MSCs From marrow aspirates of CLL patients propagate better and resist senescence in vitro when cultured in 5% O₂ rather than in ambient O₂. (A) Marrow mononuclear cell suspensions from 7 different CLL patients were cultured in DMEM-10%FBS, at 5% CO₂ in 21% O₂ or 5% O₂ at 37°C. MSCs outgrowth and expansion was monitored over time by microscopy and viability determined via Trypan blue exclusion. From the 7 samples tested, only 2 (CLL1 and CLL2) generated MSC colonies at 21% O₂. The expansion curves of MSCs in 21% O₂ are depicted on the left and those generated in 5% O₂ are depicted on the right. (B) MSCs generated in 5% O₂ were examined for expression of selected surface antigens by flow cytometry. The phenotypic characterization of MSCs from a representative patient sample is shown. The shaded histogram is the fluorescence of MSCs stained with an isotype control antibody, whereas the open histogram depicts the fluorescence of MSCs stained with fluorochrome-conjugated antibody specific for the antigen listed below each histogram. (C-E) MSCs generated from marrow aspirates of CLL patients in 5% O₂ (between passage 2 and 5) were plated at various seeding densities, as indicated at the bottom of the histograms, in parallel cultures that subsequently were placed in 21% O₂ or 5% O₂. The black bars provide the data of cultures exposed to 5% O₂ and the open bars depict the data from MSCs cultured in 21% O₂. (C) Provides the mean number of viable MSCs obtained after 20 days in each of 4 independent experiments, performed using MSCs from the marrow aspirates of each of 3 different patients (mean ± SEM). (D) Provides the BrdU incorporation of MSCs after 6 days (n = 3; mean ± SEM) as indicated on the y-axis representing the absorbance at 450 to 690 nm. (E) Provides the viability of the MSCs at each condition in (C) based on Trypan blue exclusion (mean ± SEM). Statistically significant differences for each panel are indicated by the asterisk (*P < .05; Student t test). (F-H) MSCs generated in 5% O₂ were plated in parallel cultures at 300 cells/cm² and exposed to either 21% O₂ or 5% O₂. (F) Provides representative pictures of MSCs cultured in 5% or 21% O₂ after 9 days (100× magnification). (G) Provides representative photomicrographs of MSCs cultured in 5% or 21% O₂ for 16 days after staining for SA-β-Gal, an indicator of senescent MSCs (left panels) and the fraction of SA-β-Gal⁺ stained cells over the total cell number counted in 5 microscopic fields per O₂ concentration (n = 3; mean ± SD; far right panel; *P < .05). (H) Provides the immunoblot of lysates prepared from MSCs cultured in 5% or 21% O₂, as indicated at the top of each lane. The blots were probed with antibodies specific for p16^INK4 (top row), p21^WAF/Cip (middle row), or β-actin (bottom row), which was used to control for the amount of protein added. The histograms (right) provide densitometry analyses on immunoblots for p16^INK4 and p21^WAF/Cip in lysates of MSCs grown in 5% O₂ (black bars) or 21% O₂ (open bars) for 4 and 9 days. p21^WAF/Cip and p16^INK4 band intensities were normalized to β-actin levels, and are expressed relative to the level of each protein found in 5% O₂ at the same time point. Error bars indicate ± SD of the mean.