Severe Hypoxia Defines Heterogeneity and Selects Highly Immature Progenitors Within Clonal Erythroleukemia Cells

We showed that resistance to severe hypoxia defines hierarchical levels within normal hematopoietic populations and that hypoxia modulates the balance between generation of progenitors and maintenance of hematopoietic stem cells (HSC) in favor of the latter. This study deals with the effects of hypoxia (0.1% oxygen) in vitro on Friend's murine erythroleukemia (MEL) cells, addressing the question of whether a clonal leukemia cell population comprise functionally different cell subsets characterized by different hypoxia resistance. To identify leukemia stem cells (LSC), we used the Culture Repopulating Ability (CRA) assay we developed to quantify in vitro stem cells capable of short‐term reconstitution (STR). Hypoxia strongly inhibited the overall growth of MEL cell population, which, despite its clonality, comprised progenitors characterized by markedly different hypoxia‐resistance. These included hypoxia‐sensitive colony‐forming cells and hypoxia‐resistant STR‐type LSC, capable of repopulating secondary liquid cultures of CRA assays, confirming what was previously shown for normal hematopoiesis. STR‐type LSC were found capable not only of surviving in hypoxia but also of being mostly in cycle, in contrast with the fact that almost all hypoxia‐surviving cells were growth‐arrested and with what we previously found for HSC. However, quiescent LSC were also detected, capable of delayed culture repopulation with the same efficiency as STR‐like LSC. The fact that even quiescent LSC, believed to sustain minimal residual disease in vivo, were found within the MEL cells indicates that all main components of leukemia cell populations may be present within clonal cell lines, which are therefore suitable to study the sensitivity of individual components to treatments.