Figure 7. Reprogramming efficiency of adult mature B cells into iPS cells.

(A) Schematic representation for experiment attempting to measure reprogramming efficiency. 3*10^⁶ CD19+ adult B cells were infected with retrovirus encoding C/EBPα-NeoR construct and after 24 hours we sorted IgM+IgD+ mature adult B cells and plated them as single cells in 96 wells plates preplated with OP9 stromal cell line. Cells were grown in conditioned medium + Dox + LIF throughout the experiment. On day 6, culture wells were subjected to puromycin and neomycin selections for 5 days, which allowed only the growth of transgenic B cells infected with C/EBPα. On day 20, the wells containing drug resistant cells were screened for Nanog-GFP expression by FACS analysis. Wells that scored positive were subsequently passaged on MEFs in ES media and grown into iPS cell lines. (B) Established cell lines from each experiment were retrospectively confirmed to carry different B cell receptor rearrangements and to contain C/EBPα pro-viral transgenes. (C) Efficiency was determined by dividing the number of GFP+ wells by the number of wells that contained neomycin and puromycin resistant cells.