Notch Mediated Maintenance and Expansion of Human Mesenchymal Stem Cells

描述(申请人提供)：人间充质干细胞(HMSC)是一种成体干细胞，具有分化为软骨母细胞(软骨)、成骨细胞(骨)、成肌细胞(肌肉)、神经母细胞(神经组织)和脂肪母细胞(脂肪)的潜能。由于它们在治疗骨骼疾病和促进骨骼修复方面的治疗潜力，这些细胞受到了极大的关注，尽管保持它们的多能性和体外扩增这些细胞被证明是非常困难的，限制了它们在临床上的使用。最近，我们发现依赖于RBPj的Notch信号通路是小鼠骨骼发育过程中间充质干细胞(MSC)增殖和分化的重要调节因子，这导致了Notch信号系统是否能够调节hMSC的维持和/或扩增。为了开始解决这些问题，我们建议测试这一新的假设，即临时的、受控的Notch激活通过Hes1和Sox2因子促进hMSCs的维持和扩张，同时保持其软骨分化、成骨分化和成脂分化潜能。在第一个特定目标中，我们设计了实验，以确定i)Notch激活是否维持和扩增hMSCs，ii)Hes1是调控hMSCs维持和扩张的关键Notch靶基因，iii)持续的Notch和/或Hes1信号永久阻止处于未分化状态的hMSCs，以及iv)Jagged1诱导的hMSCs暂时的Notch激活将维持和扩大hMSCs的群体，同时保持其多向分化能力。在第二个特定目的中，我们将测试i)Jagged1诱导的Notch信号是否直接调节多能干细胞因子Sox2的表达，ii)Jagged1对Sox2的诱导需要依赖于RBPj的Notch靶基因Hes1，以及iii)Notch对hMSC维持和扩张的调控需要Sox2。完成这些目标将建立一种新的方法，利用Notch途径来维持和扩大hMSCs，这项技术将有助于产生足够数量的干细胞，用于骨骼修复和再生。阐明Notch维持和扩增的hMSCs的分化潜能，并了解Notch诱导的hMSC维持和扩增的分子机制是建立这种方法作为潜在治疗考虑的关键步骤。 公共卫生相关性：最近，我们发现RBPj：依赖的Notch信号通路在小鼠骨骼发育过程中是间充质干细胞/祖细胞(MSC)增殖和分化的重要调节因子。本申请中概述的计划将建立使用Notch途径维持和扩增人MSCs的方法，是一项产生足够数量的干细胞用于骨骼修复和再生的新技术。阐明Notch维持和扩增的hMSCs的分化潜能，并了解Notch诱导的hMSC维持和扩增的分子机制是建立这种方法作为潜在治疗考虑的关键步骤。

DESCRIPTION (provided by applicant): Human mesenchymal stem cells (hMSC) are adult stem cells with the potential to differentiate into chondroblasts (cartilage), osteoblasts (bone), myoblasts (muscle), neuroblasts (neural tissue) and adipoblasts (fat). These cells have been studied with great interest due to their therapeutic potential for treating skeletal disease and facilitating skeletal repair, although maintaining their multipotency and expanding these cells ex vivo has proven to be very difficult limiting their use in clinical settings. Recently, we identified the RBPj? - dependent Notch signaling pathway as an important regulator of mesenchymal stem/progenitor cell (MSC) proliferation and differentiation during mouse skeletogenesis, leading to questions of whether the Notch signaling system can regulate hMSC maintenance and/or expansion. To begin addressing these questions, we propose to test the novel hypothesis that temporary, controlled Notch activation promotes the maintenance and expansion of hMSCs via Hes1 and Sox2 factors while preserving their chondrogenic, osteogenic, and adipogenic differentiation potential. In the first specific aim we have designed experiments to determine whether i) Notch activation maintains and expands hMSCs, ii) Hes1 is a critical Notch target gene regulating maintenance and expansion of hMSCs, iii) sustained Notch and/or Hes1 signaling permanently arrests hMSCs in an undifferentiated state, and iv) temporary Jagged1 induced Notch activation of hMSCs will maintain and expand this cell population while preserving their multipotent differentiation capacity. In the second specific aim we will test whether i) Jagged1 induced Notch signaling directly regulates expression of the multipotent stem cell factor, Sox2, ii) Jagged1 induction of Sox2 requires the RBPj?-dependent Notch target gene, Hes1, and iii) Notch regulation of hMSC maintenance and expansion requires Sox2. Completion of these aims will establish a novel methodology for utilizing the Notch pathway to maintain and expand hMSCs, a technology that will aid in generating an adequate number of stem cells to be useful in skeletal repair and regeneration. Elucidating the differentiation potential of Notch-maintained and -expanded hMSCs and understanding the molecular mechanisms involved in Notch-induced hMSC maintenance and expansion are critical steps in establishing this approach as a potential therapeutic consideration. PUBLIC HEALTH RELEVANCE: Recently, we identified the RBPj:-dependent Notch signaling pathway as an important regulator of mesenchymal stem/progenitor cell (MSC) proliferation and differentiation during mouse skeletogenesis. The plan outlined in this application, which will establish methods for using the Notch pathway to maintain and expand human MSCs (hMSCs), is a novel technology for generating an adequate number of stem cells to be useful in skeletal repair and regeneration. Elucidating the differentiation potential of Notch-maintained and - expanded hMSCs and understanding the molecular mechanisms involved in Notch-induced hMSC maintenance and expansion are critical steps in establishing this approach as a potential therapeutic consideration.