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

描述（由申请人提供）：造血干细胞（HSC）位于专门的壁龛中，并从周围的基质细胞接收信号，但它们的身份及其对HSC的影响在很大程度上是未知的。我们的工作重点是定义分泌的生长因子，由基质细胞产生，并触发HSC体外扩增。我们从胚胎第15天（E15）胎肝中鉴定出一种新的细胞群，具有早期T细胞的表面表型，并支持HSC的体外扩增。这项资助的主要目的是纯化和表征几种E15.5 FL T细胞亚群在培养中扩增HSC的能力，以确定在成年小鼠骨髓和脾脏中是否发现类似的细胞，以确定这些细胞是否是HSC生态位的一部分，并检查能够支持HSC的成人和胎儿T细胞群体中基因表达之间的差异和相似性，开发HSC扩增的细胞和分子模型。我们的DNA微阵列实验显示，在其它蛋白质中，IGF - 2和血管生成素样蛋白2（Angptl 2）和3（Angptl 5）在这些E15 FL早期T细胞中特异性表达.然后，我们开发了含有低水平SCF、TPO、FGF-1、IGF- 2和Angptl 2或Angptl 5的无血清HSC培养系统;如通过竞争性再增殖分析所测量的，在培养10天后，长期再增殖HSC（LT-HSC）的数量增加>24。Angptl 2和Angptl 5在很大程度上未被研究，并且先前未被建议作用于造血或干细胞。在这里，我们将测试IGF-2、TPO、Angptl 2和其他生长因子是否各自是离体最大HSC扩增所需的，因此这些中的每一种是否可能激活HSC中的不同信号传导途径。平行地，我们将采用高分辨率细胞追踪程序来确定Angptl 2、IGF-2、Tpo和随后的其他因子是否直接刺激HSC自我更新或阻止HSC凋亡或分化。Angptl 2或Angptl 5的受体和信号转导途径是未知的;我们将使用在我的实验室中开发和广泛使用的两种表达克隆方案来分离Angptl 2和Angptl 5细胞表面受体，然后广泛地表征它们。我们的研究可以导致造血干细胞在骨髓移植中治疗白血病和其他癌症的临床应用的重大改进;许多应用，特别是使用脐带血的移植，受到造血干细胞数量少和相容供体不可用的阻碍。造血干细胞也是一个有前途的细胞靶点，用于开发基因疗法，治疗各种遗传性免疫缺陷综合征和先天性代谢缺陷。在基因治疗的情况下，离体扩增HSC的能力将大大增强这些临床应用，从而允许选择在特定染色体位置整合了转基因的那些HSC。

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.