Abstract
Under normal physiological conditions, hematopoietic stem cells (HSC) are sequestered in a hypoxic microenvironment in the bone marrow (BM), suggesting that low oxygen levels may play a fundamental role in the maintenance of normal stem cell function and protect these cells from the damaging effects of reactive oxygen species (ROS). In vitro culture of human BM CD34+ cells under hypoxic conditions has been shown to result in expansion of SCID-repopulating cells (SRC) as compared to culture under normoxic conditions (JCI112 (1); 126, 2003). We investigated whether culture of human mobilized CD34+ cells under low oxygen conditions (5% O2) could improve lentiviral transduction efficiency in SRC compared with culture under atmospheric O2 conditions (21%). G-CSF mobilized CD34+ cells from 4 healthy volunteers were prestimulated for 48 hours in the presence of cytokines (SCF, Flt-3 ligand and thrombopoietin) and subsequently transduced in fibronectin coated plates for 24 hours with SIN-lentiviral vectors carrying the GFP gene under the control of an EF1α promoter. In 3 experiments, cells were used for in vitro assays, including ROS, phenotypic, cell cycle, clonogenic and apoptosis assays. In one experiment, cells were injected intravenously in the tail vein of sublethally irradiated NOD/SCID IL2rγ −/− mice after transduction. Intracellular ROS levels increased more significantly in human CD34+ cells cultured for 3 days in 21% O2 compared with cells cultured in 5% O2. When cultures were maintained more than 3 days, ROS levels were similar between the 2 conditions. The levels of expansion of CD34+ cells compared with baseline were similar in hypoxia (3.9-fold) and normoxia (3.5-fold) (p=0.47). In contrast, the expansion of CD34+CD38− cells, a subpopulation enriched in HSCs, was greater in hypoxia (3.8-fold) than in normoxia (2.2-fold) (p=0.02). After 3 days of culture, the total number of colony-forming cells (CFC) increased 1.1-fold and 1.3-fold under hypoxic and normoxic conditions, respectively (p=0.32) compared with freshly isolated CD34+ cells. The level of O2 had no significant effect on lineage commitment of the CFC. At baseline, the majority (59.5%) of the CD34+ cells were in the G0 phase of the cell cycle. After 3 days in culture under hypoxic or normoxic conditions, the percentages of cells in G0 were 5.5% and 3.5%, respectively (p=0.03). The differences in percentages of cells in the G1 and G2/S/M phases of the cell cycle were not statistically different. The percentages of CD34+ apoptotic cells were similar between hypoxic (32.8%) and normoxic (29.5%) conditions (p=0.18). The pO2 also had no impact on CD34+ cell death (12.2% at 5% O2 and 11.7% at 21% O2, p=0.9). When considering the bulk of CD34+ cells after transduction with GFP-lentiviral vectors, there was no statistically significant difference in the percentages of GFP+ cells under hypoxia (22.3%) or normoxia (21%) (p=0.88). In contrast, when CD34+ cells cultured under hypoxia were injected into NOD/SCID IL2rγ −/− mice at the end of the transduction period, improved human cell engraftment and lentiviral transduction efficiency were detected 2 months after transplantation compared with CD34+ cells cultured under normoxia. Human cell engraftment in the mouse BM, as determined by flow cytometry using a human specific CD45 antibody, was 84% in the hypoxic group (n=4) and 54% in the normoxic group (n=4) (p=0.04). The level of O2 had no significant impact on the lineage commitment of the SRC, with a majority of CD45+CD15+ granulocytes in both groups. The percentage of GFP+CD45+ cells was 54% (hypoxia) and 43% (normoxia) (p=0.02), indicating an improved transduction efficiency of SRC under hypoxic conditions. Overall, these data indicate that human CD34+ cells cultured under low oxygen conditions maintain a more primitive phenotype and have an increased susceptibility to lentiviral transduction compared with cells cultured in 21% O2 conditions. Improved engraftment and transduction efficiency do not appear to be related to decreased apoptosis in lower O2 concentrations; instead, increased ROS production in higher O2 concentrations could lead to increased cell signaling and differentiation. Use of low O2 levels for in vitro transduction of human CD34+ cells could have important clinical implications in gene therapy.
Harnessing hypoxia as an evolutionary driver of complex multicellularity