Functional heterogeneity within rhodamine123lo Hoechst33342lo/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 G0 and G1 of the cell cycle. Materials and Methods A combination of supravital dyes rhodamine123 (Rh), Hoechst33342 (Ho), and pyronin (PY) was used to isolate the G0 and G1 subsets of PHSC. A competitive repopulation assay was used to evaluate their in vivo function. Results We confirmed that the RhloLin−Kit+Sca-1+ PHSC were relatively quiescent when compared with the more mature RhhiLin−Kit+Sca-1+ HSC and RhhiLin−Kit+Sca-1− progenitors. In addition, cells with RhloLin−Kit+Sca-1+, RhloHoloLin−Sca-1+, or RhloHospLin−Sca-1+ phenotypes identified the same cell population. We further subfractionated the RhloHolo/spLin−Sca-1+ PHSC using PY into PYlo and PYhi subsets. Limiting dilution analysis revealed that the frequency of long-term in vivo competitive repopulating units (CRU) of the PYloRhloHolo/sp PHSC was 1 in 10 cells, whereas there was at least a three-fold lower frequency in those isolated at the G1 phase (PYhi). 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. Conclusion 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 RhloHolo/sp PHSC population but is of limited use in dissecting the relatively more mature hematopoietic stem/progenitor cell population.