Reprogramming Fibroblasts to iPSCs

A schematic diagram of the procedure is described in Figure 4. Episomal plasmids and methods were described previously^{15 (link)}. Plasmid combination #19 (pEP4-E-O2S-E-T2K, pEP4-E-O2S-E-N2K and pCEP4M2L) was used for most reprogramming unless mentioned otherwise. Plasmids and EBNA mRNA were electroporated into fibroblast cells on Amaxa apparatus according to company instructions. One million cells were used in each electroporation, which were then plated into two 6-well plates. E8 + hydrocortisone media were used for the first 5–10 days, according to cell survival and proliferation after electroporation. When confluency was reached ~20%, hydrocortisone was removed. ES-like iPS cell colonies usually appear after ~25 days. Cells were then picked into individual wells with E8 (TGFβ or NODAL). Cells were passaged for ~ 15 passages before subcloning with Y27632 on Matrigel or vitronectin.