stroma for hematopoietic stem csll (HSC) expansion

描述(申请人提供)：造血干细胞(HSCs)存在于特殊的壁龛中，接受来自周围基质细胞的信号，但它们的身份和它们对HSCs的影响在很大程度上是未知的。我们的工作重点是确定由基质细胞产生并在体外触发HSC扩增的分泌性生长因子。我们从胚胎第15天(E15)的胎肝中鉴定出一种新的细胞群，该细胞群具有早期T细胞的表面表型，支持HSCs的体外扩增。这项资助的主要目的是纯化和鉴定几个E15.5FL T细胞亚群在培养中扩增HSC的能力，确定是否在成年小鼠的骨髓和脾中发现类似的细胞，确定这些细胞是否属于HSC生态位的一部分，并研究能够支持HSC的成人和胎儿T细胞群体中基因表达的异同，以建立HSC扩增的细胞和分子模型。我们的DNA芯片实验表明，在其他蛋白质中，IGF-2和血管生成素样蛋白2(Angpt2)和3(AngptIS)在这些E15 FL早期T细胞中特异表达。然后我们建立了一种含有低水平SCF、TPO、成纤维细胞生长因子-1、IGF-2和Angpt2或AngptIS的无血清HSC培养体系；通过竞争性再繁殖分析，培养10天后长期再填充HSC(LT-HSC)的数量增加了24。Angptl2和AngptIS在很大程度上还没有被研究过，以前也没有被建议对造血或干细胞起作用。在这里，我们将测试IGF-2、TPO、Angptl2和其他生长因子是否都是最大限度地体外扩增HSC所必需的，因此是否每种因素都可能激活HSC中不同的信号通路。同时，我们将采用高分辨率的细胞跟踪程序来确定Angptl2、IGF-2、TPO以及后来的其他因素是否直接刺激HSC自我更新或阻止HSC的凋亡或分化。Angpt1 2或AngptIS的受体和信号转导途径(S)尚不清楚；我们将使用实验室开发并广泛使用的两种表达克隆方法来分离Angpt1 2和AngptIS细胞表面受体(S)，并对其进行广泛的表征。我们的研究可以使造血干细胞在治疗白血病和其他癌症的骨髓移植中的临床应用得到重大改进；许多应用，特别是使用脐带血的移植，由于造血干细胞数量少和供者相合而受到阻碍。HSCs也是一个很有希望的细胞靶点，可以开发基因疗法来治疗各种遗传性免疫缺陷综合征和先天性代谢错误。在体外扩增造血干细胞的能力将极大地增强这些临床应用，在基因治疗的情况下，允许选择那些在特定染色体位置整合了转基因的造血干细胞。

DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSCs) reside in specialized niches and receive signals from surrounding stromal cells, but their identities and the effects they have on HSCs are largely unknown. Our work focuses on defining secreted growth factors that are produced by stromal cells and that trigger HSC expansion ex vivo. We identified a novel cell population from Embryonic Day 15 (E15) fetal livers with the surface phenotype of early T cells and that supports ex vivo expansion of HSCs. Major aims of this grant are to purify and characterize several E15.5 FL T cell subpopulations for their ability to expand HSCs in culture, to determine whether similar cells are found in the adult mouse bone marrow and spleen, to determine whether these are part of the HSC niche, and to examine differences and similarities between gene expression in adult and fetal populations of T cells capable of supporting HSCs in order to develop a cellular and molecular model of HSC expansion. Our DNA microarray experiments showed that, among other proteins, IGF - 2 and Angiopoietin- like proteins 2 (Angptl2) and 3 (AngptIS) are specifically expressed in these E15 FL early T cells. We then developed a serum- free HSC culture system containing low levels of SCF, TPO, FGF-1, IGF- 2 and Angptl2 or AngptIS; as measured by competitive repopulation analyses there was a >24 increase in numbers of long-term repopulating HSCs (LT-HSC) after 10 days of culture. Angptl2 and AngptIS are largely unstudied and have not previously been suggested to act on hematopoiesis or stem cells. Here we will test whether IGF-2, TPO, Angptl2, and other growth factors are each required for maximum HSC expansion ex vivo and thus whether each of these likely activates different signaling pathways in HSCs. In parallel we will adopt a high-resolution cell tracking procedure to determine whether Angptl2, IGF-2, Tpo, and later other factors directly stimulates HSC self- renewal or prevents HSC apoptosis or differentiation. The receptors for Angptl2 or AngptIS and signal transduction pathway(s) are unknown; we will use two expression cloning protocols developed and extensively utilized in my laboratory to isolate the Angptl2 and AngptIS cell surface receptor(s), and then to characterize them extensively. Our studies can lead to major improvements in the clinical use of HSCs in bone marrow transplantation for treatment of leukemia and other cancers; many applications, especially transplantation using cord blood, have been hindered by the low numbers of HSCs and by the unavailability of compatible donors. HSCs are also a promising cell target for developing gene therapies for treating a broad variety of inherited immunodeficiency syndromes and inborn errors of metabolism. The ability to expand HSCs ex vivo would greatly enhance these clinical applications, in the case of gene therapy allowing selection of those HSCs that have integrated a transgene in a specific chromosomal location.