An outline of the culture schedule used to establish immortalized cell lines from iPS cells.

Human iPS cells (4x104) were cultured in the presence of feeder cells with cytokines in a 100 mm dish. Attached cells indicate cells attached to the dish or to the feeder cells. Detached cells indicate cells detached from the dish or from the feeder cells. VEGF, vascular endothelial growth factor. IGF-II, insulin-like growth factor-II. SCF, stem cell factor. EPO, erythropoietin. DEX, dexamethasone.
aOP9 feeder cells were used again for a further period.
bAttached cells were cultured again on OP9 feeder cells.
cAll detached cells collected from the dish were cultured again in fresh medium.
dUsed OP9 cells were discarded together with any attached cells and a fresh batch of OP9 cells were used for the next culture step.
eAll detached cells collected from the dish were infected with lentivirus expressing HPV16-E6/E7, and were then cultured again.
fThe medium was changed twice per week.
gDetached cells from the dish were either all re-cultured or some re-cultured and the remainder stored for further analyses.
hFrom this time point, the detached cells could proliferate without feeder cells.
iVery few attached cells were detected.
jAt approximately day 131, the requirement for essential factors for proliferation was evaluated.