Pep4 e02s ck2m en2l

Equine fibroblasts were transfected with different combinations of episomal reprogramming plasmids that have demonstrated to reprogram human cells [38 (link)–40 (link)]: pCXLE-hOCT3/4-shp53-F (addgene N°27077), pCXLE-hSK (addgene N°27078), pCXLE-hUL (addgene N°27080), pEP4 E02S CK2M EN2L (addgene N°20924) and pCXWB-EBNA1 (addgene N°37624). Either the 100 μl tip NEON system or FuGENE6 transfection reagent (Roche, 1814443) was used for the transfection of the plasmids, following the manufacturer´s instructions in both cases. The equine fibroblasts were transfected with different conditions of plasmid concentrations, plasmid combinations and NEON settings, which are detailed in S3 Table. All the conditions were evaluated twice and the EGFP-N1 plasmid was used as control of transfection in each procedure. Once transfected, the cells were cultured in gelatin coated p100 in DMEM 10% SFB medium for 5 days. After that time, they were trypsinized and plated in different dilutions over iMEFs in eqHES medium.

Four urine-derived renal progenitor cell samples were reprogrammed into iPSCs (four lines) using an integration-free episomal based transfection system without pathway inhibition. Briefly, urine-derived renal progenitor cells were nucleofected with two plasmids pEP4 E02S ET2K (Addgene plasmid #20927) and pEP4 E02S CK2M EN2L (Addgene plasmid #20924) expressing a combination of pluripotency factors including OCT4, SOX2, LIN28, c-MYC, KLF4, and NANOG using the Amaxa 4D-Nucleofector Kit (Lonza, Swiss) according to the manufacturer’s guidelines and as described previously^{41 (link)}. Please see supplemental materials and methods for full description.