An outline of the culture schedule used to establish immortalized cell lines from hematopoietic stem/progenitor cells.

CD34+ cells (1×105) were cultured in the absence of feeder cells with cytokines in the well of a 24-well plate or in a 100 mm dish. SCF, stem cell factor. EPO, erythropoietin. FLT3-L, FLT3 ligand. DEX, dexamethasone.
aAll cells collected from the dish were infected with lentivirus expressing HPV16-E6/E7 and then cultured again.
bAll cells collected from the dish were cultured again.
cVery few attached cells were detected.
dThe medium was changed twice per week.
eDetached cells collected from the dish were either all cultured again or only some were re-cultured and the remainder were stored for further analysis.
fAt approximately Day 110, the cultures were evaluated for the essential factor(s) for proliferation.