Low O2 cultures expand HSCs from unfractionated WBMCs and embryonic tissues. (A) Schematic diagram of low O2 WBMC cultures. Unfractionated mouse WBMCs were seeded into PVA-based media in 24-well plates (5 × 106 cells per well) in 1 ml of media and cultured for 4 weeks at 20%, 5%, or 1% O2. (B) The mean number of live cells per well within the WBMC-derived cultures described panel in A on day-28 (n = 6). (C) The mean number of CD201+CD150+KSL cells per well within the WBMC-derived cultures described in panel A on day-28 (n = 6). (D) 16-week donor peripheral blood (PB) chimerism from 4-week-old WBMC-derived cultures incubated in 20%, 5%, or 1% O2. Five thousand cells from each culture were transplanted along with 1×106 WBMCs into lethally irradiated recipients (mean ± SD; n = 3-5). (E) Twelve-week donor peripheral blood chimerism after secondary transplantation of WBMCs from primary recipient mice described in panel D (Mean ± SD; n = 3-4). (F) Schematic diagram of the CyTOF time course during 5% O2 WBMC cultures (left) and the frequency of immunophenotypic cell populations at the indicated time points (right). See supplemental Figure 5B for cell immunophenotypes. (G) Schematic diagram of the CyTOF time course during 5% O2 whole spleen cell cultures (left) and the frequency of immunophenotypic cell populations for 28-day cultures (right). supplemental Figure 5B shows cell immunophenotypes. (H) Schematic diagram of selective HSC expansion assay for mouse embryonic tissues. (I) The mean number of CD201+CD150+KSL cells generated from E11.5 to 12.5 embryonic tissue (excluding extraembryonic tissues and fetal liver) and from E11.5 to 16.5 heart, yolk sac, placenta, and fetal liver-derived cultures. The starting cell number are indicated in brackets seeded in 1 mL of media. Statistical analysis was performed using ANOVA.; ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001. n.s., not significant.
