The generation of human iPS cells used in the current manuscript have been described in detail elsewhere  following the procedures established by Yu et al. huES (H9 cells and H1 cells) and human iPS cells (hiPSC2a, hiPSC3a and hiPSC6a) were maintained on MEFs in DMEM/F12 (Invitrogen, Carlsbad, CA), supplemented with 1 mM L-glutamine (Millipore, Billerica, MA, http://www.millipore.com), 1% nonessential amino acids (Millipore, Billerica, MA), 0.1 μM ß-mercaptoethanol (Sigma Chemical, St. Louis, MO), 20% (vol/vol) Knockout serum replacement (KSR; Invitrogen, Carlsbad, CA) and 4 ng/mL bFGF. For feeder-free cultures, cells were cultured on a Matrigel-coated dish in medium conditioned by mitomycin C-treated MEF supplemented with 4 ng/mL bFGF or in mTeSR1 (Stemcell Technologies, Vancouver, Canada, http://www.stemcell.com). Cells were passaged with Accutase (Millipore, Billerica, MA) or enzyme-free Cell Dissociation Buffer (PBS-based: Invitrogen, Carlsbad, CA) before becoming confluent by incubating at room temperature until cells detached from the surface. For passage from Matrigel to hE-cad-Fc coated plates it was crucial to remove cells either using Cell Dissociation Buffer or minimal digestion with Accutase (<3 mins). All media contained 100 units/mL penicillin and 100 μg/mL streptomycin (Millipore, Billerica, MA). Cell karyotype and FISH analyses were performed by Cell Line Genetics, LLC (Madison, WI)
Expression of fusion protein and preparation of hE-cad-Fc-coated dishes
To construct hE-cad-Fc, the cDNA that encodes human E-cadherin extracellular domain was amplified by nested PCR with KOD plus polymerase (TOYOBO, Osaka, Japan, http://www.toyobo.co.jp) from the cDNA of A431 cells. The specific primer pairs were used for amplification: 5'- AAG CAC CTG TGA GCT TGC G -3' and 5'- AAG TCC TGG TCC TCT TCT CCG C -3' for 1st PCR; 5'- AAG CTT CCA CCA TGG GCC CTT GGA GCC GCA GC -3' and 5'- GCG GCC GCT CTT CCT ACA GAC GCC GGC GGC CCC -3' for nested PCR. The cDNA of human E-cadherin fragment and mutated mouse IgG1 Fc domain (T252M-T254S) , which has a high affinity to Protein A, were ligated with pRC/CMV (Invitrogen, Carlsbad, CA) fragment, which was digested with HindIII and XbaI to generate the expression vector "pRC-hECFC." 293T cells were transfected with "pRC-hECFC" using Lipofectamine and PLUS reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's directions. After selection of a highly expressing clone, 1C8, with 400 μg/mL G418 (Invitrogen, Carlsbad, CA), conditioned media were collected. The fusion proteins were loaded onto a rProtein A FF column (GE Healthcare Life Sciences, Pittsburgh, PA). The column was washed with 20 mM phosphate buffer (pH 7.0), and the bound proteins were eluted using 0.1 M sodium citrate (pH 2.7) followed by neutralization with a 1/5 volume of 1.0 M Tris-HCl (pH 9.0). Eluates were dialyzed against PBS containing 0.9 mM CaCl2 and 0.9 mM MgCl2 for 3 days. To prepare the hE-cad-Fc-coated surface, purified hE-cad-Fc solution was directly added to non-treated polystyrene plates at a concentration of 20 μg/ml unless otherwise described. After 2 h incubation at 37°C, plates were washed with PBS once and then cells were seeded.
Alkaline phosphatase detection and immunocytochemistry
Alkaline phosphatase activity was determined using an alkaline phosphatase detection kit (Millipore, Billerica, MA). For immunocytochemistry, cells were fixed with 8% formaldehyde/PBS solution for 10 min and made permeable with 0.2% Triton X-100 (Fisher Scientific International, Hampton, NH, http://www.fishersci.com) for 2 min at room temperature. Fixed cells were incubated with 1% BSA/PBS for 1 h at room temperature and then stained with anti-mouse Oct-3/4 polyclonal antibody (H-134; Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), anti-phospho-histone H3 (Ser10) antibody (Millipore, Billerica, MA), or anti-activated caspase-3 antibody (BD Pharmingen, San Diego, CA, http://www.bdbiosciences.com) for 2 h, followed by Alexa Fluor-conjugated secondary antibodies (Invitrogen, Carlsbad, CA) for 1 h. Nuclei were counterstained with 0.5 μg/mL DAPI (Invitrogen, Carlsbad, CA). Samples were observed by fluorescence microscopy. For BrdU staining, cells were incubated with 10 μM BrdU (Sigma-Aldrich, St Louis) for 75 min at 37°C and then fixed and permeabilized using the same method as above. The nuclear DNA was denatured by incubating in 2N HCl for 30 min at room temperature. After washing with PBS, cells were stained with anti-BrdU antibody (clone BU1/75 ICR1; Abcam, Cambridge, U.K., http://www.abcam.com), followed by Alexa Fluor-conjugated secondary antibody, as above.
Flow cytometry analysis
For analysis cell-surface SSEA-4 expression, cells were treated with Accutase (Millipore, Billerica, MA) and stained with phycoerythrin-conjugated anti-SSEA-4 antibody (Millipore, Billerica, MA). For E-cadherin expression, cells were treated with Accutase or Cell Dissociation Buffer and stained with phycoerythrin-conjugated anti-E-cadherin antibody (R&D systems, Minneapolis, MN, http://www.rndsystems.com). Stained cells were analyzed using Guava EasyCyte system (Millipore, Billerica, MA).
Total RNA was isolated with RNeasy Plus Mini Kit (Qiagen, Valencia, CA, http://www.qiagen.com). First-strand cDNA was synthesized using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen, Carlsbad, CA), and PCR was carried out with Taq polymerase in supplier's reaction buffer containing 1.5 mM MgCl2. Oligonucleotide primer sequences are available by request. Amplicons were analyzed by 2% agarose gel electrophoresis.
Adhesion and growth assays
For the adhesion assay, cells were seeded at a density of 1.0 × 105 cells/well into 24-well plates pre-coated with Matrigel, mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) or hE-cad-Fc. After 3 h of culture, medium and non-adherent cells were removed and cells were washed with culture medium. Adherent cells were stained with Cell Proliferation Reagent WST-1 (Roche Applied Science, Indianapolis, IN, http://www.roche-applied-science.com). After incubation for 2.5 h, absorbance at 450 nm was measured using a microplate reader (BioTek Instruments, Winooski, VT, http://www.biotek.com). For the cell growth assay, cells were seeded into a 96-well plate coated with hE-cad-Fc at a density of 3,000 cells/well or coated with Matrigel at a density of 1,000 cells/well. The cell number was evaluated once per day by the same method as for adhesion assay.
Teratoma formation assay
Cells were collected by Accutase treatment and injected into immunocompromised Rag2-/-Il2rg-/- mice (Taconic, Hudson, NY, http://www.taconic.com) subcutaneously or in the hind limb muscle. After 9 to 10 weeks, teratomas were surgically dissected from the mice. Samples were fixed in 4% zinc-formaldehyde and embedded in paraffin. Sections were cut at 4 μm and processed with hematoxylin and eosin staining. The Medical College of Wisconsin's IACUC approved all animal procedures used in this study.
Total protein was extracted with lysis buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Nonidet P40, 1% Triton X-100, 1 mM CaCl2). Samples were separated by electrophoresis on 12% polyacrylamide gels and electrophoretically transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA, http://www.bio-rad.com). Blots were probed with anti-E-cadherin antibody (BD Transduction Laboratories, San Diego, CA, http://www.bdbiosciences.com) or anti-ß-actin antibody (Sigma), followed by horseradish peroxidase-conjugated secondary antibodies, and developed by SuperSignal West Pico substrate (Thermo Scientific, Waltham, MA, http://www.thermo.com).
Coating efficiency of hE-cad-Fc onto polystyrene surface
Purified hE-cad-Fc protein was labelled with peroxidase labelling kit-NH2 (Dojindo Molecular Technologies, Inc., Rockville, MD). Labelled protein was diluted with unlabelled hE-cad-Fc (1/400) and then serially diluted with PBS. Diluted samples were added to a 96-well plate and incubated for 1 h at 37°C. Surfaces were washed with PBS three times and incubated with 100 μl of tetramethylbenzidine (TMB) substrate at room temperature. After addition of 100 μl of 1N HCl, absorbance at 450 nm was measured using a microplate reader. The amount of immobilized hE-cad-Fc was calculated from a standard curve of labelled hE-cad-Fc captured by anti-mouse IgG antibody-immobilized surface.
Values are reported as means ± SD or SEM. Statistical significance was assessed using the paired Student's t-test. The probability level accepted for significance was P < 0.05.