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Background/Aims: Although tonsil-mesenchymal stem cells (T-MSCs) have been studied as a new autologous or homologous source of MSCs, research on specific markers of MSCs and localization for purified T-MSC isolation has not yet been reported. This study investigates the expression of W5C5 (SUSD2) in tonsil stromal cells and the colony-forming ability and differentiation potential of W5C5+ cells to determine the usefulness of W5C5+ MSCs as a marker that can be used for the purification of T-MSCs. In addition, the location of W5C5+ cells expressed in the tonsil tissues is examined. Methods: T-MSCs were isolated from the tonsillar tissues of 12 patients undergoing tonsillectomy. The colony-forming ability, surface markers, proliferation potential, and differentiation capacities of purified W5C5+ MSCs, W5C5- MSCs, and unselected T-MSCs were evaluated. The location of the W5C5+ cells in the tonsillar tissues was also investigated by immunohistochemistry. Results: W5C5 was expressed in 2.5±0.4% of fresh human tonsil stromal cells. W5C5+ cells formed many colonies, but W5C5- cells did not form any colonies. The colony-forming number of W5C5+ cells (74.4 ± 9.8) was significantly higher than that of unselected tonsil stromal cells (23.6 ± 3.7). However, the differences in proliferation potential, surface marker expression, and differentiation potential between W5C5+ T-MSCs and unselected T-MSCs were not significant. W5C5+ cells were identified in the perivascular area around the blood vessels. Conclusion: W5C5+ T-MSCs possessed typical MSC properties with high colony-forming efficiency, and niches of W5C5+ T-MSCs were located in the perivascular area of tonsil tissues. These findings suggest that W5C5 is a useful single marker for the isolation of purified T-MSCs. © 2016 The Author(s) Published by S. Karger AG, Basel [ABSTRACT FROM AUTHOR]