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Notch Activation of Notch2 Selected Mesenchymal Stem Cells Demonstrate Superior Stem Cell Multipotency, Proliferation, and Enhanced Differentiation Ex Vivo +Dong, Y; Rutkowski, T; O’Keefe, RJ; Hilton, MJ University of Rochester Medical Center, Rochester, NY [email protected] Introduction: Human mesenchymal stem cells (hMSCs) 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 this heterogeneous group of cells ex vivo has proven to be very difficult limiting their use in clinical settings. Recently, the Notch signaling pathway was identified as an important regulator of MSC proliferation and differentiation during mouse skeletogenesis leading to questions whether Notch signaling can be utilized as a tool to maintain stem cell mutipotency and expand cell numbers ex vivo. Notch signaling is initiated when the ligands Jagged1, 2 or Delta-like 1, 3, 4 bind to the single-pass transmembrane cell surface Notch receptors (Notch1-4) on neighboring cells. This interaction induces cleavage of the Notch receptors, via the gamma-secretase complex, releasing the Notch intracellular domain (NICD) to the nucleus activating the canonical Notch transcriptional effector, RBPJk. Canonical Notch signaling is well recognized as a critical factor controlling stem cell differentiation and self-renewal in the hematopoietic, neural, pancreatic, intestinal, and skeletal muscle systems. Therefore, we set out to demonstrate that specific components of the Notch signaling pathway can be utilized to select a subpopulation of hMSCs that can maintain greater multipotency over multiple passages and can be expanded more readily ex vivo. Materials and Methods: To determine the role of Notch signaling on hMSC maintenance and expansion ex vivo, we cultured Lonza bone marrow derived hMSCs and Notch2 selected hMSCs on IgG or Jagged1 (Jag1) coated plates for mutiple passages. Real-time RT-PCR and flow cytometric analyses were used for measuring gene expression and stem cell surface marker expression. Notch2 positive hMSCs were selected using both flow cytometry and immonomagentic bead sorting methods. BrdU labeling was performed for cell proliferation analysis and colony forming unit – fibroblast (cfu-f) assays were used to evaluate hMSC multipotency following several cell passages. hMSCs were initially cultured on Jagged1 or IgG protein-coated plates for stem cell maintenance and expansion assays, although the cells were removed from the coated dishes and seeded on standard cell culture plates for osteogenic differentiation assays. Alkaline phosphatase (AP) staining and real-time RT-PCR assays for osteogenic markers Col1a1, AP, and Oc were performed. Results: To determine whether the Notch pathway could be utilized to promote the maintenance and expansion of hMSCs derived from bone marrow, we first analyzed the expression of all Notch receptors and each of the Hes/Hey target genes using hMSCs cultured over multiple passages. We identified Notch2 and Hes1 as being the most highly expressed components of the Notch pathway, data consistent with work from our previous developmental mouse studies. Interestingly, Notch2 and Hes1 expression levels were significantly reduced as hMSCs were cultured for multiple passages (p1 – p10), concurrent with the dramatic reduction in stem cell regulatory gene expression (Sox2, Oct4, and Nanog). Flow cytometric analyses of these cells at p2 and p10 demonstrates that p10 hMSCs only retain about 40% of the hMSCs cell surface marker CD105, suggesting that hMSCs lose their stem cell phenotype during normal culturing and passaging of cells. To identify whether Notch activation can both enhance hMSCs proliferation and maintain multipotency, we cultured hMSCs for up to 10 passages on plates coated with recombinant protein for the extracellular domain of the Notch ligand, Jagged-1, or control proteins. Gene expression analyses showed that Jagged1 induced Notch activation enhances the expression of stem cell transcriptional regulators, Sox2, Oct4 and Nanog, as well as, the Notch target gene Hes1 (Figure 1A). Cell proliferation assays using BrdU labeling also demonstrated that Jag1 mediated Notch activation increases proliferation by more than 20% as compared to controls using total hMSCs. On the other hand, when we cultured Notch2 selected hMSCs on Jag1 coated plates; cell proliferation rates were increased by 2.5 fold compared to control using total hMSCs (Figure 1B). Cfu-f assays indicate that following Jagged1 mediated Notch activation the number hMSC colonies was increased following p1, p5, and p10 for Notch2 selected hMSCs as compared to total and Notch2 negative A: Real time PCR B: BrdU ELISA Figure 1. Notch activation maintains hMSC mutipotency and enhances cell proliferation. A: Real-time RT-PCR shows Jagged1 activated Notch signaling induces the expression of stem cell transcriptional regulators, Sox2, Oct4 and Nanog, and the Notch target gene, Hes1, and Notch receptor, Notch2. B: BrdU ELISA shows enhanced hMSC proliferation of Notch2 selected hMSCs (Notch2+) cultured on Jagged1 coated plates as compared to total and Notch2 negative (Notch2 -) hMSCs. Notch2Passage 2 Notch2+ Notch2- Notch2+ Passage 5 Figure 2: Notch2 selected hMSCs show enhanced osteogenesis. Notch2 selected hMSCs were cultured on Jagged1 coated plates for 2 and 5 passages. Alkaline phosphatase staining was performed when the cells were reseeded in osteogenic media for 12 days in the absence of the Jagged1 recombinant protein. hMSCs, suggesting an enhanced maintenance of multipotency. Osteoblast differentiation assays demonstrate that when Notch2 selected hMSCs are removed from Jagged1 mediated Notch activation they have enhanced skeletal differentiation as compared to Notch2 negative hMSCs regardless of how long the cells were maintained on Jagged1 coated plates (Figure 2). Discussion: Our data clearly indicates that the Notch pathway is an important regulator of hMSC maintenance and expansion ex vivo. These data further suggest that the Notch2 positive sub-set of hMSCs represents a uniquely multipotent and expandable population of hMSCs with a greater differentiation capacity. We therefore believe that Notch signaling activation can be used as a tool for hMSC maintenance and expansion ex vivo and that Notch2 selected stromal cells have enhanced osteoblast differentiation capacity and can be rapidly expanded ex vivo for the use in bone tissue repair. We are currently trying to identify whether Notch2 selected and expanded hMSCs may also show enhanced chondrogenic differentiation, as our previous work in the mouse has demonstrated that Notch activation maintains and expands MSCs without affecting lineage allocation. These data will be crtical to determine all of the potential clinical uses for Notch2 selected, maintained, and expanded hMSCs. Additional, studies are also underway to determine whether these cells can demonstrate enhanced bone formation in various mouse models as compared to traditionally isolated bone marrow derived hMSCs. Poster No. 1762A • ORS 2011 Annual Meeting