Small Molecules Improve Episomal Reprogramming Figure 2. Developing a feeder-free condition for episomal reprogramming. (A) Effects of MEF feeder cells, matrigel and culture media on episomal reprogramming. Transfected human foreskin fibroblasts were plated to MEF feeder cell-seeded or matrigel-coated 10-cm dishes, and subjected to different reprogramming culture conditions. Alkaline phosphatase positive (AP+) colonies were counted on day 18–21 post-transfection. The number of AP+ colonies was from ,0.336106 input cells. Data shown are mean 6 SEM (n = 3). N2B27: DMEM/F12 medium supplemented with N-2 and B-27; N2B27-100: N2B27 medium supplemented with 100 ng/ml bFGF. (B) Bright-field images of a piPSC colony from test 2 and a typical human ESC-like iPSC colony from test 1, 3 or 4. Scale bars: 100 mm. (C) Quantitative RT-PCR analysis of OCT4 and NANOG expression in piPSC clone 1 to 4 (c1 to c4, p3). Total: both endogenous and transgene expression. Human H1 ESCs (H1ESC, p32) were used as a control. Data shown are mean 6 SEM (n = 3). (D) Temporal requirement of small molecule treatment for feeder-free episomal reprogramming (test 4) (Fig. 2A). Transfected human foreskin fibroblasts were plated to matrigel-coated 10-cm dishes. PCALH was added to the N2B27-100 medium for different days at stage 2 of reprogramming. Alkaline phosphatase positive iPSC colonies were counted on day 22 post-transfection. The number of iPSC colonies was from ,0.336106 input cells. Data shown are mean 6 SEM (n = 3). doi:10.1371/journal.pone.0017557.g002 neonatal and adult skin fibroblasts. When picked and expanded in mTeSR1, these iPSCs showed typical human ESC morphology (e.g. compact colonies, high nucleus-to-cytoplasm ratios and prominent nucleoli), and had normal karyotypes (Fig. 3 and Fig. S3). Most iPSC colonies showed no transgene expression or genomic integration, and had completely lost episomal vectors after about 14 passages as demonstrated by PCR and RT-PCR analysis (Fig. 3C and Fig. S3C). They expressed typical human ESC-specific antigens (SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81), down-regulated the expression of the fibroblast marker CD44 (Fig. S3D), and reactivated the expression of the endogenous plurip- otent genes (OCT4, NANOG, SOX2 and LIN28) (Fig. 3D). Both the OCT4 and NANOG promoters were demethylated in these iPSCs, similar to human ESCs and in contrast to the parental fibroblasts and piPSCs (Fig. 3E). When injected into immunocompromised mice, they formed teratomas consisting of derivatives of all three germ layers, demonstrating the pluripotency of these iPSCs (Fig. 3F and Fig. S3E). Episomal reprogramming of different human somatic cell types Using human fibroblasts, we have successfully established a feeder-free small molecule-aided episomal reprogramming meth- od. Though the reprogramming efficiency was high enough to enable routine iPSC derivation from human adult fibroblasts, we sought to further improve the efficiency by modifying the episomal vectors. Our previous work demonstrated that the balance between the expression of different transgenes had great impact on the reprogramming efficiency [19]. Since the transgene expression from different episomal vectors differs, we tested the two episomal vector combinations that were previously shown to PLoS ONE | www.plosone.org 4 March 2011 | Volume 6 | Issue 3 | e17557