of DNA was used: combination 7F-1 (7.3 mg of pEP4EO2SCK2- MEN2L and 3.2 mg of pEP4EO2SET2K); combination 7F-2 (3.0 mg of pEP4EO2SEN2K, 3.2 mg of pEP4EO2SET2K and 2.4 mg of pCEP4-M2L); combination 5F (2.8 mg of pEP4EO2- SEN2L and 3.2 mg of pEP4EO2SET2N). To test the effect of transformation-deficient LMYC on episomal reprogramming, the coding region of LMYC was cloned via PCR from human ESCs and used to replace the coding region of c-MYC in the vector pCEP4-M2L of combination 7F-2. Reprogramming of human foreskin fibroblasts was carried out as described above. Briefly transfected fibroblasts (,1.06106 cells per nucleofection) were plated directly to 3610-cm matrigel- coated dishes in fibroblast culture medium. On day 2 post- transfection, the fibroblast medium was replaced with chemically defined N2B27-100 supplemented with PCALH. Culture medium was refreshed every two days. mTeSR1 was used to expand iPSCs on day 13 post-transfection. The total number of iPSC colonies was counted on day 21 post-transfection. Reprogramming of human adult adipose tissue-derived cells was carried out similarly to that of human fibroblasts. Briefly, nucleofected adipose tissue-derived cells (VPE-1001 with program A-33, Amaxa, ,0.76106 cells per nucleofection) were plated directly to 2610-cm matrigel-coated dishes in fresh adipose tissue- derived cell culture medium. N2B27-100 supplemented with PCALH was used to support reprogramming between day 2 and 11 post-transfection followed by mTeSR1 for iPSC expansion. The total number of iPSC colonies was counted on day 21 post- transfection. Reprogramming of cord blood cells was similar to that of fibroblasts and adipose tissue-derived cells. Due to the limited number of cord blood CD34+ cells and to improve the initial cell survival following nucleofection, frozen CD34+ cells from STEMCELL Technologies were thawed and expanded in the CD34+ cell expansion medium for about 7 days. The CD34+ cells showed robust growth in culture (,75 fold expansion). Following expansion, about 16106 cells were transfected with episomal vectors via nucleofection (VPA-1003 with program T-16, Amaxa), and plated to one fibronectin/matrigel-coated 6-well plate in CD34+ cell expansion medium. The fibronectin coating was used together with matrigel to facilitate the cord blood cell recovery from nucleofection. N2B27-100 supplemented with PCALH was used to support reprogramming between day 2 and 11 post- transfection followed by mTeSR1 for iPSC expansion. The total number of iPSC colonies was counted on day 17 post-transfection. RT-PCR expression analysis, PCR analysis of episomal vectors, bisulfite-sequencing analysis, flow cytometry analysis and karyotyping PCR, RT-PCR, flow cytometry analysis were performed as previously described [2,19]. For PCR analysis, episomal DNA was purified from human cells using QIAprepH Spin Miniprep Kit (Qiagen, Valencia, CA) with Proteinase K (NEB, Ipswich, MA) digestion as previously described [37]. Genomic DNA was isolated with the traditional phenol/chloroform extraction method. All the PCR reactions were carried out with pfx DNA polymerase (Invitrogen) with the amplification buffer used at 2X and the enhancer solution used at 3X, and with the following program: initial denaturation for 5 minutes at 94uC; 32 cycles of 94uC for 15 seconds, 55uC for 30 seconds, 68uC for 1 minute; and followed by 68uC for 7 minutes. For each PCR reaction, the episomal DNA from 16104 cells equivalent or about 0.1 mg genomic DNA was added as template. Episomal DNA and genomic DNA from nontransfected fibroblasts were used as negative controls, while those from piPSCs were used as positive controls. For RT-PCR Small Molecules Improve Episomal Reprogramming analysis, total RNA was prepared as described in the RNeasy Mini Kit (Qiagen) with on-column DNase I digestion. About 1 mg total RNA from each sample was used for Oligo(dT)20-primed reverse transcription as described in the product protocol (SuperScriptTM III First-Strand Synthesis System, Invitrogen). The PCR reactions for regular RT-PCR analysis of transgene expression were carried out with the pfx DNA polymerase (Invitrogen) as described above. The cDNA from nontransfected fibroblasts and human H1 ESCs were used as negative controls, while that from piPSCs was used as positive controls. For each sample, 2 ml of diluted cDNA (1:4) was added as template in PCR reactions. Quantitative PCR reactions were carried out with Power SYBRHGreen PCR Master Mix (LightCyclerH 480 II system, Roche, Indianapolis, IN). The cDNA from human H1ESC was used as a relative standard. For each sample, 1 ml of diluted cDNA (1:8) was added as template in PCR reactions. The expression of genes of interest was normalized to that of GAPDH in all samples. The methylation status of OCT4 and NANOG promoters were analyzed using bisulfite sequencing with MethylCodeTM Bisulfite Conversion Kit (Invitrogen) [19]. All primers were in Table S1 and antibodies were in Table S2. Standard G-banding chromosome analysis was carried out in the Cytogenetics Lab at WiCell Research Institute (Madison, WI). Teratoma formation To examine the in vivo differentiation potential of human iPSCs derived with the new episomal method, iPSCs grown on matrigel in mTeSR1 were transferred to MEF feeder cells for one passage. Cells were collected with collagenase treatment, and injected into hind limb muscles of 6-week-old immunocompromised SCID- beige mice (one 10-cm dish with 50 to 80% confluence per injection per mouse) (Harlan, Madison, WI). Two injections were performed for each iPSC clone. After six to eight weeks, teratomas were obtained from all injections, which were similar in size as those from human ESCs and iPSCs generated with previous methods. The teratomas were dissected and fixed in 10% formalin (Fisher, Pittsburgh, PA). Samples were embedded in paraffin and processed with hematoxylin and eosin staining in the Experimental Pathology Department of McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, WI. Supporting Information Figure S1 Episomal reprogramming vector maps. (A) Combination 7F-1 contains two vectors: pEP4EO2SCK2MEN2L and pEP4EO2SET2K. This combination expresses all seven transgenes: OCT4, SOX2, NANOG, LIN28, c-MYC, KLF4 and SV40LT. (B) Combination 7F-2 contains three vectors: pEP4EO2- SEN2K, pEP4EO2SET2K and pCEP4-M2L. This combination also expresses all seven transgenes: OCT4, SOX2, NANOG, LIN28, c-MYC, KLF4 and SV40LT. (C) Combination 5F contains two vectors: pEP4EO2SEN2L and pEP4EO2SET2N. This combina- tion expresses five transgenes: OCT4, SOX2, NANOG, LIN28 and SV40LT. pEF: the eukaryotic elongation 1a promoter; pCMV: the cytomegalovirus immediate-early promoter; IRES2: internal ribosome entry site 2. (TIF) Figure S2 Developing a feeder-free condition for epi- somal reprogramming. (A) Flow cytometry expression analysis of human ESC-specific cell surface markers (SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81) and a fibroblast marker CD44 in piPSCs (p6). Unfilled: isotype control; filled: antigen staining. (B) PCR analysis of reprogramming vectors in the episomal DNA isolated from piPSCs (p7). Lane 1: transgene OCT4 (T-OCT4); Lane 2: PLoS ONE | www.plosone.org 8 March 2011 | Volume 6 | Issue 3 | e17557