piPSCs or iPSCs were obtained from fibroblasts in its absence. Truncated mutants of SV40LT such as its transformation domain (N-terminal 147 amino acids) and p53-binding domain failed to replace the full-length SV40LT (unpublished data). It remains an intriguing question how the combination of the SV40LT expression and small molecule treatments enables the prolonged retention of oriP/EBNA-1-based reprogramming vectors and transgene expression. More importantly, due to this distinct reprogramming intermediate stage, it will be of great interest to characterize the iPSCs obtained with this method in details, e.g. the mutation rate arising during reprogramming and the epigenetic memory retention. This will help address the question whether the quality of iPSCs is significantly affected by their derivation methods. In summary, using defined media, we have successfully established a robust feeder-free episomal reprogramming method, which applies to different somatic cell types readily available from human donors. This method can be used for the derivation of footprint-free iPSCs from many donor samples, and can be easily adapted to the production of clinical-grade human iPSCs. Materials and Methods Ethics Statement All human primary cells were generated in vitro from tissue samples from human donors with appropriate written informed consent given to the commercial providers. All mouse work was conducted according to relevant national and international guidelines under the approval of the Cellular Dynamics International Animal Care and Use Committee. As a private company, our animal facility does not provide a permit number or approval ID since mouse is not a protected species. Cell culture Human ESCs and iPSCs were maintained on irradiated MEFs in DMEM/F12 culture medium supplemented with 20% KnockOut serum replacement, 0.1 mM non-essential amino acids, 1 mM GlutaMAX (all from Invitrogen, Carlsbad, CA), 0.1 mM b-mercaptoethanol (Sigma, St. Louis, MO) and 100 ng/ ml zebrafish basic fibroblast growth factor (zbFGF) [19]. MEF- conditioned human ESC medium was prepared as previously described [35]. Human newborn foreskin fibroblasts (Cat# CRL- 2097, ATCC, Manassas, MA) and adult skin fibroblasts (Cat# CRL-2106, ATCC) were cultured in DMEM (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS, HyClone Laboratories, Loan, UT), 0.1 mM non-essential amino acids, 1 mM GlutaMAX, 0.1 mM b-mercaptoethanol and 4 ng/ml zbFGF. Human adult adipose tissue-derived stem cells (Cat# ASC-F, ZenBio, Research Triangle Park, NC) was cultured in MesenCult-XF basal medium supplemented with 16Mesen- Cult-XF supplement (STEMCELL Technologies, Vancouver, BC, Canada) and 2 mM GlutaMAX on collagen (60 mg per 10-cm dish)/fibronectin (18 mg per 10-cm dish)-coated 10-cm dishes. Human cord blood CD34+ cells were obtained from STEMCELL Technologies (Cat# CB008F-S), and expanded on fibronectin- coated plates in CD34+ cell expansion medium - StemSpan SFEM (STEMCELL Technologies) supplemented with EX- CYTE growth enhancement media supplement (1:1000, Milli- pore, Billerica, MA), 2 mM GlutaMAX, 250 ng/ml SCF, 250 ng/ ml FLT3L, 100 ng/ml TPO, 20 ng/ml IL-3, 50 ng/ml IL-6 and 10 ng/ml sIL6-R (all from Peprotech, Rocky Hill, NJ). The feeder-free culture of human ESCs and iPSCs on matrigel (BD Biosciences, Bedford, MA) in mTeSR1 (STEMCELL Technologies) was carried out as previously described with Small Molecules Improve Episomal Reprogramming modifications in the passaging procedure [36]. Briefly EDTA splitting method was employed. When human ESCs and iPSCs reach confluence, cells were washed once with PBS free of Ca2+ and Mg2+, and incubated with 0.5 mM EDTA for 8 minutes at 37uC (2 ml/well of 6-well plate). After incubation, the EDTA solution was removed and fresh mTeSR1 (2 ml/well of 6-well plate) was added dropwise to each well for cell detachment. Most cells came off the plate with gentle shaking. Dissociated cells were then immediately dispersed into freshly prepared matrigel plates prefilled with mTeSR1. To improve cell attachment and survival, the ROCK inhibitor HA-100 (10 mM, Santa Cruz Biotechnology, Santa Cruz, CA) was added to mTeSR1 for 1 day during passaging. With this method, human ESCs and iPSCs were passaged every 3 to 4 days at a splitting ratio of 1:8 for optimal growth. Improve episomal reprogramming of human fibroblasts with small molecules Episomal reprogramming vectors containing expression cas- settes for human OCT4, SOX2, NANOG, LIN28, c-MYC, KLF4 and SV40LT transgenes were as previously described [19]. Specifically, vector pEP4EO2SCK2MEN2L and pEP4EO2SET2K (combina- tion 7F-1, Fig. S1A) were used for reprogramming optimization. About 7.3 mg of vector pEP4EO2SCK2MEN2L and 3.2 mg of pEP4EO2SET2K were co-transfected into ,1.06106 human neonatal foreskin fibroblasts via nucleofection (VPD-1001 with program U-20, Amaxa, Walkersville, MD). Transfected fibroblasts were plated directly to 3610-cm MEF-seeded or matrigel-coated dishes in fibroblast culture medium. On day one post-transfection, the fibroblast medium was replaced with MEF-conditioned human ESC medium supplemented with 100 ng/ml zbFGF (CM100), or chemically defined N2B27 medium (N2B27), or N2B27 medium supplemented with 100 ng/ml zbFGF (N2B27-100). The N2B27 medium consists of DMEM/F12 culture medium supplemented with N-2 supplement (16, Invitrogen), B-27 supplement (16, Invitrogen), 0.1 mM non-essential amino acids, 1 mM Gluta- MAX, and 0.1 mM b-mercaptoethanol. Where applied, small molecules PD0325901 (P, 0.5 mM), CHIR99021(C, 3 mM), A-83- 01(A, 0.5 mM) (all from Stemgent, San Diego, CA), hLIF (L, 1000 U/ml, Millipore) and HA-100 (H, 10 mM) were added to reprogramming culture. Culture medium was refreshed every two days. Alkaline phosphatase staining (Cat# SCR004, Millipore) was performed in subsets of reprogramming experiments to facilitate the identification of iPSCs. Briefly, cells were fixed in 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 30 minutes at room temperature before staining according to manufacturer’s protocol. Authentic iPSC colonies were stained positive for alkaline phosphatase, and showed morphology typical of human ESCs (e.g. compact colonies, high nucleus-to-cytoplasm ratios and prominent nucleoli) (Fig. S4). Episomal reprogramming of human adult skin fibroblasts was carried out similarly to that of foreskin fibroblasts. To characterize the newly derived iPSCs, iPSC colonies were picked directly onto matrigel-coated 12-well plates in mTeSR1. EDTA splitting method was employed to facilitate iPSC expansion and removal of differentiated cells. Complete loss of episomal vectors was generally achieved around passage 14 for fibroblast-derived iPSC clones. Optimizing episomal vector and transgene combinations for reprogramming human fibroblasts, adult adipose tissue-derived cells and cord blood cells Episomal reprogramming vector combinations tested were shown in Figure S1. For each nucleofection, the following amount PLoS ONE | www.plosone.org 7 March 2011 | Volume 6 | Issue 3 | e17557