MSCs can provide survival support to CLL cells in vitro. (A) CLL cells were cultured in the presence of increasing concentrations of MSC conditioned media (CM), which was prepared in 5% O₂, and exposed to either 21% or 5% O₂ for 3 days, at which point the CLL cells were collected for viability measurements by flow cytometry. Data from 4 different CLL patients are presented (mean ± SEM), which were normalized to day 0. The initial absolute viability of all samples at day 0 was of 66% ± 6% (mean ± SEM). Asterisks indicate significant difference between CM conditions and media control values in 5% O₂ and 21% O₂ (1-way ANOVA and Tukey posthoc test; ***P < .001, **P < .01, *P < .05). (B) CLL cells were cultured for 3 days in the presence of increasing amounts of MSC CM, which was prepared by incubating MSCs either in 5% O₂ or 21% O₂ for 6 days. CLL-cell viability was assessed by flow cytometry. Data on cells obtained from each of 3 different CLL patients are presented (mean ± SEM), which were normalized to day 0. The initial absolute viability of all samples at day 0 was of 87% ± 2% (mean ± SEM). Asterisks indicate significant difference measured by 1-way ANOVA and Tukey posthoc test (**P < .01, *P < .05). (C) The presence of CXCL12 was quantified in MSC CM prepared by incubating MSCs either in 5% O₂ or 21% O₂ for 6 days by ELISA. Left: CXCL12 concentration in the CM; right: CXCL12 secretion rate per 1 × 10⁵ MSCs was estimated by dividing CXCL12 concentration in the CM by the number of MSCs collected at the end of the 6-day culture period. The data presented were obtained from MSCs derived from each of 2 patients, each tested in 2 independent experiments (mean ± SEM). Asterisks indicate significant difference measured by Student t test. (*P < .05). (D) CXCR4 expression levels on CLL cells (n = 6) cultured for 24 hours in 5% or 21% O₂ were assessed by flow cytometry, which was gated on CD19^PosCD5^Pos cells. CXCR4 expression is presented as the absolute mean fluorescence intensity (MFI), which was the MFI of CD19⁺CD5⁺ cells stained for CXCR4 minus the MFI of the same cells stained with an isotype control antibody. Student t test was used to determine statistical significance. (E-F) MSCs generated in 5% O₂ were plated in parallel cultures at 1000 cells/cm² and subsequently placed in 5% or 21% O₂ for 2 to 3 days before the addition of CLL cells (1 × 10⁶ cells/mL). CLL cells from 4 different patients (3 ZAP-70^Neg, 1 ZAP-70^Pos) were seeded in duplicate either alone, directly on MSCs (E), or separated from the MSCs by transwell (TW) porous membrane (0.4 μm; F). CLL-cell viability was assessed after 0, 3, 6, 10, or 14 days and normalized to that of the cells on day 0 (mean ± SEM; n = 4). The initial absolute viability of all samples at day 0 was of 74% ± 5% (mean ± SEM). In panel E an asterisk indicates the significant difference between the percentage live CLL cells on MSCs in 21% O₂ and 5% O₂ (Student t test; P < .05). In panel F asterisks (*) or (**) indicate a P < .05 or P < .01, respectively (Student t test) for the differences between CLL cells cultured alone or with MSCs. (G) CLL cells from 4 different patients (3 ZAP-70^Neg and 1 ZAP-70^Pos) were cocultured with MSCs that were derived from 2 different ZAP-70^Neg patients (bottom) or 2 ZAP-70^Pos CLL patients (top) for 14 days, at which point the cells were collected for viability assessment by flow cytometry. The viability data presented have been normalized to day 0 (mean ± SEM). The initial absolute viability of all samples at day 0 was 74% ± 6% for the 4 samples plated on MSCs from ZAP-70^Pos patients, and of 73% ± 10% for the 4 samples plated on MSCs from ZAP-70^Neg patients (mean ± SEM). Asterisks indicate significant difference measured by Student t test. (*P < .05; **P < .01).