Characteristic of side population cells in parotid glands

Objectives: Stem cell therapy is expected to be able to reconstruct lost organs in salivary gland disorders. Recently, it has been reported that hematopoietic stem cells or bone marrow progenitor cells are assumed to be in the ‘side population' (SP) when stained with Hoechst 33342 dye, but few SP cells of the parotid gland have been reported to be stained. The aim of this study was to investigate characteristics of SP cells obtained from the parotid gland.

Methods: Mice parotid gland tissues and bone marrow tissue as a control were obtained from four-week-old Std/ddy mice. Both tissue samples were incubated in phosphate-buffered saline solution containing 3 mg/mL collagenase and 4 mg/mL dispase for 1 hr at 37°C, to divide single cells. The cells were re-suspended in Hanks' balanced solution, including 10 mM HEPES and 2% fetal bovine serum. Subsequently, aliquots were prepared for further staining and flow cytometric analysis. After the cell concentrations were adjusted to exactly 10⁶ cells/mL, the cells were stained with 5 µg/mL Hoechst 33342 at 37°C for 90 min. With a BD FACSAria, the SP cells stained by Hoechst 33342 at low side were sorted and purified from the main population (MP) cells. To evaluate cell characteristics, we also carried out immunochemical staining. The cells were stained with directly conjugated anti-mouse CD45, Ly6A/E (Sca-1), and CD117 (c-kit) (BD Pharmingen) at 4°C for 30 min, and then analyzed by FACSAria.

Results: The parotid SP cells accounted for approximately 0.3% of the cell population, and bone marrow SP cells for approximately 0.2%, respectively. The SP cells were confirmed positive for sca-1 and c-kit and negative for CD34 in SP cells.

Conclusion: These results suggested that SP cells in parotid glands could have the characteristics of stem cells.