Figure 3: Long-term expansion of fopMKs.

(a) Schematic representation of the culture conditions for long-term MK-FOP. The combination of TPO (20 ng ml⁻¹) and SCF (50 ng ml⁻¹) allowed mature MK expansion for 90 days and beyond. (b) The cumulative mature MK (CD41a/CD42a double positive cells) fold increase relative to day 0 hiPSC cell input is shown for the hiPSC lines #1 (n=4) and #3 (n=5) over 90 days in culture (mean±s.e.m.). (c,d) The corresponding percentages of CD41a+ and CD42a+ cells monitored by flow cytometry from whole cultures are shown over the 90 day period (mean±s.e.m.). (e) Representative flow cytometry dot plots for CD41a and CD42a expression from the hiPSC#3 line. (f,g) Representative phase contrast picture of a day 70 hiPSC#3 fopMK long-term culture and associated Romanowsky staining of fixed cells (scale bars 50 μm). White arrowheads: clumps of actively growing small cells; red arrowheads: single big cells in suspension culture and polyploid cells identified by Romanowsky staining. (h) Cell size distribution from fopMK and LT-fopMK cultures is shown as box plots (n=50, 65 respectively). (i) Endogenous and transgenic expression levels of the 3-TFs were independently monitored by RT-qPCR throughout MK-FOP long-term cultures (mean±s.e.m. from hiPSC lines #1 and #3; n=3). The average range of expression levels in cbMKs (mean±s.e.m.; n=5) is shown as a benchmark (in red).