Growth factors and engineered stroma for HSC expansion

用于 HSC 扩增的生长因子和工程基质

• 批准号: 6757436
• 负责人: Harvey F Lodish
• 金额: $ 28.5万
• 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 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6757436
• 关键词: biotechnology; bone marrow; cell adhesion; cell differentiation; cell growth regulation; cell line; cell population study; cell proliferation; cytokine receptors; fibrillin; flow cytometry; gene expression; genetic manipulation; genetically modified animals; growth factor receptors; hematopoietic growth factor; hematopoietic stem cells; insulinlike growth factor; interleukin 6; laboratory mouse; liver cells; protein structure function; stem cell factor; thrombopoietic factor; tissue /cell culture

DESCRIPTION (provided by applicant): Our principal hypotheses are that many as yet unidentified morphogens/growth factors control fetal and adult hematopoietic stem cell (HSC) survival and proliferation, and that genetic modifications of stromal cell lines can enhance their ability to support HSC expansion in culture without undergoing differentiation to lineage-restricted progenitors. We identified several potentially important secreted proteins specifically expressed in AFT024, a mouse fetal liver stromal cell line that maintains HSC stem cell activity: Pleiotrophin; Deltalike; the fibrillin-like protein T16; Cytokine Receptor-like Factor, and three members of the Proliferin gene family, Proliferin- 1; Mrp4, and proliferin- related protein (PRP). Using stable expression of siRNAs to block their production in AFT024 and AFT024 - FIt3L cells we will determine the function of these and other signaling proteins, such as M-CSF, in HSC expansion and differentiation. Using Fc fusion proteins we will determine whether HSCs have receptors for these novel growth/differentiation factors and whether their receptors might provide additional HSC- specific surface markers. Our preliminary data indicates positive effects of added Flt3L and IGF-2 on HSC maintenance and expansion in vitro. Thus, in parallel we will determine whether forced overexpression in AFT024 and AFT024 - Flt3L cells of several proteins including thrombopoietin; Stromal cell - derived factor 1; stem cell factor; IL-6; 3 Wnts; and IGF-2 enhances their ability to support expansion of HSCs in culture. As appropriate knock- out mice selectively missing one or more of these factors will be made and analyzed for HSCs and hematopoiesis. By comparing the ability of other stromal cell lines, including OP-9, MS-5, and S 17, to support maintenance and expansion of fetal liver and adult bone marrow HSCs, together with our existing transcriptional profiling data, we should identify other secreted/ surface proteins that potentially affect HSC expansion or differentiation positively or negatively, and in later years will test their role in HSC biology. Our long- term aim is to engineer a cloned line that supports robust, continuous expansion of fetal liver or bone marrow HSCs in culture. In continued collaboration with Prof. George Daley, we will test the ability of our novel growth factors/morphogens and genetically altered stromal cell lines to support the generation of transplantable HSCs from cultured human and mouse ES cell lines.

描述（由申请人提供）：我们的主要假设是，许多尚未确定的形态因素/生长因子控制胎儿和成人造血干细胞（HSC）的生存和增殖，并且基质细胞系的遗传修饰可以增强其在培养中支持HSC扩张的能力，而无需分化对谱系构成的养分群体的分化。我们确定了在AFT024中特异性表达的几种潜在重要的分泌蛋白，这是一种维持HSC干细胞活性的小鼠胎儿肝脏基质细胞系：pleiotiotiotirophirophin; deltalike;纤维蛋白样蛋白T16；细胞因子受体样因子和增殖蛋白基因家族的三个成员，增殖蛋白1； MRP4和相关蛋白（PRP）。使用siRNA的稳定表达来阻止其在AFT024和AFT024 -FIT3L细胞中的产生，我们将确定这些和其他信号蛋白（例如M -CSF）在HSC膨胀和分化中的功能。使用FC融合蛋白，我们将确定HSC是否具有用于这些新生长/分化因子的受体，以及它们的受体是否可以提供其他HSC特异性表面标记。我们的初步数据表明添加的FLT3L和IGF-2对HSC维持和体外扩张的积极影响。因此，同时我们将确定在包括血小板蛋白在内的几种蛋白质的Aft024和Aft024 -FLT3L细胞中是否强制过表达；基质细胞 - 衍生因子1；干细胞因子； IL-6; 3个Wnt； IGF-2增强了他们支持培养中HSC扩展的能力。在适当的敲除小鼠中，将对HSC和造血作用进行选择性丢失和多个这些因素。通过比较包括OP-9，MS-5和S 17在内的其他基质细胞系的能力，支持胎儿肝和成人骨髓HSC的维持和扩展，以及我们现有的转录分析数据，我们应该确定其他分泌/表面蛋白质，这些蛋白质可能会潜在地影响HSC的扩张或差异化或差异化或差异化，并在HSC中测试HSC的角色。我们的长期目标是设计一条克隆线，该线路支持培养中胎儿肝或骨髓HSC的稳健，连续扩展。在与乔治·戴利（George Daley）教授的持续合作中，我们将测试新型生长因子/形态剂和基因改变的基质细胞系的能力，以支持从培养的人和小鼠ES细胞系中产生可移植的HSC。