Cell Culture. The hESC line H9 was grown in feeder-free conditions in six-well Nunclon surface plates (Nunc, USA) coated with Matrigel (R&D systems, USA) and maintained in mTESR1 media (Stem Cell Technologies, USA). Cells were passaged at a 1:3~4 ratio using dispase (Invitrogen, USA). All Matrigel plates were coated with a 1:80 dilution in Advanced DMEM-F12 (Gibco, USA) and incubated at room temperature for at least 1 hr before use.
Generation of pancreas endocrine cells (PEC) from hESCs. Human ESCs were passaged with Accutase (Sigma, USA) and plated at a density of 100,000 cells/cm2 in mTeSR1 media with 10 μM Y27632 (Selleckchem, USA) on RPMI1640 (Gibco, USA), Matrigel (R&D systems, USA) and collagen IV (R&D systems, USA) (5:2:1) mixed gel coated-plate (Corning, USA). In the restriction of definitive endoderm (DE) stage (S1), cells were cultured for 24 hrs in RPMI1640 with B-27 supplement (1:50, Gibco, USA), N-2 supplement (1:50, Gibco, USA), 100 ng/ml Activin A (R&D systems, USA) and 50 ng/ml Wnt3a (R&D systems, USA), 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), 30ng/ml FGF7, 5 ng/ml Wnt3a, 0.75μM Dorsomophin (Sigma, USA), 2% FBS for 3 days. And in the stage (S3) of pancreatic progenitors (PPs), cells were cultured in Advanced DMEM-F12 supplemented with B27 supplement (1:100), 2 μM Retinoic acid (Sigma, USA) , 0.25 μM Cyclopamine (Selleckchem, USA), 30 ng/ml FGF7 (R&D systems, USA), 50 ng/ml Noggin (R&D systems, USA), 0.3 μM IL-5 (R&D systems, USA), and 6 μM SB431542 (Selleckchem, USA) for 3 days. At the end of stage 3, media were changed to DMEM (Gibco, USA) supplemented with B27 supplement (1:100), 50ng/ml Exendin-4 (R&D systems, USA), 6 μM SB431542, 50 ng/ml Noggin, and 10 mM Nicotinamide (Sigma, USA). 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, USA), 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 Eclipse Ti-S phase contrast microscope (Nikon, Japan).
Quantitative real-time PCR analyses. Total RNA was isolated using an RNeasy extraction kit. RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen, USA) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed with SYBR Green real-time PCR master mix (TOYOBO, Japan) on a Bio-Rad iQ5 Real-Time PCR detection system (Bio-Rad, USA). 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 1h at room temperature. Cells were counterstained with 4´,6-diamidino-2-phenylindole (DAPI) for visualization of cell nuclei and observed using a Zeiss LSM 510 confocal microscopy (Zeiss, German) and the Zeiss LSM Image Browser Software (Zeiss, German). 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, USA) 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 with BD FACSAria (BD Biosciences, USA), and the data was analyzed using the Flowjo software Version 10 (TreeStar, USA). The sources and concentrations of primary, secondary antibodies and isotype controls are listed in supplementary Table S2.
Dithizone staining. The dithizone stock solution was prepared by adding 3 ml ethanol and 50 µl concentrated ammonium hydroxide to 50 mg dithizone (Sigma, USA). The clear dark-red solution was then diluted with PBS up to 30 ml and stored at −20°C. Next the stock solution was diluted 1 : 20 in PBS. Cells were washed with PBS for three times and incubated in working solution for 10 minutes at 37°C. Finally, cells were examined under a Nikon Eclipse Ti-S microscope (Nikon, Japan).
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, 30 mM KCl or 30 μM Forskolin). 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, USA) according to the Bradford method. Ultra-sensitive human C-peptide ELISA kit (Mercodia, Sweden) has been used and the assays done according to manufacturer’s instructions.
Transmission electron microscopy (TEM). 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., USA). 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, Japan) operating at 80 kV (H7650 Electron Microscopy) and photographed using an AMT XR16M CCD Digital Camera and AMT Capture Engine Software Version 600.259 (Advanced Microscopy Techniques Corp, USA).
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, USA). 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, USA).
CCK-8 Assay. Cell proliferation was assessed by Cell Counting Kit-8 (Dojindo, Japan) assay. PPs from stage 3 were seeded at 2000 cells/well into 96-well plates with 100 μL culture medium and were incubated at 37 ˚C overnight. The 10 μL of CCK-8 solution was added to the cells at specific time points and cells were incubated for 2 hr at 37°C. The optical density (OD) value of each well was measured using a SpectraMax M5 microplate reader (Molecular Devices, USA) at the wavelength of 450 nm.
Statistics. Data are shown as mean ± SD. For most statistic evaluation, 2-tailed Student’s t test was applied for calculating statistical probability in this study. Multi-group comparisons were conducted using the two-way ANOVA. 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.