Small molecules
Several small molecular compounds were used to differentiate iPSCs into IPCs. In most cases, the small molecules inhibited the signaling pathways in cells. The differentiation process was divided into three steps to mimic the process of pancreatic development in the embryonic stage. The first step was the formation of a definitive endoderm, the second step was its differentiation into the pancreatic endoderm and pancreatic progenitor cells, and the third step was the induction of specific differentiation of the cells into IPCs. Therefore, the differentiation-inducing small molecules were processed at each step, and the results were confirmed by comparing and analyzing the combinations of small molecules (Table 1).
Cell culture
iPSCs were kindly provided by the Center for Stem Cell Research of Asan Institute (Seoul, Korea). The cells were maintained on a vitronectin-coated dish with essential 8TM basal medium (Gibco, NY, USA). For differentiation into a definitive endoderm, iPSCs were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 1% B27TM supplement (minus insulin, Invitrogen) with or without human activin A (100 ng/mL) (PeproTech, Rocky Hill, NJ), 3 µM CHIR99021 (Sigma-Aldrich, MO, USA), and 5 µM LY294002 (Sigma-Aldrich) for 24 h, and then in RPMI 1640 medium containing 1% B27 with activin A (100 ng/mL) for 48 h. The iPSCs were subsequently incubated in Improved MEM Zinc Option culture medium (Invitrogen) containing 1% B27 with 1 µM Dor (Sigma-Aldrich), 4 µM RA, and 10 µM SB431542 (Tocris Bioscience, Bristol, UK), with or without FGF2 (50 ng/mL, PeproTech), 3 µM FR180204 (Sigma-Aldrich, MO, USA), and 0.25 µM SANT-1 (Sigma-Aldrich, MO, USA) for differentiation into pancreatic progenitor cells. The cells were then cultured in differentiation-inducing medium for 6 d, and the medium was changed daily. Finally, for differentiation into IPCs, 10 µM forskolin (Fors, Sigma-Aldrich, MO, USA), 10 µM dexamethasone (Dexa, Sigma-Aldrich, MO, USA), and 10 µM nicotinamide (Nico, Sigma-Aldrich, MO, USA) were added to the medium, and the cells were cultured for 10 d.
Teratoma analysis
The iPSCs were harvested and dissociated into a single cell suspension using TrypLETM Express (Gibco, NY, USA). The cells (2 × 106 cells) were subcutaneously injected dorsally into 8-week-old mice with severe combined immunodeficiency (SCID). They were maintained under non-specific pathogen-free (SFP) conditions at an experimental animal facility in Asan Institute. They were euthanized after the development of tumors >1 cm3, or following an observation period of 40 d. The tumor-containing tissue was fixed with 4% paraformaldehyde (PFA), embedded in paraffin, and serially sectioned (4 µm sections). The tissue sections were stained with hematoxylin and eosin and subjected to histological analysis using a microscope.
Immunostaining
Cells were fixed with 4% PFA for 30 min at room temperature (RT) and then washed thrice with phosphate-buffered saline (PBS). The cells were permeabilized with 0.1% Triton X-100 for 5 min, and then washed with PBS. For blocking, cells were incubated with normal horse serum (1:30) for 30 min, and then incubated overnight at 4℃ with the following primary antibodies: rabbit anti-Oct4, 1:50 (abcam, MA, USA); mouse anti-Nanog, 1:200 (abcam, MA, USA); rabbit anti-Sox2, 1:1000 (abcam, MA, USA); mouse anti-SSEA4, 1:200 (abcam, MA, USA); mouse anti-FoxA2, 1:100 (abcam, MA, USA); goat anti-CXCR4, 1:300 (abcam, MA, USA); mouse anti-insulin, 1:200 (Santa Cruz, TX, USA); rabbit anti-glucagon, 1:200 (Santa Cruz, TX, USA); and goat anti-Pdx1, 1:10000 (abcam, MA, USA). After washing with PBS, the cells were incubated with Alexa Fluor 488- or 594-conjugated donkey or goat antibodies directed against rabbit, goat, or mouse IgG at 1:250 dilutions at RT for 1 h. Nucleotides were stained with Hoechst 33342 (Thermo Scientific, Germany).
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was isolated using TRIzol reagent (Invitrogen), and used to synthesize first strand cDNA using SuperScript III First-Strand Synthesis SuperMix (Invitrogen). The SYBR pre-mixed system SsoAdvancedTM Universal SYBR Green Supermix (BIO-RAD, CA), and specific primers were used to determine the level of each mRNA transcript to relative to that of 18sRNA. Primer sequences used were the following: Sox17, forward 5'-CGCTTTCATGGTGTGGGCTAAGGACG-3', reverse 5'-TAGTTGGGGTGGTCCTGCATGTGCTG-3'; FoxA2, forward 5'- ACTGGAGCAGCTACTTAGCAGAGC-3', reverse 5'-TCATGGAGTTCATGTTGGCGTAG-3'; CXCR4, forward 5'-GGTGGTCTATGTTGGCGTCT-3', reverse 5'-TGGAGTGTGACAGCTTGGAG-3'; Pdx1, forward 5'-GCATCCCAGGTCTGTCTTCT-3', reverse 5'-ATCCCACTGCCAGAAAGGTT-3'; Ngn3, forward 5'-CTTGCTGCTCAGGAAATCCC-3', reverse 5'-CTTCTGGTCGCCAAGTTCAG-3'; Nkx6.1, forward 5'-CGTTGGGGATGACAGAGAGT-3', reverse 5'-TGGGATCCAGAGGCTTATTG-3'; Sox9, forward 5'-TACGACTACACCGACCACCA-3', reverse 5'-TCAAGGTCGAGTGAGCTGTG-3'; NeuroD, forward 5'-GCCGACGGAGATTAGGAGAA-3', reverse 5'-TCTTGTCCTGACACTGGCAT-3'; insulin, forward 5'-ATCCTGGATCTCAGCTCCCT-3', reverse 5'-CTCACAGCCCTTCAGAGACA-3'; glucagon, forward 5'-ATTGCTTGGCTGGTGAAAGG-3', reverse 5'-TATAAAGTCCCTGGCGGCAA-3'; and 18sRNA, forward 5'-GAGCCTGCGGCTTAATTTGA-3', reverse 5'-AACTAAGAACGGCCATGCAC-3'. PCR was performed at 45 cycles of 20 s at 98℃, 20 s at 55℃, and 20 s at 72℃. The relative level of each gene mRNA transcript to the 18sRNA level was analyzed by the 2-ΔΔCT method.
Insulin release assay
Differentiated cells were pre-incubated in Krebs-Ringer bicarbonate HEPES buffer without glucose (Krebs buffer; 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM HEPES, and 0.5% BSA) at 37℃ for 2 h. Then, the cells were sequentially incubated with 2.8 mM, 20 mM, and 30 mM glucose and 30 mM Kcl-Krebs buffer for 1 h. The insulin secretion of the Krebs buffer samples and differentiation-induced media was measured using an insulin ELISA kit (ALPCO, NH, USA).
Flow cytometry
Differentiated cells were dissociated to single cells using Accutase, fixed with 4% PFA, and permeabilized with Perm buffer III (BD Biosciences). The cells were incubated with normal horse serum for 10 min and then with mouse anti-insulin, rabbit anti-glucagon, and rabbit anti-Ngn3 antibody for 30 min at RT. The cells were then stained with Alexa Fluor 488-conjugated donkey antibodies directed against mouse or rabbit IgG for 30 min at RT, and flow cytometry was performed using FACSAria II (Becton Dickinson).
Statistics and reproducibility
Statistical tests performed for specific data sets are described in the corresponding figure legends. Two-tailed unpaired t-tests (Student’s t-test) were used to measure standard deviation (SD). Two-way ANOVA test for multiple comparisons was used to calculate the significance, including P values. All statistical tests were performed using GraphPad Prism Software v8.