ROCK inhibitor increases proacinar cells in adult salivary gland organoids

Salisphere-derived adult epithelial cells have been used to improve saliva production of irradiated mouse salivary glands. Importantly, optimization of the cellular composition of salispheres could improve their regenerative capabilities. The Rho Kinase (ROCK) inhibitor, Y27632, has been used to increase the proliferation and reduce apoptosis of progenitor cells grown in vitro. In this study, we investigated whether Y27632 could be used to improve expansion of adult submandibular salivary epithelial progenitor cells or to affect their differentiation potential in different media contexts. Application of Y27632 in medium used previously to grow salispheres promoted expansion of Kit+ and Mist1+ cells, while in simple serum-containing medium Y27632 increased the number of cells that expressed the K5 basal progenitor marker. Salispheres derived from Mist1CreERT2; R26TdTomato mice grown in salisphere media with Y27632 included Mist1-derived cells. When these salispheres were incorporated into 3D organoids, inclusion of Y27632 in the salisphere stage increased the contribution of Mist1-derived cells expressing the proacinar/acinar marker, Aquaporin 5 (AQP5), in response to FGF2-dependent mesenchymal signals. Optimization of the cellular composition of salispheres and organoids can be used to improve the application of adult salivary progenitor cells in regenerative medicine strategies. Keywords: Salivary gland, Salisphere, Organoid, Proacinar differentiation, Y27632