Factor-dependent cells capable of sustained growth in liquid medium supplemented with recombinant IL3 were generated by serial replating.
These cells were plated either in semi-solid medium for the determination of their serial replating potential or in liquid culture for detection of the expression of key regulators of B cell differentiation; in both cases, the cells were cultured in the presence of SCF, Flt3-L and IL-7 as described in Figure 7A.
5×103 cells/plate were replated in methyl-cellulose, thus permitting determination of the serial replating potency.
Serial replating was performed by collecting methylcellulose and washing cells in complete media then resuspending a fixed proportion (1/10 1/1,000) of the cells in 1 ml of methylcellulose as described above.
Furthermore, secondary transplantation of leukemia cells and serial replating assays demonstrated that leukemia-initiating capacity and self-renewal are not affected by the loss of endogenous MLL1 HMT activity.
Retroviral transduction of murine bone marrow cells, serial replating and transplantation assays were performed as previously described [7], [14].
As shown in Fig 2B (left), in agreement with other reports (Harrison et al, 2010; Cordenonsi et al, 2011), non-induced cells had on average 0.6% of MFE, remaining constant throughout serial replating to M4.
Our finding that the effects of Hh inhibition on leukemic growth persisted through serial replating without further exposure to drug suggests that Hh signaling is involved in the regulation of long-term self-renewal as well.
We used a complementary in vitro assay with serial replating and colony-formation, as well as serial in vivo transplantation in order to assess the effects of Hh inhibition on long-term self-renewal.
Remarkably, p185BCR/ABL also conferred increased engraftment, although it did not increase serial replating efficiency, as shown above.
