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.