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Fig. 2 | BMC Cancer

Fig. 2

From: Etoposide-mediated interleukin-8 secretion from bone marrow stromal cells induces hematopoietic stem cell mobilization

Fig. 2

Primary culture of human bone marrow stromal cells and results of the cytotoxicity assays, cytokine arrays, and apoptosis and cell cycle analyses. a Mononuclear cells were collected from a healthy donor during bone marrow harvest. After 1–2 weeks of primary culture, adherent cells showed spindle-shaped morphology and reached 65–70% confluence. b Flow cytometry indicated that these cells were positive for the human bone marrow stromal cell (hBMSC) markers CD73, CD90, and CD105 and negative for the hematopoietic stem cell markers CD34 and CD45. These results indicate that hBMSCs were properly isolated. c Cytotoxic concentration (CC) 10, CC 25, and CC 50, defined as the concentrations sufficient to cause the death of 10, 25, and 50% of viable hBMSCs, were calculated for various concentrations of cyclophosphamide and etoposide. d hBMSCs were cultured in normal saline (control group, n = 4), cyclophosphamide (dose of CC10, n = 5), or etoposide (dose of CC10, n = 5) for 24 h. Human cytokine analysis was performed with the conditioned media. The level of IL-8, a mobilization-associated cytokine, was significantly higher in the etoposide-treated group than that in the cyclophosphamide-treated group (p = 0.021 after Bonferroni correction). f Expansion of etoposide-treated hBMSCs was significantly lower than that of cyclophosphamide-treated hBMSCs in both P1 and P2 (control, n = 7; cyclophosphamide, n = 7; etoposide, n = 7; both, p < 0.001 after Bonferroni correction). g No differences in the numbers of early apoptotic and necrotic cells or late apoptotic cells were observed among the groups (control, n = 4; cyclophosphamide, n = 7; etoposide, n = 7). As a negative control, hBMSCs treated only with normal saline were used. The values within the figures represent the mean ± standard error in repeated experiments. All experimental data of representative figures are presented as Supplementary Material 6: Fig. S3. h Etoposide-treated hBMSCs showed a higher proportion of cells arrested in the G0/G1 phase of the cell-cycle than the cyclophosphamide-treated and untreated hBMSCs (control, n = 3; cyclophosphamide, n = 3; etoposide, n = 3; p = 0.03 and p = 0.01 after Bonferroni correction, respectively). Note: * p < 0.05 after Bonferroni correction. Note: Values are reported as the mean ± standard error of the mean (SEM). Abbreviations: P1, passage 1; P2, passage 2; CC, cytotoxic concentration

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