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: 6895286
• 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/6895286
• 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，许多尚未鉴定的形态发生素/生长因子控制胎儿和成人造血干细胞 (HSC) 的存活和增殖，并且基质细胞系的遗传修饰可以增强其支持 HSC 扩增的能力在培养物中不经历分化为谱系限制的祖细胞。我们鉴定了几种在 AFT024 中特异性表达的潜在重要的分泌蛋白，AFT024 是一种维持 HSC 干细胞活性的小鼠胎肝基质细胞系：多效蛋白；类似三角洲；原纤维蛋白样蛋白T16；细胞因子受体样因子，以及 Proliferin 基因家族的三个成员 Proliferin-1； Mrp4 和增殖蛋白相关蛋白 (PRP)。使用 siRNA 的稳定表达来阻断它们在 AFT024 和 AFT024 - FIt3L 细胞中的产生，我们将确定这些信号蛋白和其他信号蛋白（例如 M-CSF）在 HSC 扩增和分化中的功能。使用 Fc 融合蛋白，我们将确定 HSC 是否具有这些新型生长/分化因子的受体，以及它们的受体是否可能提供额外的 HSC 特异性表面标记。我们的初步数据表明添加 Flt3L 和 IGF-2 对 HSC 体外维持和扩增有积极作用。因此，我们将同时确定 AFT024 和 AFT024 - Flt3L 细胞中是否强制过度表达包括血小板生成素在内的几种蛋白质；基质细胞-衍生因子1；干细胞因子； IL-6; 3 Wnt； IGF-2 增强了它们支持 HSC 在培养物中扩增的能力。适当时，将制备选择性缺失一种或多种这些因子的敲除小鼠并分析HSC和造血功能。通过比较其他基质细胞系（包括 OP-9、MS-5 和 S 17）支持胎儿肝脏和成人骨髓 HSC 维持和扩增的能力，以及我们现有的转录谱数据，我们应该识别其他分泌的/ 表面蛋白可能对 HSC 扩张或分化产生积极或消极的影响，并将在以后的几年中测试它们在 HSC 生物学中的作用。我们的长期目标是设计一个克隆系，支持胎儿肝脏或骨髓 HSC 在培养物中稳健、持续的扩增。在与 George Daley 教授的持续合作中，我们将测试我们的新型生长因子/形态发生素和基因改变的基质细胞系支持从培养的人类和小鼠 ES 细胞系生成可移植 HSC 的能力。