Cell Culture. The (hESC line H9 was grown in feeder-free conditions in six-well Nunclon surface plates (Nunc) coated with Matrigel (BD Biosciences) and maintained in mTESR1 media (Stem Cell Technologies). Cells were passaged at a 1:3 ~ 4 ratio using dispase (Invitrogen). All Matrigel plates were coated with a 1:80 dilution in Advanced DMEM-F12 (Life Technologies) and incubated at room temperature for at least 1 hr before use.
Generation of pancreas endocrine cells (PEC) from ESCs. Human ESCs were passaged with Accutase (Sigma) and plated at a density of 100,000 cells/cm2 in mTeSR with 10 µM Y27632 (Selleck) on RPMI1640 (Gibco), Matrigel (BD) and collagen IV (BD) (5:2:1) mixed gel coated-plate (Corning). In the restriction of definitive endoderm (DE) stage (S1), cells were cultured for 24 hrs in RPMI1640 with B27 supplement (1:50, Gibco), N2 supplement (1:50, Gibco), 100 ng/ml Activin A (R&D) and 50 ng/ml Wnt3a (R&D), and then treated with 100 ng/ml Activin A and 0.2% FBS for 2 days. In the stage (S2) to get primitive gut tube (PGT), the culture medium was replaced with RPMI1640 supplemented with B27 supplement (1:50), N2 supplement (1:50), 30 ng/ml FGF7, 5 ng/ml Wnt3a, 0.75 µM Dosomophine, 2% FBS for 3 days. And in the stage (S3) of pancreatic progenitors (PP), cells were cultured in Advanced DMEM-F12 supplemented with B27 supplement (1:100), 2 µM RA, 0.25 µM Cyclop, 30 ng/ml FGF7, 50 ng/ml Noggin, 0.3 µM IL-5, and 6 µM SB431542 for 6 days. At the end of stage3, media were changed to DMEM (Gibco) supplemented with B27 supplement (1:100), 50 ng/ml Exendin-4, 6 µM SB431542, 50 ng/ml Noggin, and 10 mM Nicotinamide. For 3D culture, cells at stage 3 were digested with Accumax and replated at a density of 3 × 105/ml in ultra-low attachment 6-well plates (Corning), and the plates were placed on a 3D orbital shaker set at rotation rate of 80 rpm in a 37℃ incubator, 5% CO2. Cells were photographed during differentiation using a Nikon phase contrast microscope (Nikon Microscopes).
Quantitative real-time PCR analyses. Total RNA was isolated using an RNeasy extraction kit. RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed with SYBR Green real-time PCR master mix (TOYOBO) on a Bio-Rad iQ5 Real-Time PCR detection system (Bio-Rad). The data were analyzed using the delta-delta Ct method. The primers are listed in supplementary Table S1.
Immunofluorescent Staining. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature and blocked with 10% goat or donkey serum for 1 h, followed by incubation with primary antibodies at 4 ºC overnight. Labeled isotype-specific secondary antibodies were added and incubated 1 h at room temperature. Cells were counterstained with 4´,6-diamidino-2-phenylindole (DAPI) for visualization of cell nuclei and observed using a confocal microscopy (PerkinElmer) and Volocity Software (PerkinElmer). Antibodies used in this study were summarized in supplementary Table S2.
Flow cytometry. Single cell suspensions were obtained by dissociation with Accutase for 3–5 min. Cell surface antigen staining was performed in PBS at 4 ℃. Intracellular staining was performed with the BD Cytofix/Cytoperm™ Kit (BD Biosciences) according to the manufacturer’s instructions. Briefly, cells were fixed and permeabilized with BD Cytofix/Cytoperm solution for 20 min at 4 °C. Intracellular antigen staining was performed in BD Perm/Wash solution.
The stained cells were analyzed or sorted with BD FACSCalibur (BD Biosciences), and the data was analyzed using the Flowjo software (TreeStar). The sources and concentrations of primary, secondary antibodies and isotype controls are listed in supplementary Table S2.
C-peptide release assay. The pancreatic endocrine cells were used for the C-peptide release assay as previously described. Briefly, after a 1 hour-wash in KRBH medium, 300 µl of basal media that contains 2 mM D-glucose (Sigma) were added to each well of 12-well dishes. After 1 hour incubation, the basal media were changed into 300 µl of stimulation media (20 mM D-glucose or 30 mM KCl). The cultures were incubated at 37 °C in a 5% CO2 environment for 30 min. For each experiment, 6 wells of supernatants were pooled together and stored at -20 °C until assay, meanwhile the cells were harvested for protein determination using the Bio-Rad Protein Assay K (Bio-Rad) according to the Bradford method. Ultra-sensitive human C-peptide ELISA kit (Mercodia) has been used and the assays done according to manufacturer’s instructions.
Transmission electron microscopy. The cell samples were rinsed with PBS and fixed in 3% glutaraldehyde/0.1 M sodium cacodylate, pH 7.4 overnight. Following three rinses with sodium cacodylate buffer, the samples were postfixed for 1 hour in 1% osmium tetroxide/0.1 sodium cacodylate buffer. After rinsing in deionized water, samples were dehydrated and embedded in Polybed 812 epoxy resin (Polysciences, Inc., Warrington, PA). The samples were sectioned perpendicular to the substrate at 70 nm using a diamond knife. Ultrathin sections were collected on 200 mesh copper grids and stained with 4% aqueous uranyl acetate for 15 min, followed by Reynolds’ lead citrate for 7 min. Samples and stained sections were observed using a H7650 transmission electron microscope (HITACHI) operating at 80 kV (H7650 Electron Microscopy) and photographed using an AMT XR16M CCD Digital Camera and AMT Capture Engine Software Version 600.259 (AMT).
Western blotting. Cells were harvested in lysis buffer (50 mM Tris-HCl, pH 7.4, 0.25 mM sodium-deoxycholate, 150 mM NaCl, 2 mM EDTA, 0.1% sodium dodecyl sulfate, 1% Triton X-100) containing protease and phosphatase inhibitors (Roche). Lysates were sonicated for 30 s, maintained on ice for 30 min, and then spun at 15,000 rpm for 15 min at 4 ˚C. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and probed with antibodies listed in supplementary Table S2. Proteins were detected by enhanced chemiluminescence HRP substrate (Millipore).
Statistics. Data are shown as means and SEMs. For most statistic evaluation, 2-tailed Student’s t test was applied for calculating statistical probability in this study. P values less than 0.05 were considered to be statistically significant. For all statistics, data from at least three independent samples or repeated experiments were used.