FIG. 5.

TW3R human feeders support efficient and stable gene transfer to human iPSCs by a piggyBac vector. (A) BC1 iPSCs were co-transfected with a vector expressing transposase gene and a piggyBac vector expressing GFP and puromycin resistance gene. The transfected BC1 cells were then plated onto TW3R feeders for recovery and puromycin selection. (B) GFP positive colonies were observed 11 days after transfection. In the absence of puromycin, a majority of the colonies did not express GFP. (C) FACS analysis showed that with puromycin selection on TW3R for 11 days, nearly 90% of the TRA-1-60+ iPSCs became GFP+. Moreover, this transgene expression can be stably maintained on TW3R feeders, as the FACS analysis on day 29 showed 96% TRA-1-60 positive iPSCs maintained GFP expression. (D) piggyBac GFP labeled BC1 iPSCs maintained expression of pluripotency markers such as SSEA4, TRA-1-60, NANOG, and OCT4 in addition to GFP expression. FACS, fluorescence-activated cell sorting. Color images available online at www.liebertonline.com/scd