Growth factors and engineered stroma for HSC expansion

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子/生长因子控制着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在培养中支持HSC扩增的能力，而无需分化为谱系受限的祖细胞。我们在AFT024（一种维持HSC干细胞活性的小鼠胎肝基质细胞系）中特异性表达了几种潜在的重要分泌蛋白：Pleiotrophin；Deltalike;纤维蛋白样蛋白T16；细胞因子受体样因子和增殖素基因家族的三个成员，增殖素- 1；Mrp4和增殖素相关蛋白（PRP）。在AFT024和AFT024 - FIt3L细胞中，利用sirna的稳定表达来阻断其产生，我们将确定这些和其他信号蛋白（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特异性表面标记物。我们的初步数据表明，添加Flt3L和IGF-2对体外HSC维持和扩增有积极作用。因此，我们将同时确定AFT024和AFT024 - Flt3L细胞中是否强迫过表达几种蛋白质，包括血小板生成素；基质细胞衍生因子1；干细胞因子；il - 6;3实际上wnt;和IGF-2增强它们在培养中支持hsc扩增的能力。适当的敲除小鼠选择性地缺失一个或多个这些因素，并分析造血干细胞和造血。通过比较其他基质细胞系（包括OP-9、MS-5和s17）支持胎儿肝脏和成人骨髓造血干细胞维持和扩增的能力，以及我们现有的转录谱数据，我们应该确定其他可能对造血干细胞扩增或分化产生积极或消极影响的分泌/表面蛋白，并在以后的几年里测试它们在造血干细胞生物学中的作用。我们的长期目标是设计一种克隆系，能够在培养中支持胎儿肝脏或骨髓造血干细胞的稳健、持续扩增。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/形态因子和转基因基质细胞系的能力，以支持从培养的人类和小鼠胚胎干细胞系中产生可移植的造血干细胞。

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.