Functional heterogeneity within rhodamine123(lo) Hoechst33342(lo/sp) primitive hemopoietic stem cells revealed by pyronin Y.
OBJECTIVE
The aim of this study was to determine the function of primitive hematopoietic stem cells (PHSC) at phases G(0) and G(1) of the cell cycle.


METHODS
A combination of supravital dyes rhodamine123 (Rh), Hoechst33342 (Ho), and pyronin (PY) was used to isolate the G(0) and G(1) subsets of PHSC. A competitive repopulation assay was used to evaluate their in vivo function.


RESULTS
We confirmed that the Rh(lo)Lin(-)Kit(+)Sca-1(+) PHSC were relatively quiescent when compared with the more mature Rh(hi)Lin(-)Kit(+)Sca-1(+) HSC and Rh(hi)Lin(-)Kit(+)Sca-1(-) progenitors. In addition, cells with Rh(lo)Lin(-)Kit(+)Sca-1(+), Rh(lo)Ho(lo)Lin(-)Sca-1(+), or Rh(lo)Ho(sp)Lin(-)Sca-1(+) phenotypes identified the same cell population. We further subfractionated the Rh(lo)Ho(lo/sp)Lin(-)Sca-1(+) PHSC using PY into PY(lo) and PY(hi) subsets. Limiting dilution analysis revealed that the frequency of long-term in vivo competitive repopulating units (CRU) of the PY(lo)Rh(lo)Ho(lo/sp) PHSC was 1 in 10 cells, whereas there was at least a three-fold lower frequency in those isolated at the G(1) phase (PY(hi)). We found a dose-dependent PY-mediated cytotoxicity that at moderate concentration affected most of the murine hematopoietic compartment but spared the early HSC compartment.


CONCLUSIONS
Our data confirm that the HSC compartment is hierarchically ordered on the basis of quiescence and further extend this concept to PY-mediated cytotoxicity. PY supravital dye can be used to reveal functional heterogeneity within the Rh(lo)Ho(lo/sp) PHSC population but is of limited use in dissecting the relatively more mature hematopoietic stem/progenitor cell population.
Hüttmann
A
A
Queensland Institute of Medical Research, Herston, Queensland, Australia.
Liu
S L
SL
Boyd
A W
AW
Li
C L
CL
D000818:Animals; D001562:Benzimidazoles; D001854:Bone Marrow Cells / Q000166:cytology; D016923:Cell Death / Q000187:drug effects / Q000528:radiation effects; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D016193:G1 Phase / Q000502:physiology; D006410:Hematopoiesis; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects*; D016130:Immunophenotyping; D051379:Mice; D008810:Mice, Inbred C57BL; D017319:Photosensitizing Agents / Q000494:pharmacology; D011754:Pyronine / Q000494:pharmacology; D016192:Resting Phase, Cell Cycle / Q000502:physiology; D020112:Rhodamine 123
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D017319:Photosensitizing Agents; D020112:Rhodamine 123; C017807:bisbenzimide ethoxide trihydrochloride; D011754:Pyronine