Figure 5. Generation and Characterization of Reprogramming Factor-free hiPSCs.

(A) Schematic overview of Cre-mediated excision of the transgenes to generate reprogramming factor-free hiPSCs. IPS PDB^2lox cells were derived with FUW-tetO-loxP lentiviral vectors transducing three reprogramming factors, OCT4, KLF4, and SOX2.

(B) Southern blot analysis for proviral integrations of parental fibroblasts (PDB), provirus-carrying PDB^2lox clones (PDB^2lox-17 and PDB^2lox-21), and the indicated PDB^1lox clones after Cre-recombinase-mediated excision of the transgenes. Puro indicates PDB^1lox clones, which were isolated by puromycin selection; GFP indicates PDB^1lox clones isolated by FACS sorting for EGFP (as shown in [A]). Genomic DNA was digested with XbaI and probed for proviral integrations using ³²P-labeled DNA probes against OCT4, KLF4, and SOX2. PDB^1lox clones indicated in blue were disregarded because of remaining transgene integrations based on the MfeI digest shown in Figure S5.

(C) Cytogenetic analysis of hiPSC lines PDB^1lox-17Puro-5 and PDB^1lox-21Puro-12 shows normal karyotype after Cre-mediated excision of the transgenes.

(D) Summary of the generation of factor-free hiPSCs.