Small Molecules Improve Episomal Reprogramming Figure 1. Improving episomal reprogramming efficiency with small molecules. (A) Effects of PD0325901 (P, 0.5 mM), CHIR99021(C, 3 mM), A-83-01(A, 0.5 mM), hLIF (L, 1000 U/ml) and HA-100 (H, 10 mM) on episomal reprogramming. Transfected human foreskin fibroblasts were plated to MEF feeder cells. MEF-conditioned human ESC medium supplemented with 100 ng/ml bFGF (CM100) was used to support reprogramming. Different combinations of small molecules and hLIF were added throughout reprogramming starting on day 4 post-transfection. (B) Temporal requirement of small molecule treatment for improved episomal reprogramming. PCALH was added to CM100 for different days during reprogramming. Alkaline phosphatase positive iPSC colonies with typical human ESC morphology (e.g. compact colonies, high nucleus-to-cytoplasm ratios and prominent nucleoli) were counted on day 22–23 post-transfection. The number of iPSC colonies was from ,0.336106 input cells. Data shown are mean 6 standard error (SEM) (n = 3). (C) Extensive differentiation of the newly derived iPSCs (p3) in the presence of small molecules. When expanded in human ESC medium or MEF-conditioned human ESC medium supplemented with small molecules on MEF feeder cells, the iPSCs derived with the continuous presence of small molecules exhibited extensive differentiation. The addition of bFGF in the culture medium had no effect. Black arrow: undifferentiated iPSC colonies; white arrows: differentiated colonies. Scale bars: 100 mm. doi:10.1371/journal.pone.0017557.g001 compact morphology typical of human ESC-like iPSC colonies (Fig. 2B). They could be expanded for more than 10 passages in the N2B27 medium supplemented with small molecules. Flow cytometry analysis of these cells, however, showed no expression of human ESC-specific cell surface antigens (SSEA-3, SSEA-4, Tra- 1-60 and Tra-1-81), while the expression of a fibroblast marker CD44 was present (Fig. S2A). Quantitative RT-PCR analysis also failed to detect any expression of the endogenous OCT4 and NANOG, two essential markers for human pluripotent stem cells (Fig. 2C). These results suggested that the colonies were partially reprogrammed iPSCs, not human ESC-like iPSCs as those derived with the human ESC medium (test 1) or mouse ESC-like iPSCs, thus illustrating the important influence of reprogramming culture conditions on the pluripotent state of iPSCs [28–29]. Interestingly, the piPSCs contained abundant episomal vectors and maintained high-level transgene expression even after multiple passages in the N2B27 medium supplemented with small molecules (Fig. 2C and Fig. S2B), suggesting a likely mechanism that these small molecules improved episomal reprogramming was through the retention of episomal vectors and transgene expression. Removal of small molecules led to occasional emergence of human ESC-like iPSCs after two weeks’ culture of piPSCs in the chemically defined human ESC medium mTeSR1. Thus, although the N2B27 medium failed to yield authentic iPSCs, modifications could be made in the reprogramming protocol to enable the derivation of human ESC-like iPSCs using the N2B27 medium. To this end, we divided the reprogramming process into three stages: transfection of human somatic cells with episomal repro- gramming vectors (stage 1), reprogramming using N2B27 medium supplemented with small molecules (stage 2), and expansion with mTeSR1 medium (stage 3) (Fig. 2A). When the N2B27 medium supplemented with small molecules was used at stage 2 to support reprogramming, only rare conversion of piPSCs to human ESC-like iPSCs could be observed, suggesting that the transgene expression during expansion in mTeSR1 was insufficient to reactivate the expression of the endogenous pluripotent genes in most piPSCs. Thus we examined whether it was possible to improve episomal reprogramming by adding additional cytokines in the N2B27 medium supplemented with small molecules (stage 2). Of factors that are involved in the proliferation of human ESCs, bFGF and TGF-b/Activin/Nodal signaling are of particular importance. As inhibition of TGF-b/Activin/Nodal signaling by A-83-01 facilitated reprogramming (Fig. 1A), we tested the effect of bFGF on episomal reprogramming. Indeed, addition of high concentration bFGF to the N2B27 medium yielded reasonable number of human ESC-like iPSC colonies (test 3) (Fig. 2A). This result was consistent with previous observations that high concentration bFGF supported human ESC growth through multiple pathways besides MEK. Importantly, removal of MEF feeder cells by replacing with matrigel yielded even higher reprogramming efficiency (test 4) (Fig. 2A), consistent with the previous hypothesis that the MEF feeder cells contained small molecule-mitigating activities. Time-course exper- iments showed a requirement for an optimal time window of small molecule treatment (Fig. 2D). Thus, using small molecules and defined media (test 4) (Fig. 2A), we have established a feeder free episomal reprogramming method with significantly improved efficiency (,220 iPSC colonies from 16106 input fibroblasts, ,70-fold increase) (Fig. 2D). Characterizing iPSCs generated with the feeder-free small molecule-aided episomal reprogramming method With the newly developed feeder-free reprogramming condi- tion, we have successfully derived human ESC-like iPSCs from PLoS ONE | www.plosone.org 3 March 2011 | Volume 6 | Issue 3 | e17557