Y-27632, a ROCK inhibitor, delays senescence of putative murine salivary gland stem cells in culture

Abstract

ObjectiveA loss of functional salivary glands often occurs after radiotherapy for head and neck tumour, and causes many problems in oral health. Recently, the use of salispheres, which consist of salivary gland stem cells (SGSCs), has been suggested as therapy for these problems. However, an insufficient number of cells can be obtained and cultured for cell transplantation. In the present study, salispheres were propagated and passaged by suspension culture to acquire a sufficient number of SGSCs for cell therapy. DesignThe relationship between sphere formation and the degree of cellular senescence was investigated by analysing senescence-associated β-galactosidase activity and the expression of senescence-related markers such as CDKN2A (p16) and p21. Because the sphere formation potential of SGSCs was decreased and the senescence of the cells was increased after passaging subculture, Y-27632, a Rho-associated kinase inhibitor, was used to treat the passaging subculture to aid the proliferation of the cells in culture. ResultsThe number of spheres was higher in the Y-27632 treatment group than in the control group, and the expression of c-Kit, a proliferation marker, was also increased. In addition, the expression of p16 and p21 proteins tended to be lower in the Y-27632 group. ConclusionY-27632 suppresses the expression of senescence-related proteins and enhances cellular proliferation. This study points to the possibility of scaling-up the therapeutic use of SGSCs, which requires a large amount of cells.