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

描述（由申请人提供）：造血干细胞（Hematopoietic stem cells, hsc）存在于特定的壁龛中，并接受来自周围基质细胞的信号，但它们的身份及其对hsc的影响在很大程度上是未知的。我们的工作重点是确定基质细胞产生的分泌生长因子，并触发HSC体外扩增。我们从胚胎第15天（E15）的胎儿肝脏中鉴定出一种新的细胞群，其表面表型为早期T细胞，并支持造血干细胞的体外扩增。格兰特的主要目的是净化和描述几个E15.5 FL T细胞亚群的能力扩大肝星状细胞在文化、确定类似的细胞在成年小鼠骨髓和脾脏,确定这些是HSC利基市场的一部分,并检查基因表达之间的相同点与不同点在成人和胎儿T细胞的数量能够支持肝星状细胞为了培养的细胞和分子模型HSC扩张。我们的DNA微阵列实验表明，在其他蛋白中，IGF - 2和血管生成素样蛋白2 （Angptl2）和3 （AngptIS）在这些E15 FL早期T细胞中特异性表达。然后，我们开发了一种无血清的HSC培养系统，其中含有低水平的SCF、TPO、FGF-1、IGF- 2和Angptl2或AngptIS；通过竞争性再繁殖分析，培养10天后，长期再繁殖的hsc （LT-HSC）的数量增加了bbbb24。Angptl2和AngptIS在很大程度上没有被研究过，以前也没有被认为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体外最大限度的HSC扩增所必需的，以及它们是否都可能激活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和其他因素是否直接刺激HSC自我更新或阻止HSC凋亡或分化。Angptl2或AngptIS的受体和信号转导途径尚不清楚；我们将使用我实验室开发并广泛使用的两种表达克隆方案分离Angptl2和AngptIS细胞表面受体，然后对它们进行广泛的表征。我们的研究可以导致造血干细胞在骨髓移植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，由于造血干细胞数量少和无法获得兼容的供体而受到阻碍。造血干细胞也是开发基因疗法治疗多种遗传性免疫缺陷综合征和先天性代谢错误的一个有希望的细胞靶点。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.