The Use of Hoechst Staining To Identify Hematopoietic and Other Adult Stem Cells.

BACKGROUND: Hoechst staining has been used to identify hematopoietic stem cells, but it may also be useful in identifying other adult stem cells. Here we report our efforts to purify and characterize stem cells in the stromal vascular fraction of adipose tissue with Hoechst staining, also comparing our results with those of our studies using bone marrow.

METHODS: Stromal vascular fractions (SVFs) of adipose tissue and whole bone marrow (BM) were harvested from C57BL/6N mice, as were stem cells. Then the cells were stained with Hoechst 33342 and analyzed by flow cytometry and the number of cells in the side population (SP) counted. Moreover, surface antigens of SP cells were analyzed by flow cytometry using antibodies against CD44, 45, 45R, Sca-1, and c-kit, respectively, for 30 min on ice. Finally, the morphologic characteristics of cells in the SP of both BM and SVF were observed using electron microscopy.

RESULTS: The percentage of SP cells in BM was about 0.05 to 0.1% and that in the SVF was about 1.0 to 3.0%. The cell-surface antigens of BM expressed were CD44 (−), CD 45 (+), CD 45R (−), Sca-1 (+) and c-kit (+), while those of SVF were CD44 (−), CD 45 (−), CD 45R (+/−), Sca-1 (+/−) and c-kit (−). Upon electron microscopic observation, both BM and SVF cells in the SP were considered to be remarkably immature (immature cell organelles and a high N/C ratio).

CONCLUSION: The rate and expression patterns of cell-surface antigens in SP cells derived from BM were consistent with the results of previous reports. However, the same characteristics in SP cells derived from SVFs were clearly different. At present it is not clear whether cells in the SP of SVFs are adipose-derived stem cells. Indications were that there are 10 to 60 times as many immature cells in adipose tissue as in bone marrow. Moreover, it is possible that the great majority of cells in the SP of SVFs are not hematopoietic stem cells but unique adipose-derived stem cells. Finally, our studies suggest that Hoechst staining may be useful for identifying not only hematopoietic stem cells but also other adult stem cells.