These differentiating pre-B cells rapidly loose their capacity to proliferate when replated on BM stromal cells and IL-7 1. Furthermore, apoptosis is induced. AnnexinV stainings one day after removal of IL-7 revealed that overexpression of Myc alone even enhanced apoptosis, while overexpression of Pim1 alone reduced the amount of apoptotic and proapoptotic cells during differentiation (Fig. 1E). Nevertheless, overexpression of Pim1 or Myc alone was not sufficient to induce an overall increase in cell numbers

of pre-B cells in the absence of their growth factor IL-7. However, Everolimus concentration co-induction of Pim1 and Myc together in double-transduced pre-B cells allowed survival and proliferation of cells after removal of IL-7. Approximately, 1–10% of the cells began to expand by IL-7/OP9 cell-independent proliferation, as assessed by extrapolation of the growth curves shown in Fig. 1F and by limiting dilution analysis (data not shown). The Pim1/Myc overexpressing cells proliferated 2 weeks and beyond in culture, increasing the numbers of cells in culture 20-fold in one week. This proliferation was terminated upon removal of doxycycline, Selleck EPZ015666 i.e. by the termination of overexpression of Pim1 and Myc (Fig. 1F, bottom panel, gray circles). Next, we monitored potential changes of surface expression of c-kit (CD117), CD25 and IgM as the markers of

subsequent differentiation stages of pre-B cells. Overexpression of Pim1 or Myc alone did not change from the downregulation of c-kit (Fig. 2) and the upregulation of CD25 (data not shown) over time in differentiation-inducing conditions, i.e. after removal of IL-7. Overexpression of Pim1 and Myc together in pre-BI cells and subsequent induction of differentiation by the removal of IL-7 led to the downregulation of c-kit expression (Fig. 2) and to the upregulation of CD25 expression, though with a delay in time as compared with normal pre-B cells. Interestingly, cells overexpressing Myc, alone

or together with Pim1, did not acquire IgM on the surface (Fig. 2) or intracellularly (data not shown) after removal of IL-7. In contrast, overexpression of Pim1 alone in the absence of IL-7 resulted in normal percentages of IgM+ cells over time. Differentiation of pre-BI cells to later stages of B-cell development was also tested by the potential loss of their clonability on OP9 cells in the presence of IL-7, a measure of their pre-BI cell status 1. Doxycycline-induced Pim1/Myc-overexpressing cells were incubated for 1, 2, 3 or 7 days in the absence of IL-7. The cells were then transferred back onto OP9 cells in the presence of IL-7, the conditions for pre-BI cell expansion. Clonability of these differentiating pre-B cells in the absence of Pim1/Myc overexpression was lost from 1 in 6 at day 1 of differentiation down to almost 1/10 000 at day 3 (Table 1 and Supporting Information Fig. 1E).