The expansion of murine bone marrow cells preincubated in hypoxia as an in vitro indicator of their marrow-repopulating ability

Abstract

In liquid cultures of murine bone marrow cells stimulated with interleukin-3 and granulocyte/macrophage colony-stimulating factor, hypoxia (1% oxygen) induced a reversible block of hematopoiesis, maintaining the progenitors' expansion potential unreduced. Progenitors repopulating day-14 hypoxic cultures with cells or granulocyte/macrophage colony-forming units (CFU-GM) were found, on the basis of their maintenance in hypoxia (12% and 76%, respectively), to belong to different subsets, the latter being much more efficiently maintained. The maintenance in hypoxic cultures of progenitors detectable by marrow-repopulating ability (MRA) assay was 18% for MRA cell progenitors and 69% for MRA CFU progenitors. Thus, the repopulation of hypoxic cultures with cells or CFU-GM closely reflected the presence of progenitors capable of repopulating, with cells or CFU-GM, the bone marrow of lethally irradiated syngeneic animals. Progenitors repopulating hypoxic cultures were, like MRA progenitors, significantly resistant to 5-fluorouracil, progenitors repopulating cultures with CFU-GM being two-fold more resistant than those repopulating cultures with cells. We concluded that the repopulation of day-14 hypoxic cultures occurring after their transfer to air is to be considered an indicator of the maintenance of MRA progenitors in hypoxia. The relevance of these results to stem cell biology and their potential practical applications are discussed. Leukemia (2000) 14, 735-739.

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

“…Cells selected in hypoxia, in a new set of experiments, were subjected to the CRA assay [2] to measure their STR-type stem cell potential (Fig. 3).…”

“…3A). LC2 was repopulated with the standard kinetics of CRA assay [2]. Thus, the MEL cell population contained hypoxia-resistant CRC.…”

“…Before being replated into LC2, cells selected in LC1 were centrifuged, resuspended in fresh medium, and counted. CRA has been shown to reflect stem cells endowed with marrow-repopulating ability in vivo, therefore representing a simple and economic method to detect in vitro STR-HSC [2]. The maintenance of culturerepopulating cells (CRC) after selection in LC1 is quantified by the ratio of peak (day 6) cell numbers in LC2 to peak cell numbers in nonselective LC1 established with equal numbers of cells.…”

“…We showed previously that resistance to severe hypoxia defines hierarchical levels within normal hematopoietic populations; in vitro colony-forming cells (CFC) are highly hypoxia-sensitive, as opposed to their progenitors, which are hypoxia-resistant [1][2][3][4][5]. Indeed, severe hypoxia enhances maintenance in vitro of normal stem cells capable of short-term hematopoietic reconstitution (STR-HSC).…”

“…In the study reported here, targeted to deepen the characterization of the effects of severe hypoxia on leukemia progenitor and stem cells, we used the murine Friend's erythroleukemia (MEL) cell line to address the question of whether cell subsets characterized by different sensitivity to hypoxia are detectable within a clonal leukemia cell line and whether these differences define the level within the progenitor/ stem cell hierarchy. To identify LSC, we used the Culture Repopulating Ability (CRA) assay, developed in our laboratory [2] and extensively applied [3,5,7] to quantify in vitro normal STR-HSC and later adapted to study their leukemic counterpart [11,12]. We found that the MEL cell line is functionally and metabolically heterogeneous in that it comprises hypoxia-sensitive CFC as well as hypoxia-resistant culture repopulating cells (CRC), which correspond to STR-type LSC.…”