but rather represents increased viability and therefore functionality of SP cells that stain low for Hoechst. This is a valid concern to which we respond below. Data on the toxicity of H33342 have been available for decades, as it is well known that H33342 is powerful inhibitor of DNA sythesis. 4 From these early experiments, it was very evident that the effects on cell viability varied tremendously between different cell types and Hoechst dye concentration. 4,5 Work by Van Zant and Fry 5 found that most hematopoietic progenitors, with the exception of colony-forming unit– megakaryocyte (CFU-Meg) and CFU-erythroid (CFU-E), were functionally unaffected after H33342 staining. Interestingly, CFU-spleen (CFU-S), a heterogeneous population consisting of both short and long-term HSCs, was the least sensitive to Hoechst exposure under conditions similar to the ones used for our SP stain. In addition, experiments that we reported in 1997, in which we compared the long-term repopulation activity of whole SP cells with that of SP cells additionally exposed to Hoechst in the presence of the efflux inhibitor verapamil, demonstrated no differences in terms of function, as assayed by competitive transplan-tation experiments. 2 We have also compared the clonogenicity of highly purified HSCs that were either stained or not with Hoechst 33342, and our results indicate that at least in vitro, there are no significant detrimental effects associated with Hoechst exposure. 3 Even though Hoechst staining does involve a great deal of viability loss in nonhematopoietic tissues, 6 as pointed out, it does not seem to cause any functional impairment within the primitive hematopoietic compartments. Therefore, we consider that our results do represent an inherent difference in the self-renewal capacity of cells within the SP subset that is directly correlated with their ability to efflux dye. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. Dye efflux studies suggest that he-matopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species.etic stem cells do not engraft with absolute efficiencies. Hoechst 33342 on survival and growth of two tumor cell lines and on hemato-poietically normal bone marrow cells.1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population. Cappellini and colleagues 1 claim that the use of a superconducting quantum interference device (SQUID) biosusceptometer underestimates liver iron concentration (LIC) in their phase 3 study of deferasirox (DFX). LIC was measured either in deparaffinized liver samples excised …
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