Expansion of human SCID-repopulating cells under hypoxic conditions
Guénahel H. Danet, … , Dominique A. Bonnet, M. Celeste Simon
Guénahel H. Danet, … , Dominique A. Bonnet, M. Celeste Simon
Published July 1, 2003
Citation Information: J Clin Invest. 2003;112(1):126-135. https://doi.org/10.1172/JCI17669.
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Article Hematology

Expansion of human SCID-repopulating cells under hypoxic conditions

Abstract

It has been proposed that bone marrow (BM) hematopoietic stem and progenitor cells are distributed along an oxygen (O2) gradient, where stem cells reside in the most hypoxic areas and proliferating progenitors are found in O2-rich areas. However, the effects of hypoxia on human hematopoietic stem cells (HSCs) have not been characterized. Our objective was to evaluate the functional and molecular responses of human BM progenitors and stem cells to hypoxic conditions. BM lineage–negative (Lin–) CD34+CD38– cells were cultured in serum-free medium under 1.5% O2 (hypoxia) or 20% O2 (normoxia) for 4 days. Using limiting dilution analysis, we demonstrate that the absolute number of SCID-repopulating cells (SRCs) increased by 5.8-fold in hypoxic cultures compared with normoxia, and by 4.2-fold compared with freshly isolated Lin–CD34+CD38– cells. The observed increase in BM-repopulating activity was associated with a preferential expansion of Lin–CD34+CD38– cells. We also demonstrate that, in response to hypoxia, hypoxia-inducible factor-1α protein was stabilized, surface expression of angiogenic receptors was upregulated, and VEGF secretion increased in BM Lin–CD34+ cultures. The use of low O2 levels to enhance the survival and/or self-renewal of human BM HSCs in vitro represents an important advance and could have valuable clinical implications.

Authors

Guénahel H. Danet, Yi Pan, Jennifer L. Luongo, Dominique A. Bonnet, M. Celeste Simon

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Figure 1

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Expansion and division history of BM cells after 4 days in culture in hy...
Expansion and division history of BM cells after 4 days in culture in hypoxia or normoxia. Lin–CD34+ and Lin–CD34+CD38– cells were cultured in serum-free conditions for 4 days in the presence of IL-3, IL-6, SCF, Flt-3 ligand, and G-CSF. (a) Expansion. The fold increase in cell number relative to the initial cell number plated (day 0) is represented for each subpopulation cultured in normoxia (white bars) or hypoxia (black bars). *P < 0.05. (b) Division history of primitive subpopulations of Lin–CD34+ cells. Freshly isolated Lin–CD34+ cells were labeled with CFSE, cultured for 4 days in hypoxia (1.5% O2) or normoxia, and analyzed for CFSE fluorescence intensity and expression of markers associated with primitive progenitors and stem cells. Histograms (representative of three separate experiments) of CFSE fluorescence in various Lin–CD34+ cell subsets are shown after 4 days of culture in hypoxia (solid lines) or normoxia (dashed lines). Day 0 CFSE fluorescence intensity is indicated by an arrow on each histogram.