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. 2016 Jun 11;7(28):43974–43988. doi: 10.18632/oncotarget.9949

Figure 4. Proliferation of circulating MF-derived CD34+ cells is positively enhanced by IL-1β + TNF-α ± TIMP-1 combinations.

Figure 4

Circulating CD34+ cells were isolated from MF patients (n = 20) and CB units (n = 8) and cultured in the presence of the selected two-by-two pro-inflammatory factors. After 14 days, the total CFU-C output was assessed as described in Methods (A). Circulating CD34+ cells were isolated from MF patients (n = 10) and CB units (n = 8) and cultured in the presence or absence of inflammatory factors alone or combined. After 12 days, the CFU-MK growth was assessed as described in Methods (B). The results are expressed as growth fold change versus untreated CTR samples. (A) The clonogenic output of the MF-derived CD34+ cells was significantly stimulated by the IL-1β + TIMP-1 combination as compared with untreated cells or the CB-derived counterparts. No other combinations of factors two-by-two were effective. The mean number of colonies in MF-derived and CB-derived untreated samples was 59 ± 8 and 63 ± 6, respectively. (B) The MF-derived CFU-MK growth was significantly inhibited by TNF-α. By contrast, IL-1β has stimulatory activity on MK colony formation. Factors in combination did not significantly modify the growth of patients/CB CFU-MK as compared with factors alone. Factors alone or in combination did not significantly modify the CFU-MK growth of the CB counterparts. The mean number of CFU-MK in MF- and CB-derived untreated samples was 26 ± 11 and 46 ± 10, respectively. In (C and D ) are shown the results of cell-cycle analysis of MF-derived (n = 10) and CB-derived (n = 8) CD34+ cells after in vitro incubation for 24 hours in the presence or absence of various combinations of pro-inflammatory factors. Results are expressed as the percentage of cells in different phases of the cell cycle. IL-1β plus TNF-α highly promote cell cycling of CD34+ cells from MF patients. IL-1β + TNF-α + TIMP-1 and IL-1β + TNF-α + TIMP-1 + ATP were also effective (C). Conversely, no significant differences were observed when CB-derived cells were analysed (D). All data are presented as mean ± SEM. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 vs untreated cells) (#p ≤ 0.05; ##p ≤ 0.01 vs CB).