Cell transplantation At the fourth passage under standard or CDSF conditions, the mESCs were trypsinized and counted. TrypLETM Express was inactivated with an equal volume of 1 mg/ml soybean trypsin inhibitor (Sigma-Aldrich). For this purpose, no culture medium with animal serum was used. One to two million cells were centrifuged at 1,000 g for 5 min and resuspended into 25 ml PBS, which was mixed with 25 ml of 0.3 mg/ml type IA collagen. Mouse ESCs were kept on ice before being injected into NOD- SCID mice (the Jackson Laboratory, Bar Harbor, ME, http:// www.jax.org) subcutaneously. Animal health was monitored routinely until the diameter of the tumors reached several few centimeters at which time the animals reached their end points and were euthanized. This procedure was approved by the Illinois Institutional Animal Care and Use Committee. The incidence of teratoma formation was statistically validated using the Fisher’s exact probability test. Histology Before dissecting the teratomas, pictures of the experimental animals were taken. The sizes of the longest axis of the teratomas were measured. Then, the teratomas were surgically dissected out, cut into smaller pieces, and fixed in 4% paraformaldehyde (Sigma- Aldrich) at 4uC overnight, followed by dehydration and embedding in Paraplast plus (Sigma-Aldrich). Sections of 8 mm thickness were cut and processed for standard hematoxylin and eosin staining. Immunofluorescence microscopy The Oct3/4::EGFP ESC line was immunostained essentially as described previously [75,82]. Goat anti-mouse Nanog (AF2729, R & D systems) and anti-human Sox2 (sc-17320, Santa Cruz Biotechnology, Santa Cruz, CA) polyclonal antibodies and a mouse anti-SSEA1 monoclonal antibody (Developmental Studies Hybridoma Bank, University of Iowa) were used as primary antibodies. Alexa Fluor 488 goat anti-mouse IgG (H+L) and Alexa Fluor 546 donkey anti-goat IgG (H+L) polyclonal antibodies (Invitrogen) were used as secondary antibodies. Fluorescence images were taken using the same exposure time (750 msec for 505 nm and 850 msec for 595 nm) and enhanced in the same way. Aggregation chimeras Mouse ESCs cultured under CDSF conditions for 10–12 days were aggregated with zona-free morulae, which were obtained from superovulated C57BL/6 females (the Jackson Laboratory) mated with C57BL/6 males. Aggregated embryos were cultured in the KSOM medium with 1/2 amino acids (Millipore) for 14– 15 hrs as described previously [79]. Chimeric blastocysts were transferred to pseudopregnant females at the transgenic mouse facility on campus. RT-PCR A total of 1.6 mg of total RNA extracted from mESCs cultured under each condition was used to synthesize the first cDNA strand as previously described [83,85]. PCR mixtures were prepared using Phusion DNA polymerase (New England Biolab, Ipswich, MA, http://www.neb.com) according to the manufacturer’s instructions. The PCR conditions were as follows: initial denaturing at 98uC for 1 min followed by 19, 21, 23, 24 or 25 cycles of denaturing at 98uC for 10 sec, annealing at 65uC for 30 sec, extension at 72uC for 30 sec, and a final extension at 72uC for 7.5 min. The primer pairs have previously been described: Eras [38], Oct4 [86], Esg1 and Ef1a [87], Sox2 [88], and c-Myc [89]. Gsk3b Inhibition Induces Teratoma Formation Screening of factors A day after mESCs were plated under CDSF conditions, these cultures were supplemented with 10 nM retinol (Sigma-Aldrich) in 100% EtOH with or without 1 nM retinol binding protein (RBP) from human urine (Sigma-Aldrich), 10 nM all-trans retinoic acid (Sigma-Aldrich), 10 ng/ml Bmp4 (R & D Systems, Minneapolis, MN, http://www.rndsystems.com), or 3 mM Stemolecule TM CHIR99021 in DMSO (Stemgent). Before applying RBP to culture, buffer exchange was carried out with Amicon Ultra (Millipore) to remove a preservative. Mouse ESCs grown under these conditions were split with 0.02% EDTA for 3 passages when they reached confluence. At the fourth passage, these mESCs were injected into NOD-SCID mice as described above. Supporting Information Figure S1 Anatomical images of NOD-SCID mice trans- planted with mouse embryonic stem cells. (A-D, F and G): Teratomas developed from mouse embryonic stem cells (mESCs) and embryonic carcinomas (F9 in D) cultured under the conditions indicated are shown. Also, the number of weeks (wks) needed for the experimental animals to reach their end points are shown. Bars, 1cm. CDSF+FBS, CDSF culture sup- plemented with fetal bovine serum; CDSF-Standard, CDSF conditions followed by standard conditions; CDSF (F9), embry- onic carcinoma cells F9 maintained under CDSF conditions; CDSF+Bmp4, CDSF culture supplemented with Bmp4; CDSF+iGsk3b, CDSF culture supplemented with the Gsk3b inhibitor. (E): This animal was sacrificed one week after mESCs cultured under CDSF conditions followed by standard conditions were transplanted. The rectangle indicates the area shown in the inset. Bar, 1cm. The inset shows an enlarged image of a tiny mass of the mESCs depicted by dashed lines with a scale bar of 0.1 cm. See supporting information Fig. S2 for detail. (TIF) Figure S2 Mouse embryonic stem cells exhibited initial differentiation as early as one week after transplanta- tion when cultured under CDSF-Standard conditions. Mouse ESCs were cultured in CDSF for three passages followed by transfer to standard conditions for two passages prior to transplantation. (A): An epithelialized cellular mass has aggregates formed among the collagen fibers (stained pale pink) used to transplant the mESCs. Rectangles indicate the areas shown in B, C and D. Bar, 500 mm. (B-D): Two types of cells are prominent, one of which is reminiscent of keratin pearls (B and D), and the other that resembles cartilage (C and D). Bars, 20 mm. (TIF) Figure S3 Identification of factors that support the tumor-like growth of mouse embryonic stem cells maintained under CDSF conditions. (A-E): Phase contrast (top) and fluorescence (bottom) images of mESCs under CDSF conditions supplemented with each factor indicated above are shown. Bars, 20 mm. CDSF+RA, CDSF with retinoic acid; CDSF+RL, CDSF with retinol; CDSF+RL+RBP, CDSF with retinol and retinol binding protein; CDSF+Bmp4, CDSF with Bmp4; CDSF+iGsk3b, CDSF with the Gsk3b inhibitor. (TIF) Figure S4 The incidence of teratoma formation was compared among different CDSF conditions. The sizes of the teratomas formed (orange bar, left axis) and the number of days required for the experimental NOD-SCID mice to reach their end points (blue stars, right axis) were compared among mESCs cultured under the conditions indicated. Parentheses PLoS ONE | www.plosone.org 8 June 2011 | Volume 6 | Issue 6 | e21355