Role of Jagged/Notch Signaling in the Cell Fate Determination of Bone Marrow Human Mesenchymal Stem Cells.

Notch, expressed on hematopoietic progenitors plays a crucial role in hematopoiesis. Mesenchymal stem cells (MSC) express both, Notch and its ligand Jagged and are known to support self renewal of hematopoietic progenitors via cell-cell contact and cytokine secretion. The Jagged/Notch signaling pathway has been implicated in the differentiation process of MSC, however it is not completely understood and current observations are contradictory. In order to analyze the effect of Notch signaling on human MSC differentiation we constructed lentiviral vectors that contained either the GFP-marker gene, hJagged1 IRES GFP, hNotch1 intracellular domain (NICD) IRES GFP or a gene fusion between dominant negative Mastermind1 (MAML1dn - inhibitor of Notch signaling) and the Cherry reporter gene. Primary hMSC that were obtained from bone marrow of 3 different donors were transduced with respective lentivirus vectors to greater than 98%. After exposure to adipogenic and osteogenic differentiation stimuli hMSC differentiation was quantified by Alizarin red or oil red staining, alkaline phosphatase (AP) activity and expression levels of adipogenic or osteogenic markers by Real-time PCR. Jagged1 transduced hMSC demonstrated enhanced calcium phosphate deposits and enhanced AP activity and expression levels in osteogenic differentiation medium, while adipogenic differentiation was strongly inhibited as quantified by oil red staining and low mRNA expression of genes upregulated during adipogenic differentiation (pprY, Fabp4). Similarly, overexpression of NICD induced strong and rapid osteogenic differentiation while inhibiting adipogenic differentiation and reducing cell viability. Moreover, NICD overexpression upregulates the expression of endogenous Jagged1 up to 5-fold. Inhibition of Notch signaling via overexpression of MAML1dn partially blocked the effect of hJagged1 and NICD in co-transduction experiments. In another approach MSC samples obtained from 20 donors with various osteogenic differentiation potential as measured by AP activity were analyzed for Notch1 and Jagged1 expression. While there was no correlation between AP activity and Notch1 levels we observed a significant positive correlation for AP activity and Jagged1 expression. In summary, our data strongly suggest that increased Jagged/Notch signaling enhances the osteogenic differentiation of hMSC while inhibiting their adipogenic fate.