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

用于造血干 csll (HSC) 扩增的生长因子和工程化基质

• 批准号: 7627338
• 负责人: Harvey F Lodish
• 金额: $ 39.18万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7627338
• 关键词: Adopted; Adult; Affect; Apoptosis; Binding; Biological Assay; Blood Cells; Blood capillaries; Bone Marrow; Bone Marrow Transplantation; CD3 Antigens; CD4 Positive T Lymphocytes; Cell Culture System; Cell Death; Cell Line; Cell Surface Proteins; Cell Surface Receptors; Cells; Clinical; Clone Cells; Coculture Techniques; Confocal Microscopy; DNA; DNA Microarray Chip; Data; Embryo; Engineering; Fetal Liver; Fibroblast Growth Factor 1; Flow Cytometry; Frozen Sections; Gene Expression; Gene Expression Profile; Genes; Glycolipids; Goals; Grant; Growth Factor; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Hormones; Immunoassay; Immunologic Deficiency Syndromes; Inborn Errors of Metabolism; Inherited; Insulin-Like Growth Factor II; Isoelectric Focusing; Laboratories; Lead; Location; Lymphocyte; Malignant Neoplasms; Measures; Mediating; Memory; Methods; Molecular; Molecular Models; Monitor; Mus; Pan Genus; Phenotype; Population; Procedures; Proteins; Protocols documentation; RNA Interference; Research Personnel; Resolution; Scientist; Serum; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Spleen; Staining method; Stains; Stem cells; Stromal Cells; Surface; System; T-Cell Receptor; T-Lymphocyte; Testing; Tissues; Transgenes; Transplantation; Umbilical Cord Blood; Work; base; cDNA Library; capillary; cell type; clinical application; expression cloning; fetal; gene therapy; in vivo; leukemia; magnetic beads; molecular modeling; novel; prevent; programs; receptor; research study; self-renewal; spatial relationship; stem; stem cell niche

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.

描述（由申请人提供）：造血干细胞（HSC）位于专门的壁ni中，并从周围的基质细胞接收信号，但是它们的身份及其对HSC的影响在很大程度上是未知的。我们的工作着重于定义由基质细胞产生的分泌生长因子，并触发HSC扩张后体内。我们从胚胎第15天（E15）的胎儿肝脏中鉴定出具有早期T细胞表面表型的新细胞群，并支持HSC的离体扩张。这笔赠款的主要目的是净化和表征几个E15.5 fl T细胞亚群，以扩大培养物中的HSC的能力，以确定在成年小鼠骨髓和脾脏中是否发现了相似的细胞，以确定这些细胞是否是HSC Niche的一部分，并确定是否属于HSC niche的一部分，并检查基因在成年和胎儿群体中的差异和相似之处，以扩展HSSS的模型ands sssc and sssc and sssc and of SSSS的群体和胎儿的差异。我们的DNA微阵列实验表明，在其他蛋白质中，IGF -2和类似的蛋白质蛋白2（ANGPTL2）和3（ANGPTI）在这些E15 fl早期T细胞中特别表达。然后，我们开发了一个含有低水平的SCF，TPO，FGF-1，IGF-2和Angptl2或Angptis的无血清HSC培养系统；通过竞争性重生分析衡量，在培养10天后，长期重现HSC（LT-HSC）的数量增加了24。 Angptl2和Angptis在很大程度上没有研究，以前没有建议作用于造血细胞或干细胞。在这里，我们将测试IGF-2，TPO，ANGPTL2和其他生长因子是否是最大HSC膨胀需要的每个生长因子，因此每一种是否可能激活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和后来的其他因素是否直接刺激HSC自我更新或防止HSC凋亡或分化。 Angptl2或Angptis和信号转导途径的受体未知。我们将使用在我的实验室中开发和广泛使用的两个表达克隆方案来隔离Angptl2和Angptis细胞表面受体，然后广泛表征它们。我们的研究可以导致HSC在骨髓移植中的临床使用方面有重大改善，以治疗白血病和其他癌症。许多应用，尤其是使用脐带血的移植，受到HSC数量少以及兼容供体的不可用而阻碍。 HSC也是开发基因疗法的有希望的细胞靶标，用于治疗各种遗传性免疫缺陷综合症和代谢的先天误差。在基因治疗的情况下，可以选择在特定的染色体位置中整合转基因的HSC，可以大大增强HSC的外体能力，从而大大增强这些临床应用。