Hypoxia Potentiates Notch-Induced Expansion of Human Hematopoietic Stem/Progenitor Cells

Activation of Notch signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with Notch ligand Delta1 has enabled a clinically relevant ex vivo expansion of short-term HSPCs. In vitro studies have also revealed a role of low O₂ tension in HSPC regulation. A molecular link has been demonstrated in several stem/progenitor cell populations between Notch and hypoxia pathways but their interaction has not been investigated in human HSPCs. G-CSF mobilized human CD34+ cells from 4 healthy subjects were cultured in the presence of cytokines (SCF, FLT3L and TPO) in hypoxia (1.5-2% O₂) or normoxia (21% O₂) in vessels coated with fibronectin alone or combined with increasing concentrations of the immobilized ligand Delta1 (2.5, 5, 10 and 20 µg/mL). After 21 days in culture, cells were counted and characterized using CFU assays, flow cytometry for lineage (Glycophorin A+, CD13+, CD20+, CD3+ and CD41+ cells) and HSC (CD34+ CD38- CD45RA- CD90+ CD49f+ Rho^low) phenotypes, and transplantation in immunodeficient (NSG) mice. In normoxia, the total number of cells increased 118-fold compared to baseline in the absence of Delta1 with limited residual CD34+ cells (1.5 ± 0.7%), extensive differentiation toward the myeloid lineage (96.3 ± 0.3% CD13+ cells) and minimal engraftment potential in NSG mice (0.2 ± 0.2% human CD45+ cells). With increasing concentrations of Delta1 in normoxia, consistent with the hypothesis that Delta1 delays differentiation, the total number of cells increased less (41-, 25-, 11- and 7-fold relative to baseline, respectively) CD34+ cells expanded more (4-, 4-, 3- and 2-fold relative to baseline, respectively), and CFU numbers increased more (8-, 7-, 4- and 3-fold relative to baseline, respectively) than without Delta1. However, phenotypically defined HSCs were undetectable or markedly decreased at the lowest Delta1 concentrations used (2.5 and 5 µg/mL) and their numbers were maintained or only minimally increased at the highest Delta-1 concentrations tested (10 and 20 µg/mL) relative to uncultured CD34+ cells. Accordingly, only cells cultured with 10 and 20 µg/mL Delta1 resulted in levels of engraftment in NSG mice (5.5 ± 5.4% and 5.4 ± 0.9% human CD45+ cells, respectively) comparable to uncultured cells (7.0 ± 0.1% human CD45+ cells). In hypoxia, total cell counts increased less than in normoxia both without (8-fold relative to baseline) and with increasing concentrations of Delta1 (11-, 11-, 9-, 9-fold relative to baseline, respectively) due to diminished myeloid differentiation. Total CD34+ cells decreased 1.7-fold in hypoxia in the absence of Delta1, but expanded modestly in the presence of Delta1 (3-, 3-, 2- and 2-fold, respectively). CFU numbers followed a similar trend. However, in hypoxic cultures with 2.5, 5 and 10 µg/mL Delta1, phenotypically defined HSCs increased 2.5-, 6.6- and 1.3-fold, respectively, compared to uncultured cells. Importantly, hypoxia combined with 2.5, 5 and 10 µg/mL Delta1 concentrations resulted in increased human cell engraftment in NSG mice (21.2 ± 4.4%, 29.3 ± 11% and 11.8 ± 5.4% human CD45+ cells, respectively) compared to uncultured cells (7.0 ± 0.1% human CD45+ cells). When 20 µg/mL Delta1 was used in hypoxia, engraftment potential in NSG mice was decreased (1.1 ± 0.6% human CD45+ cells). We next performed limiting dilution analysis to measure the frequencies of long-term repopulating HSCs (LT-HSCs) within the CD34+ cell compartment at baseline and after 21 days in hypoxic or normoxic cultures supplemented with the optimized concentrations of Delta1 (10 µg/mL in normoxia and 5 µg/mL in hypoxia). LT-HSCs in uncultured CD34+ cells were measured at the expected frequency (1 in 7,706; 95% CI of 3,446 to 17,232). When analyzed at 3 months post-transplantation, a limited (1.5-fold) increase in LT-HSC frequency (1 in 5,090; 95% CI 2.456 to 10,550) was obtained from Delta1 normoxic cultures compared to uncultured cells. In contrast, the frequency of LT-HSCs (1 in 1,586; 95% CI 680 to 3,701) was 4.9-fold higher in hypoxic Delta1 cultures compared to uncultured cells, and 4.2-fold higher than in normoxic Delta1 cultures. Similarly, absolute numbers of LT-HSCs per 100,000 Day 0 equivalent CD34+ cells increased from 13 (baseline) to 216 (normoxia) and 694 (hypoxia). Our data indicate that hypoxia potentiates Notch-induced expansion of human HSPCs and may be of benefit in stem cell transplantation and gene therapy applications.