Bone marrow cell liquid cultures were incubated at various oxygen concentrations ranging from 0% to 18% (air). The total number of cells in culture (CT) at the end of a 6-day incubation was found to be directly proportional to the oxygen concentration. As compared with air- incubated controls, cells recovered from severely hypoxic (1% oxygen) day-5 liquid cultures showed (1) the same day-7 colony-formation efficiency in semisolid culture (neutrophilic/monocytic colonies) or in spleen; (2) a higher day-14 spleen colony-formation efficiency; (3) an enhanced radio-protection ability; and (4) an increased marrow repopulation ability, as measured by determining either total cell number in recipient marrow MRAcell, or the capacity of the latter of generating day-7 neutrophilic/monocytic colonies in secondary in vitro assays (MRACFU-NM). Taking into account CT, the absolute numbers of progenitors in culture were also computed. The results showed that, with respect to time 0, incubation in air produced an increase in the number of day-7 CFUs and a decrease in the number of the other progenitors, whereas in hypoxic cultures all types of progenitors decreased. However, as compared with air-incubated controls, all progenitors, except cells sustaining MRACFU-NM, were reduced in hypoxic cultures. The degree of reduction paralleled the position of the progenitor in the hematopoietic hierarchy, being maximum for day-7 CFUs and null for cells sustaining MRACFU-NM, which, in fact, were better preserved in hypoxic cultures.

When cultured at 1-percent O(2) for 7 days in presence of IL-3 and SCF, the CD34+ cells present in apheresis components underwent more cell divisions and better maintained their primitive progenitor cell potential. As suggested by previous results in mice, our data on human cells emphasize the potential interest of cultures at low O(2) tension (1%) for cell therapy protocols aimed at expanding primitive HPCs in autografts.

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BackgroundIncubation of chronic myeloid leukemia cells in hypoxia inhibits growth and selects BCR/Ablindependent cells with stem cell properties which are refractory to imatinib-mesylate. This study aimed to characterize the relationship of this refractoriness with glucose availability in the environment. Design and MethodsK562 or primary chronic myeloid leukemia cells were cultured at 0.1% O2, different cell densities and glucose concentrations. The stem and progenitor cell potential of these cultures at different times of incubation in relation to BCR/Ablprotein expression and sensitivity to imatinibmesylate was explored by transferring cells to growth-permissive secondary cultures in normoxia, according to the Culture-Repopulating Ability assay methodology. ResultsHypoxia-resistant cells maintained BCR/Ablprotein expression until glucose was no longer available in primary hypoxic cultures, where glucose availability appeared to regulate cell number and the balance between the enrichment of cells with kinetic properties typical of stem or progenitor cells. Cells surviving merely hypoxic conditions were, upon transfer to secondary cultures, immediately available for numerical expansion due to the maintained BCR/Ablprotein expression, and were consequently sensitive to imatinib-mesylate. Instead, BCR/Ablprotein-negative cells selected in primary cultures under oxygen/glucose shortage underwent a delayed numerical expansion in secondary cultures, which was completely refractory to imatinib-mesylate. Cells with the latter properties were also found in primary chronic myeloid leukemia explants. ConclusionsGlucose shortage in hypoxia was shown to represent the condition selecting BCR/Ablprotein-negative cells refractory to imatinib-mesylate from either chronic myeloid leukemia lines or patients. These cells, exhibiting stem cell properties in vitro, are metabolically suited to home to stem cell niches in vivo and so may represent the chronic myeloid leukemia cell subset responsible for minimal residual disease.Key words: imatinib-mesylate, chronic myeloid leukemia, glucose, hypoxia.Citation: Giuntoli S, Tanturli M, Di Gesualdo F, Barbetti V, Rovida E, and Dello Sbarba P. Glucose availability in hypoxia regulates the selection of chronic myeloid leukemia progenitor subsets with different resistance to imatinib-mesylate. Haematologica 2011;96(2):204-212. doi:10.3324/haematol.2010 Glucose availability in hypoxia regulates the selection of chronic myeloid leukemia progenitor subsets with different resistance to imatinib-mesylate