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）是成体干细胞，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）的潜力。由于这些细胞在治疗骨骼疾病和促进骨骼修复方面的治疗潜力，人们对它们进行了极大的研究，尽管维持它们的多能性和体外扩增这些细胞已被证明是非常困难的，限制了它们在临床环境中的使用。最近，我们确定了RBPj？在小鼠骨骼形成过程中，Notch信号通路是间充质干细胞/祖细胞（MSC）增殖和分化的重要调节因子，这导致了Notch信号系统是否可以调节间充质干细胞的维持和/或扩展的问题。为了开始解决这些问题，我们提出验证一个新的假设，即暂时的、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、成骨和脂肪分化的潜力。在第一个特定目标中，我们设计了实验来确定i) Notch激活是否维持和扩展hMSCs， ii) Hes1是调节hMSCs维持和扩展的关键Notch靶基因，iii)持续的Notch和/或Hes1信号永久地阻止处于未分化状态的hMSCs， iv) Jagged1诱导的hMSCs的Notch激活将维持和扩展该细胞群，同时保持其多能分化能力。在第二个具体目标中，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诱导Sox2需要RBPj？iii) Notch对hMSC维持和扩增的调控需要Sox2。这些目标的完成将建立一种利用Notch通路维持和扩增hMSCs的新方法，这种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维持和扩增的造血干细胞的分化潜力，了解notch诱导的造血干细胞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.