Cytokine receptor repertoire and cytokine responsiveness of Ho(dull)/Rh(dull) stem cells with differing potentials for G1/S phase progression.
OBJECTIVE
Subsetting of Hoechst 33342 dull (Ho(dull)) hematopoietic stem cells on the basis of rhodamine 123 (Rh) efflux utilizing an improved dual-dye efflux strategy resolves Ho(dull)/Rh(dull) stem cell subsets that differ with regard to their rate of recruitment and progression through the cell cycle upon exposure to cytokines.


METHODS
Murine bone marrow cells were isolated by negative immunomagnetic selection using lineage-directed antibodies followed by Ho and Rh staining using a dual-dye efflux method.


RESULTS
Ho(dull)/Rh(dull) stem cells that efflux Rh more efficiently (R1) exhibit a 4- to 8-hour delay in progression to S phase when stimulated by interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor (SCF) compared to Ho(dull)/Rh(medium) stem cells, which retain low levels of Rh (R2). R1 and R2 cells show a hierarchical entry into S phase upon exposure to any or all of these cytokines. The R1 subset contains proportionately more high proliferative potential colony-forming cells than the R2 subset, but equivalent levels of engraftable stem cells at 3 and 8 weeks after competitive transplantation. Both R1 and R2 cells express c-kit, IL-3R, and IL-11R, whereas IL-6R and c-fms are only expressed by R1 or R2 cells, respectively. Cytokine stimulation of R1 and R2 cells induced cell cycle progression with elevated or induced expression of c-kit, c-fms, IL-2R, and IL-6R.


CONCLUSIONS
These studies indicate that primitive marrow stem cells can be further subsetted by degree of Rh staining to reveal important functional phenotypic differences between cells with different levels of Rh staining.
Vattikuti Urology Institute, Henry Ford Health Sciences Center, Detroit, MI, USA.
G Prem Veer
GP
Reddy
Christina I
CI
McAuliffe
Lizhen
L
Pang
Peter J
PJ
Quesenberry
Ivan
I
Bertoncello
D000818:Animals; D001562:Benzimidazoles / Q000032:analysis* / Q000378:metabolism; D001692:Biological Transport; D015415:Biomarkers; D001854:Bone Marrow Cells / Q000145:classification / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016026:Bone Marrow Transplantation; D019070:Cell Lineage; D003114:Colony-Forming Units Assay; D016207:Cytokines / Q000494:pharmacology*; D005260:Female; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D016193:G1 Phase / Q000502:physiology*; D006085:Graft Survival; D006412:Hematopoietic Stem Cells / Q000145:classification* / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018189:Immunomagnetic Separation; D017370:Interleukin-11 / Q000494:pharmacology; D053703:Interleukin-11 Receptor alpha Subunit; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D019009:Proto-Oncogene Proteins c-kit / Q000096:biosynthesis / Q000235:genetics; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics; D018121:Receptors, Cytokine / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics; D018123:Receptors, Interleukin / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D053702:Receptors, Interleukin-11; D015375:Receptors, Interleukin-2 / Q000096:biosynthesis / Q000235:genetics; D016185:Receptors, Interleukin-3 / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D019947:Receptors, Interleukin-6 / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D020112:Rhodamine 123 / Q000032:analysis* / Q000378:metabolism; D016196:S Phase / Q000502:physiology*; D019089:Stem Cell Factor / Q000494:pharmacology; D013997:Time Factors; D015854:Up-Regulation / Q000187:drug effects
D001562:Benzimidazoles; D015415:Biomarkers; D016207:Cytokines; D005456:Fluorescent Dyes; C508429:Il11ra1 protein, mouse; D017370:Interleukin-11; D053703:Interleukin-11 Receptor alpha Subunit; D007377:Interleukin-3; D015850:Interleukin-6; D018121:Receptors, Cytokine; D018123:Receptors, Interleukin; D053702:Receptors, Interleukin-11; D015375:Receptors, Interleukin-2; D016185:Receptors, Interleukin-3; D019947:Receptors, Interleukin-6; D019089:Stem Cell Factor; D020112:Rhodamine 123; D019009:Proto-Oncogene Proteins c-kit; D016186:Receptor, Macrophage Colony-Stimulating Factor; C017807:bisbenzimide ethoxide trihydrochloride