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Rhodamine-123 Staining in Hematopoietic Stem Cells of Young Mice
Indicates Mitochondrial Activation Rather Than Dye Efflux
MiJung Kim,
Donna D. Cooper,
Stanley F. Hayes, and
Gerald J. Spangrude
From the Departments of Pathology, Division of Cell Biology and
Immunology, and Medicine, Division of Hematology/Oncology, University
of Utah, Salt Lake City, UT; and Microscopy Branch, National Institute
of Allergy and Infectious Diseases, National Institutes of
Health, Rocky Mountain Laboratories, Hamilton, MT.
Low-intensity fluorescence of rhodamine-123 (Rh-123) discriminates a
quiescent hematopoietic stem cell (HSC) population in mouse bone
marrow, which provides stable, long-term 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 substrate for P-glycoprotein (P-gp). To address the
mechanisms of long-term repopulating HSC discrimination by Rh-123,
mouse bone marrow stem and progenitor cells were isolated based on
surface antigen expression and subsequently separated into subsets
using various fluorescent probes sensitive to mitochondrial characteristics and/or MDR function. We determined the cell
cycle status of the separated populations and tested for HSC function using transplantation assays. Based on blocking studies using MDR
modulators, we observed little efflux of Rh-123 from HSC obtained from
young (3- to 4-week-old) mice, but significant efflux from HSC derived
from older animals. A fluorescent MDR substrate (Bodipy-verapamil, BodVer) and Rh-123 both segregated quiescent cells into a dim-staining population, however Rh-123-based separations resulted in better enrichment of HSC function. Similar experiments using two other fluorescent probes with specificity for either mitochondrial mass or
membrane potential indicated that mitochondrial activation is more
important than either mitochondrial mass or MDR function in defining
HSC in young mice. This conclusion was supported by morphologic studies
of cell subsets separated by Rh-123 staining.
Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4106-4117
© 1998 by The American Society of Hematology.
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