Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

The overall goal of this procedure is to generate induced pluripotent stem cells from human peripheral blood mononuclear cells obtained from frozen buffy coats using a cost-effective and virus free protocol. This is accomplished first by the isolation of PB MNCs from Buffy coats after whole blood centrifugation without a density gradient followed by expansion in a specific blood culture medium. The second step is to transfect the Buffy coat PB MNCs with episomal plasmids by electroporation.

The transfected PB MNCs are then plated onto fresh mouse embryonic fibroblast feeder cells. The final step is to manually pick induced pluripotent stem cells from the culture and transfer them onto new feeder cell plates for expansion. Ultimately, the efficient reprogramming of PB MNCs into induced pluripotent stem cells with a typical human morphology can be shown by immunofluorescence and measuring gene expression.