iPSC Generation and Validation:
One million cells were transfected with 2 µg of each episomal plasmid encoding OCT3/4, shP53, Lin28, SOX2, L-MYC, KLF4, and GFP(Addgene plasmids 27077, 27078, 27080, 27082 ) using the Amaxa transfection program X-005.
Transfected cells were grown in suspension for a week in hESC media (DMEM/F12 supplemented with 20% KOSR, 0.1mM NEAA, 2mM GlutaMAX, 1% Pen/Strep, 0.1 mM BME, and 12.5 ng/mL human bFGF) supplemented with 0.5mM sodium butyrate between days 2–12 post-nucleofection.
The authors report that using reprogrammed LCL\\\'s can recover donor gene expression which were previously lost during long term LCL culture maintenance. Futhermore, the findings suggest that iPSCs generated from LCLs are useful in studies regarding the genetic basic for individual phenotypic variation.