Modification of rhodamine staining allows identification of hematopoietic stem cells with preferential short-term or long-term bone marrow-repopulating ability.
We have developed a modified rhodamine (Rho) staining procedure to study uptake and efflux in murine hematopoietic stem cells. Distinct populations of Rho++ (bright), Rho+ (dull), and Rho- (negative) cells could be discriminated. Sorted Rho- cells were subjected to a second Rho staining procedure with the P-glycoprotein blocking agent verapamil (VP). Most cells became Rho positive [Rho-/Rho(VP)+ cells] and some remained Rho negative [Rho-/Rho(VP)- cells]. These cell fractions were characterized by their marrow-repopulating ability in a syngeneic, sex-mismatch transplantation model. Short-term repopulating ability was determined by recipient survival for at least 6 weeks after lethal irradiation and transplantation--i.e., radioprotection. Long-term repopulating ability at 6 months after transplantation was measured by fluorescence in situ hybridization with a Y-chromosome-specific probe, by graft function and recipient survival. Marrow-repopulating cells were mainly present in the small Rho- cell fraction. Transplantation of 30 Rho- cells resulted in 50% radioprotection and > 80% donor repopulation in marrow, spleen, and thymus 6 months after transplantation. Cotransplantation of cells from both fractions in individual mice directly showed that within this Rho- cell fraction, the Rho-/Rho(VP)+ cells exhibited mainly short-term and the Rho-/Rho(VP)- cells exhibited mainly long-term repopulating ability. Our results indicate that hematopoietic stem cells have relatively high P-glycoprotein expression and that the cells responsible for long-term repopulating ability can be separated from cells exhibiting short-term repopulating ability, probably by a reduced mitochondrial Rho-binding capacity.
Zijlmans
J M
JM
Department of Hematology, University Medical Center, Leiden, The Netherlands.
Visser
J W
JW
Kleiverda
K
K
Kluin
P M
PM
Willemze
R
R
Fibbe
W E
WE
D000818:Animals; D001853:Bone Marrow / Q000502:physiology; D001854:Bone Marrow Cells; D016026:Bone Marrow Transplantation / Q000401:mortality; D002469:Cell Separation / Q000379:methods; D005260:Female; D014644:Genetic Variation; D018380:Hematopoietic Stem Cell Transplantation / Q000401:mortality; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D017404:In Situ Hybridization, Fluorescence; D066298:In Vitro Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D016634:Radiosurgery; D012235:Rhodamines; D013154:Spleen / Q000166:cytology; D013194:Staining and Labeling / Q000379:methods*; D016019:Survival Analysis; D013950:Thymus Gland / Q000166:cytology; D018183:Transplantation Chimera; D014700:Verapamil / Q000494:pharmacology; D014916:Whole-Body Irradiation
D012235:Rhodamines; D014700:Verapamil