Notch Mediated Maintenance and Expansion of Human Mesenchymal Stem Cells

描述（由申请人提供）：人间充质干细胞（hMSC）是具有分化为成软骨细胞（软骨）、成骨细胞（骨）、成肌细胞（肌肉）、成神经细胞（神经组织）和成脂肪细胞（脂肪）潜能的成体干细胞。这些细胞由于其治疗骨骼疾病和促进骨骼修复的治疗潜力而受到极大关注，尽管维持其多能性和体外扩增这些细胞已被证明非常困难，限制了它们在临床环境中的使用。最近，我们确定了RBPj？- Notch依赖的信号通路作为小鼠骨骼发生过程中间充质干/祖细胞（MSC）增殖和分化的重要调节因子，导致Notch信号系统是否可以调节hMSC维持和/或扩增的问题。为了开始解决这些问题，我们建议测试新的假设，即暂时的，控制Notch激活促进维护和扩大的hMSCs通过Hes 1和Sox 2因子，同时保留其软骨，成骨和成脂分化潜力。在第一个具体目标中，我们设计了实验以确定i）Notch激活是否维持和扩增hMSC，ii）Hes 1是否是调节hMSC维持和扩增的关键Notch靶基因，iii）持续的Notch和/或Hes 1信号传导是否将hMSC永久地阻滞在未分化状态，和iv）暂时Jagged 1诱导的Notch激活hMSC将维持和扩增该细胞群，同时保留其多能分化能力。在第二个具体目标中，我们将测试i）Jagged 1诱导的Notch信号传导是否直接调节多能干细胞因子Sox 2的表达，ii）Jagged 1诱导Sox 2是否需要RBPj？依赖性Notch靶基因Hes 1，和iii）Notch对hMSC维持和扩增的调节需要Sox 2。这些目标的完成将建立一种利用Notch途径维持和扩增hMSC的新方法，该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hMSC的分化潜力和理解Notch诱导的hMSC维持和扩增中涉及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，我们确定了RBP 1：依赖性Notch信号通路作为一个重要的调节剂间充质干/祖细胞（MSC）的增殖和分化在小鼠骨骼发育。本申请中概述的计划将建立使用Notch途径维持和扩增人MSC（hMSC）的方法，这是一种用于产生足够数量的干细胞以用于骨骼修复和再生的新技术。阐明Notch维持和扩增的hMSC的分化潜力和理解Notch诱导的hMSC维持和扩增中涉及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.