Retinoic acid receptor signaling preserves tendon stem cell characteristics and prevents spontaneous differentiation in vitro

Stuart Webb, Chase Gabrelow, James Pierce, Edwin Gibb, Jimmy Elliott

Previous studies have reported that adult mesenchymal stem cells (MSCs) tend to gradually lose their stem cell characteristics in vitro when placed outside their niche environment. They subsequently undergo spontaneous differentiation towards mesenchymal lineages after only a few passages. We observed a similar phenomenon with adult tendon stem cells (TSCs) where expression of key tendon genes such as Scleraxis (Scx), are being repressed with time in culture. We hypothesized that an environment able to restore or maintain Scleraxis expression could be of therapeutic interest for in vitro use and tendon cell-based therapies. TSCs were isolated from human cadaveric Achilles tendon and expanded for 4 passages. A high content imaging assay that monitored the induction of Scx protein nuclear localization was used to screen ~1000 known drugs. We identified retinoic acid receptor (RAR) agonists as potent inducers of nuclear Scx in the small molecule screen. The upregulation correlated with improved maintenance of tendon stem cell properties through inhibition of spontaneous differentiation rather than the anticipated induction of tenogenic differentiation. Our results suggest that histone epigenetic modifications by RAR are driving this effect which is not likely only dependent on Scleraxis nuclear binding but also mediated through other key genes involved in stem cell self-renewal and differentiation. Furthermore, we demonstrate that the effect of RAR compounds on TSCs is reversible by revealing their multi-lineage differentiation ability upon withdrawal of the compound. Based on these findings, RAR agonists could provide a valid approach for maintaining TSC stemness during expansion in vitro, thus improving their regenerative potential for cell-based therapy.