(A) Phase contrast picture and immunofluorescence staining of hiPSC lines M^3F-1 (non-PD hiPSCs), PDA^3F-1, PDB^3F-5, PDC^3F-1, PDD^3F-1, and PDE^3F-3 for pluripotency markers SSEA4, Tra-1-60, OCT4, SOX2, and NANOG.
(B) Quantitative RT-PCR for the reactivation of the endogenous pluripotency-related genes NANOG, OCT4, and SOX2 in indicated hiPSC lines, hESCs, and primary fibroblasts. Relative expression levels were normalized to expression of these genes in fibroblasts.
(C) Methylation analysis of the OCT4 promoter region. Light gray squares indicate unmethylated and black squares methylated CpGs in the OCT4 promoter of hiPSCs and parental primary fibroblasts cells.

(A) Hematoxylin and eosin staining of teratoma sections generated from hiPSC lines A6 (non-PD hiPSCs), PDA^3F-1, PDB^3F-1, PDC^3F-1, PDD^3F-1, and PDE^3F-3 showing: top row panels, pigmented neural epithelium; second row panels, neural rosettes; third row panels, intestinal epithelium; fourth row panels, bone/cartilage; and bottom row panels, smooth muscle.
(B) Southern blot analysis of hESC line BG01, mouse embryonic fibroblast (MEF) feeder cells, and the indicated PD patient-derived hiPSCs (and non-PD hiPSC line M^3F-1) for proviral integrations of XbaI-digested genomic DNA with ³²P-labeled DNA probes against OCT4, KLF4, SOX2, and c-MYC.
(C) Table summarizing the approximate number of proviral integrations for the four reprogramming factors in hiPSCs based on Southern blot analysis shown in (B).

(A) Immunofluorescence staining of neuronal cultures derived from hESC line BG01, non-PD hiPSCs M^3F-1, and the indicated PD patient-specific hiPSCs for neuron-specific class III β-tubulin (TUJ1; green) and the dopaminergic neuron-specific marker tyrosine hydroxylase (TH; red). Neuronal cultures were derived with an embryoid body (EB)-based differentiation protocol.
(B) Immunofluorescence staining of neuronal cultures derived from hESC line H9, non-PD hiPSCs A6, and the indicated PD patient-specific hiPSCs for neuron specific class III β-tubulin (TUJ1; red) and the dopaminergic neuron-specific marker tyrosine hydroxylase (TH; green). Neuronal cultures were derived with a stromal cell coculture-based protocol.
(C) Quantification of TUJ1-positive neurons (left graph) and TH-positive dopaminergic neurons (right graph) after neuronal differentiation of hESCs (BG02, H9), non-PD hiPSCs (iPS A6, M^3F-2) and indicated PD patient-specific hiPSCs using a stromal cell coculture-based protocol (as shown in [B]). Graphs indicate the percentage of cells that stain positive relative to nuclear Hoechst staining. Error bars indicate the SEM generated from triplicates of the same experiment.

(A) Schematic drawing of the DOX-inducible lentiviral construct FUW-tetO-loxP and the genomic locus after proviral integration (2lox) and Cre-recombinase-mediated excision (1lox). The FUW-TetO-loxP vector contains a tetracycline response element (TRE) located 5′ of a minimal CMV promoter and a unique MfeI site used for diagnostic Southern blot digests. The reprogramming factors are flanked by EcoRI restriction sites. The 3′ LTR of this lentiviral vector contains a single loxP site, which is duplicated during proviral replication into the 5′ LTR. This duplication results in a transgene flanked by two loxP sites after genomic integration of the provirus (2lox). This allows the excision of the transgene in combination with the complete promoter sequences with Cre-recombinase (1lox). (WRE, woodchuck response element.)
(B) Phase contrast picture and immunofluorescence staining of hiPSC lines PDB^2lox-17 and PDB^2lox-21 for pluripotency markers SSEA4, Tra-1-60, OCT4, SOX2, and NANOG. PDB^2lox-17 and PDB^2lox-21 were derived by expression of the three reprogramming factors OCT4, SOX2, and KLF4 from the FUW-tetO-loxP virus shown in (A). In these cells, all three reprogramming factors are flanked by loxP sites at their genomic integration site.
(C) Hematoxylin and eosin staining of teratoma sections generated from PDB^2lox-17 and PDB^2lox-21 cells carrying excisable reprogramming factors.

(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.

(A) Phase contrast picture and immunofluorescence staining of reprogramming factor-free hiPSC lines PDB^1lox-17Puro-5 and PDB^1lox-21Puro-12 for pluripotency markers SSEA4, Tra-1-60, OCT4, SOX2, and NANOG.
(B) Quantitative RT-PCR for the reactivation of the endogenous pluripotency-related genes NANOG, OCT4, and SOX2 in hESCs, fibroblasts (PDB), provirus-carrying PDB^2lox clones (PDB^2lox-17 and PDB^2lox-21), and indicated PDB^1lox clones after Cre-recombinase-mediated excision of the transgenes. Relative expression levels were normalized to the expression of these genes in fibroblasts.
(C) Hematoxylin and eosin staining of teratoma sections generated from factor-free PDB^1lox-17puro-5 and PDB^1lox-21puro-26 cells.
(D) Immunofluorescence staining of neuronal cultures derived from factor-free PDB^1lox-17puro-31 and PDB^1lox-21puro-13 hiPSCs for neuron-specific class III β-tubulin (TUJ1; green) and the dopaminergic neuron-specific marker tyrosine hydroxylase (TH; red). Neuronal cultures were derived with an embryoid body (EB)-based differentiation protocol.
(E) Quantitative RT-PCR for residual transgene expression of OCT4, KLF4, and SOX2 in hESCs (BG01), primary fibroblasts (PDB), primary infected fibroblasts (PDD^3F ± DOX), hiPSCs (M3^F3-1), PD-derived hiPSCs (PDA^3F-1, PDB^3F-5, PDC^3F-1, PDD^3F-1, PDE^3F-3), provirus-carrying PDB^2lox clones (PDB^2lox-17 and PDB^2lox-21), and the reprogramming factor-free PDB^1lox clones (PDB^1lox-17Puro-5, PDB^1lox-17Puro-31, PDB^1lox-21Puro-12, PDB^1lox-21Puro-20). Relative expression levels are normalized to DOX-induced expression in primary infected fibroblasts.
(F) Comparison of gene expression profile of hESCs (BG01, H9), factor-carrying PDB^2lox lines (PDB^2lox-5, PDB^2lox-17, PDB^2lox-21, PDB^2lox-22), and factor-free PDB^1lox lines (PDB^1lox-17Puro-5, PDB^1lox-17Puro-10, PDB^1lox-21Puro-20, PDB^1lox-21Puro-26). All genes (n = 1434) showing at least 2-fold differential expression between factor-free hiPSCs and factor-carrying hiPSCs cells were ordered by this ratio and hierarchically clustered by sample (using uncentered correlation and average linkage). All log2 ratios are relative to expression in fibroblasts. Numbers shown in dendrogram indicate Pearson correlation between clusters. The expression profile of factor-free hiPSCs is higher correlated to hESCs than the expression profile of factor-carrying hiPSCs as tested by Fisher’s Z transformation (p < 1e-7). Confidence of the hierarchical clustering was computed with multiscale bootstrap resampling, generating in more than 99% a cluster between the hESCs and all factor-free PDB^1lox lines.
(G) Venn diagram displaying the number of differentially expressed genes (p < 0.05 determined by moderated t test, corrected for false discovery rate) between provirus-carrying PDB^2lox lines (PDB^2lox-5, PDB^2lox-17, PDB^2lox-21, PDB^2lox-22) compared to hESCs (H9, BG01), or reprogramming factor-free PDB^1ox lines (PDB^1lox-17Puro-5, PDB^1lox-17Puro-10, PDB^1lox-21Puro-20, PDB^1lox-21Puro-26) compared to hESCs (H9, BG01), respectively.