have a wide host cell range, enabling the application of this method to many human cell types. Second, it does not require viral packaging. Third, no repeated treatments with reprogram- ming factors are needed. A single transfection of episomal vectors is sufficient for the derivation of human iPSCs. Moreover, higher transfection efficiency can be achieved with these vectors due to the oriP/EBNA-1-mediated nuclear import and retention of vector DNA [20]. Fourth, the oriP/EBNA-1 vectors replicate once per cell cycle and are generally present at low copy number per cell, thus minimizing DNA rearrangement and genome integration [21]. Last, the removal of episomal vectors from human iPSCs can be accomplished by simple cell culture without any additional manipulation, due to the silencing of the viral promoter driving EBNA-1 expression in iPSCs, and the inherent instability of oriP/EBNA-1 episomal state - stably established episomes are lost from cells at a rate of ,5% per cell generation due to defects in vector synthesis and partitioning [22]. However, despite these advantages, our original episomal method yielded low reprogramming efficiency (,3 iPSC colonies from ,16106 input human foreskin fibroblasts), and used mouse embryonic fibroblast (MEF) feeder cells, which seriously limit the industrial and therapeutic applications of this method. In this report, we have made significant improvement of the episomal reprogramming method. Using chemically defined media, we have established a small molecule-aided feeder-free reprogramming condition for the efficient derivation of footprint- free human iPSCs from skin fibroblasts, adipose tissue derived cells and cord blood cells. This method can be readily adapted to the derivation of clinical-grade human iPSCs. Of particular interest, iPSC derivation with this method appeared to progress through a distinct intermediate stage. It will be interesting to find out how this small-molecule aided episomal reprogramming method compares to other reprogramming methods in terms of the quality of iPSCs they generate. Results Identifying small molecules for improved episomal reprogramming We have previously developed an oriP/EBNA-1-based episomal reprogramming approach for the derivation of footprint-free human iPSCs [19]. The reprogramming efficiency with this method, however, was prohibitively low (,3 iPSC colonies from ,16106 input fibroblasts). This low reprogramming efficiency was likely due to the loss of oriP/EBNA-1 vectors (.25% per cell generation) and DNA methylation-mediated transgene silencing during the first two weeks post-transfection, a time window required for successful iPSC generation [23]. Thus, small molecules that could either accelerate reprogramming process, or reduce episomal vector loss and transgene silencing during the first two weeks post-transfection, were expected to improve episomal reprogramming. To identify such small molecules, we tested chemical compounds that were previously implicated in reprogramming and epigenetic modifications, using a two-vector episomal combination containing expression cassettes for all seven transgenes (OCT4, SOX2, NANOG, LIN28, c-MYC, KLF4 and SV40LT) (7F-1, Fig. S1A), human foreskin fibroblasts as donor cells and traditional reprogramming conditions (MEF feeder cells and human ESC medium). We found that the episomal reprogramming efficiency could be greatly enhanced with the addition of a MEK inhibitor PD0325901, a GSK3b inhibitor CHIR99021, and a TGF-b/Activin/Nodal receptor inhibitor A- 83-01 (Fig. 1A). Previous studies showed that TGF-b signaling inhibitors together with the MEK inhibitor PD0325901 resulted in Small Molecules Improve Episomal Reprogramming .100-fold increase in the viral reprogramming efficiency [24]. As shown in Figure 1A, the TGF-b signaling inhibitor A-83-01, either alone or together with the MEK inhibitor PD0325901, had minimal effect on episomal reprogramming. All three inhibitors PD0325901, CHIR99021, and A-83-01 were required to achieve the maximal increase in reprogramming efficiency. Human leukemia inhibitory factor (hLIF), though did not significantly improve episomal reprogramming efficiency, increased the proliferation of reprogramming intermediates and the reprogram- ming pace. The ROCK inhibitor HA-100, though had minimal effect on its own, further increased the episomal reprogramming efficiency in the presence of PD0325901, CHIR99021, A-83-01 and hLIF. The increase in the episomal reprogramming efficiency correlated with the duration of small molecule treatment (Fig. 1B). Early treatment, i.e. between day 1 and 5 post-transfection, though not sufficient on its own, appeared to be important for the maximal effect of small molecules on episomal reprogramming. The observation that episomal reprogramming could be significantly improved by the continuous presence of PD0325901, CHIR99021, A-83-01 and hLIF raised an interesting question about the identity of iPSCs generated. Based on the gene expression and culture requirements, recent studies suggest that human ESCs are in a pluripotent state similar to mouse epiblast-derived stem cells (EpiSCs), and different from mouse ESCs derived from earlier blastocyst stage, which were proposed to be in a na¨ıve ground state of pluripotency [25]. In the presence of PD0325901, CHIR99021, A-83-01 and hLIF, human ESCs and human ESC-like iPSCs differentiated rapidly. On the contrary, these molecules were able to expand mouse ESC-like human iPSCs from reprogramming cultures that were not previously exposed to these small molecules [26]. These mouse ESC-like human iPSCs readily differentiated following withdrawal of the small molecules. Surprisingly, the human episomal iPSCs obtained in the continuous presence of small molecules expanded well under conditions for human ESCs (without small molecules), but underwent extensive differentiation when cultured in the same condition used for their derivation, i.e. in the presence of small molecules (Fig. 1C), suggesting that these iPSCs were in a pluripotent state similar to human ESCs, not mouse ESCs. The seemingly conflicting results could be explained by the presence of activities that mitigated the effectiveness of small molecules in the MEF feeder cells and human ESC medium used for reprogramming (e.g. ligands for TGF-b signaling), which might enable the generation of human ESC-like pluripotent state in the presence of small molecules. Developing a feeder-free episomal reprogramming condition with defined media Since the MEF feeder cells and human ESC medium used for reprogramming likely contains small molecule-mitigating activi- ties, it is possible to further improve episomal reprogramming by using matrix and defined culture media. Such defined reprogram- ming conditions will not only reduce the quality variations associated with feeder cells and KnockOut serum replacement in the human ESC medium, but also can be readily adapted to the production of clinical-grade human iPSCs. To find defined media that could support episomal reprogramming, we initially tested the N2B27 medium since it has a simple formulation and was able to support the proliferation of human ESCs when supplemented with cytokines [27]. As shown in Figure 2A, the N2B27 medium supplemented with small molecules gave rise to nearly 6-fold higher number of colonies stained positive for alkaline phosphatase (a human pluripotent stem cell marker) (test 2 vs. test 1). These colonies (piPSC for partially reprogrammed iPSCs) had a mouse ESC-like domed morphology, which differs from the flattened PLoS ONE | www.plosone.org 2 March 2011 | Volume 6 | Issue 3 | e17557