RHODAMINE-123 STAINING IN HEMATOPOIETIC STEM-CELLS OF YOUNG MICE INDICATES MITOCHONDRIAL ACTIVATION RATHER THAN DYE EFFLUX

Low-intensity fluorescence of rhodamine-123 (Rh-123) discriminates a q uiescent hematopoietic stem cell (HSC) population in mouse bone marrow , which provides stable, longterm hematopoiesis after transplantation. Rh-123 labels mitochondria with increasing intensity proportional to cellular activation, however the intensity of staining also correlates with the multidrug resistance (MDR) phenotype, as Rh-123 is a substra te for P-glycoprotein (P-gp). To address the mechanisms of long-term r epopulating HSC discrimination by Rh-123, mouse bone marrow stem and p rogenitor cells were isolated based on surface antigen expression and subsequently separated into subsets using various fluorescent probes s ensitive to mitochondrial characteristics and/or MDR function. We dete rmined the cell cycle status of the separated populations and tested f or HSC function using transplantation assays. Based on blocking studie s using MDR modulators, we observed little efflux of Rh-123 from HSC o btained from young (3- to 4-week-old) mice, but significant efflux fro m HSC derived from older animals. A fluorescent MDR substrate (Bodipy- verapamil, BodVer) and Rh-123 both segregated quiescent cells into a d im-staining population, however Rh-123-based separations resulted in b etter enrichment of HSC function. Similar experiments using two other fluorescent probes with specificity for either mitochondrial mass or m embrane potential indicated that mitochondrial activation is more impo rtant than either mitochondrial mass or MDR function in defining HSC i n young mice. This conclusion was supported by morphologic studies of cell subsets separated by Rh-123 staining. (C) 1998 by The American So ciety of Hematology.