Small Molecules Improve Episomal Reprogramming Figure 3. Characterization of iPSCs derived using the small molecule-aided feeder-free condition. (A) Bright-field image of iPSCs derived from human adult skin fibroblasts (iPS(SK46) clone 2). Scale bar: 100 mm. (B) G-banding chromosome analysis of iPS(SK46) clone 2 (p17). (C) PCR analysis of reprogramming vectors in iPSCs. E: episomal DNA; G: genomic DNA; NF: neonatal foreskin fibroblasts (p5); iPSF7 clone 1 to 3: iPSCs derived from neonatal foreskin fibroblasts (p26); AF: adult skin fibroblasts (p6); iPS(SK46) clone 1 to 3: iPSCs derived from adult skin fibroblasts (p22). piPSC derived from human foreskin fibroblasts (p4) were used as controls. T-OCT4: transgene OCT4; T-SOX2: transgene SOX2; T-NANOG: transgene NANOG; T- LIN28: transgene LIN28; T-c-MYC: transgene c-MYC; T1-KLF4: transgene KLF4 (1); T2-KLF4: transgene KLF4 (2); T-SV40LT: transgene SV40LT; OCT4: endogenous OCT4. 32 PCR cycles were used for all primer sets. (D) Quantitative RT-PCR analysis of the endogenous OCT4, NANOG, SOX2 and LIN28 expression in iPSC clones. Data shown are mean 6 SEM (n = 3). (E) Bisulfite-sequencing analysis of the methylation status of the OCT4 and NANOG promoters in iPSC clones. Open circles indicate unmethylated, and filled circles indicate methylated CpG dinucleotides. (F) Hematoxylin and eosin staining of teratoma sections of iPSC(SK46) clone 2. Teratomas were obtained from all iPSC clones. Left panel: neural tissue (ectoderm); middle panel: cartilage (mesoderm); right panel: gut epithelium (endoderm). Scale bars: 100 mm. doi:10.1371/journal.pone.0017557.g003 be functional [19] (Fig. S1A and S1B): a two-vector combination 7F-1, which was used for the studies above, and a three-vector combination 7F-2. Both combinations contain the expression cassettes for all seven transgenes (OCT4, SOX2, NANOG, LIN28, c- MYC, KLF4 and SV40LT). As shown in Figure 4A, the three-vector combination 7F-2 consistently yielded more iPSC colonies than the two-vector combination 7F-1. More importantly, the replace- ment of c-MYC in the three-vector combination 7F-2 with transformation-deficient LMYC further improved the episomal reprogramming efficiency (,1000 iPSC colonies from 16106 input fibroblasts) (Fig. 4A), which is consistent with earlier studies [30]. Thus with LMYC, the small molecule-aided episomal reprogramming method is robust for human skin fibroblasts. Since skin fibroblast might contain higher number of somatic mutations, they might not be an ideal cell type for the production of clinical-grade human iPSCs. We tested episomal reprogram- ming of additional cell types that are readily available from human donors, specifically, adipose tissue derived cells and cord blood cells. As shown in Figure 4B and 4C, the small molecule-aided episomal reprogramming method could be easily adapted to these two cell types. For adipose tissue-derived cells, the three-vector combination 7F-2 only yielded very few iPSCs, although many piPSC colonies were present. Removal of c-MYC and KLF4 (combination 5F: OCT4, SOX2, NANOG, LIN28 and SV40LT), however, gave rise to numerous iPSC colonies (,1200 iPSC colonies from 16106 input human adult adipose tissue-derived cells) (Fig. 4B), thus stressing the importance of finding the right transgene combination for each cell type in order to achieve the optimal reprogramming efficiency. Cord blood cells, compared to other somatic cell types, have unique advantages as donor cells for the production of clinical-grade human iPSCs, since these cells potentially contain less somatic mutations, and are readily available. With the small molecule-aided episomal reprogramming method, high reprogramming efficiency was also achieved with these cells (,800 iPSC colonies from 16106 input post-expansion cord blood cells, i.e. ,600 iPSC colonies per 16104 input freshly isolated cord blood CD34+ cells with 75-fold expansion during 7- day culture) (Fig. 4C). Discussion In this report, using a combined genetic and chemical approach, we have successfully established a robust feeder-free episomal reprogramming method using defined media. Though developed with fibroblasts, this method was readily adapted to adipose tissue-derived cells, cord blood cells, and potentially other cell types easily accessible from human donors. Additional features can be introduced into episomal vectors to further improve reprogramming efficiency. For example, the current episomal vectors have elements necessary for bacterial propagation, which contain many CpG islands known to contribute to transgene silencing [31]. It is possible to minimize transgene silencing by PLoS ONE | www.plosone.org 5 March 2011 | Volume 6 | Issue 3 | e17557