Notch Signaling through Jagged-1 is Necessary to Initiate Chondrogenesis in Human Bone Marrow Stromal Cells, but Must Be Switched Off to Complete chondrogenesis

¹ UK Centre for Tissue Engineering, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK.; Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK.; North West Embryonic Stem Cell Centre, Faculty of Life Sciences, University of Manchester, Manchester, M13 9NT, UK.
² UK Centre for Tissue Engineering, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK.; Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK.
³ UK Centre for Tissue Engineering, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK.
⁴ UK Centre for Tissue Engineering, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK.; North West Embryonic Stem Cell Centre, Faculty of Life Sciences, University of Manchester, Manchester, M13 9NT, UK.
⁵ Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK.

^* To whom correspondence should be addressed. E-mail: timothy.e.hardingham{at}manchester.ac.uk.

	Abstract

We investigated Notch signaling during chondrogenesis in human bone-marrow stromal cells (hMSC) in 3D cell aggregate culture. Expression analysis of Notch pathway genes in 14 day chondrogenic cultures showed that the Notch ligand, Jagged-1 (Jag-1) sharply increased in expression peaking at day 2 and then declined. A Notch target gene, HEY-1, was also expressed with a temporal profile, which closely followed the expression of Jag-1 and this preceded the rise in type II collagen expression that characterized chondrogenesis. We demonstrated that the shut-down in Notch signaling was critical for full chondrogenesis, as adenoviral hJag-1 transduction of hMSC, which caused continuous elevated expression of Jag-1 and sustained Notch signaling over 14 days, completely blocked chondrogenesis. In these cultures there was inhibited production of extracellular matrix and the gene expression of aggrecan and type II collagen were strongly suppressed and this may reflect the retention of a prechondrogenic state. The JAG-1 mediated Notch signaling was also shown to be necessary for chondrogenesis, as DAPT added to cultures days 0-14, or just days 0-5 inhibited chondrogenesis, but DAPT added from day 5 did not. The results thus showed that Jag-1 mediated Notch signaling in hMSC was necessary to initiate chondrogenesis, but must be switched off for chondrogenesis to proceed.

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