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.
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