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来源/生长因子控制着胎儿和成人造血干细胞(HSC)的存活和增殖，基质细胞系的基因改造可以增强它们在培养中支持HSC扩张的能力，而不会分化为谱系受限的祖细胞。我们鉴定了几个在AFT024中特异表达的潜在重要分泌蛋白，AFT024是一种维持HSC干细胞活性的小鼠胎肝基质细胞系：Pleiotroin；Deltalike；纤维素样蛋白T16；细胞因子受体样因子；以及增殖相关蛋白(PRP)家族的三个成员，增殖蛋白-1、MRP4和增殖相关蛋白(PRP)。利用稳定表达的siRNAs在AFT024和AFT024-FIt3L细胞中阻断其产生，我们将确定这些信号蛋白和其他信号蛋白，如M-CSF，在HSC扩增和分化中的功能。利用Fc融合蛋白，我们将确定HSC是否具有这些新的生长/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的维持和体外扩增有积极的作用。因此，我们将同时确定在AFT024和AFT024-Flt3L细胞中强制过表达几种蛋白质，包括血小板生成素、基质细胞衍生因子1、干细胞因子、IL-6、3WNTS和IGF-2是否增强了它们支持HSC在培养中扩增的能力。在适当的情况下，选择性地缺失这些因子中的一个或多个的基因敲除小鼠将被制造出来，并对其进行造血干细胞和造血分析。通过比较其他基质细胞系，包括OP-9、MS-5和S 17，支持胎肝和成人骨髓HSC维持和扩增的能力，以及我们现有的转录图谱数据，我们应该识别其他可能对HSC扩增或分化产生积极或消极影响的分泌/表面蛋白，并在未来几年测试它们在HSC生物学中的作用。我们的长期目标是设计一种克隆品系，以支持胎肝或骨髓HSCs在培养中强劲、持续地扩增。在与George Daley教授的持续合作下，我们将测试我们的新型生长因子/形态因子和基因改变的基质细胞系支持从培养的人和小鼠ES细胞系生成可移植的HSC的能力。

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.