EBV immortalized human lymphoblastoid cell lines as a source for iPSCs generation by nucleofection

To date lymphoblastoid cell lines (LCLs), that constitute the source of several worldwide biobank linked to patient's clinical history, genotype and phenotype data and molecular/functional studies, represent a severely underused bioresource to obtain induced pluripotent stem cells (iPSCs). The overall reprogramming efficiency and poor success rate constitute the limit of use of this cellular resource, and only few groups have reported the successful reprogramming of LCLs. We report an nucleofector based, optimized and reliable method for LCLs reprogramming protocol using known episomal plasmids and mouse embryonic fibroblast (MEF) conditioned culture medium. Methods iPSC reprogramming was performed in accordance with the protocol by S. Kumar et al () but with some modifications. In detail: SF Cell Line 4D-Nucleofector X kit and SF-EO100 pulse programme was utilised for nucleofection; from day 3 to day 10 complete WiCell medium (added of ?MeOH 0,1mM, ?-FGF 1,25ng/?l, NaBut 0,5mM) was utilized to maintain and expand nucleofected cell; from day 11 to day 21 Wi Cell complete medium was gradually replaced with WiCell medium without NaBut. Complete WiCell was obtained conditioning base WiCell (DMEM/F12, KO sierum 20%, MEM NEAA 0,1mM, L-glutamine 2mM, L-ascorbic acid 50ng/?l) with MEF. Reprogrammed iPSC lines were confirmed by immunochemistry. Results The utilized Nucleofection protocol allowed to obtain 40-50% transfection efficiency as shown by the GFP positive cell number detected. Adherent cells appeared already from day 3 after transfection while iPSC-like colonies appeared 2-3 weeks after transfection. In the same time lapse non adherent colonies like to the embryoid bodies also appeared. The iPSC-like colonies detached by an EDTA based, enzyme free method, showed the expression of the specific stem cell proteins such as the OCT-3/4 and SOX-2 when examined by immunofluorescence. The number of colonies obtained from 1.0*106 cells was of 30-60. Using this protocol, we achieved 100% success rate and high reprogramming efficiency. Conclusions To encourage the use of LCLs to generate iPSCs it is crucial to improve and/or to develop new effective reprogramming protocols. Our optimized method, goes in this direction. The availability of iPSC deriving from LCLs of well characterized patients may become crucial in discovering molecular mechanism and new therapeutics strategy for Mendelian or complex human disease.