Cell culture and iPSC-derived pancreatic islet cells (iPIC) differentiation
Ff-I14s04 and QHJI-14s04 were kindly provided by the Center for iPS Cell Research and Application (CiRA), Kyoto University. Ff-I14s04 is derived from the same clone as QHJI but cultured and stocked for non-clinical use. QHJI-14s04 is a stock for clinical use. Cells were maintained on iMatrix-511 (Nippi)-coated dishes in StemFit AK03N (Ajinomoto) at 37 °C in a humidified 5% CO2 incubator. Cells were passaged every 3 or 4 days by non-enzymatic dissociation using 0.5 mM EDTA (Thermo Fisher Scientific) and subjected to differentiation experiments, usually after over 2 weeks of running culture. The use of human iPSCs was approved by the ethical review committee of Kyoto University and Takeda Pharmaceutical Company Limited. For differentiation culture to generate iPIC, we performed 2D monolayer to static aggregate culture based on our previous report10 and 3D stirred-floating aggregate culture. The details for 3D floating culture are provided below.
Stage 1
Dissociated undifferentiated iPSCs were resuspended at a density of 6 × 106 cells in a spinner type 30 mL bio-reactor (Biott) suspended in AK03N containing 10 μM Y-27632 (FUJIFILM Wako) and stirred at a speed of 70 rpm throughout culture. The next day, aggregated cells were cultured in DMEM (high glucose, GlutaMAX Supplement, pyruvate; Thermo Fisher Scientific) or RPMI 1640 Medium (Thermo Fisher Scientific) supplemented with 1% (v/v) penicillin/streptomycin (P/S, FUJIFILM Wako), 1 × B-27 (Thermo Fisher Scientific), 1% Pluronic® F-68 (Poloxamer 188, Merck Millipore) to reduce fluid mechanical damage, 5–10 ng/ml45 Activin A (PeproTech), 3 μM CHIR99021 (Axon Medchem), and 1% DMSO (FUJIFILM Wako). The following day, CHIR99021 was removed from the medium, and culture was continued for another 2 days.
Stage 2
Cells were cultured with MCDB 131 medium (Thermo Fisher Scientific) supplemented with 1% P/S, 0.5 × B27, 1% Pluronic® F-68 and 50 ng/ml keratinocyte growth factor (KGF, R&D Systems), 4.44 mM glucose (added to yield a final concentration of 10 mM, FUJIFILM Wako), 1.5 g/L NaHCO3 (FUJIFILM Wako), and 1% GlutaMAX (Thermo Fisher Scientific) for 4 days.
Stage 3
Continuing culture was performed with improved MEM (iMEM, Thermo Fisher Scientific) containing 1% P/S, 0.5 × B27, 1% Pluronic® F-68, 50 ng/ml KGF, 100 ng/ml Noggin (FUJIFILM Wako), 0.5 μM 3-keto-N-aminoethyl-N’-aminocaproyldihydrocinnamoyl cyclopamine (KAAD-cyclopamine, Toronto Research Chemicals), and 10 nM 4-[(E)-2-(5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl] benzoic acid (TTNPB, Santa Cruz Biotechnology) for 3 days.
Stage 4
Cells were exposed to iMEM containing 1% P/S, 0.5 × B27, 1% Pluronic® F-68, 100 ng/ml KGF, 50 ng/ml epidermal growth factor (EGF, R&D Systems), 10 mM nicotinamide (STEMCELL Technologies), 0.1 μM TR05991851 (Takeda original ROCK inhibitor), 0.5 μM phorbol 12,13-dibutyrate (PdBU, Merck Millipore), and 5 ng/mL activin A for 4 days.
Stage 5
Cells were treated with iMEM with 1% P/S, 0.5 × B27, 1% Pluronic® F-68, 0.25 μM SANT-1 (Merck), 50 nM retinoic acid (Merck), 10 μM ALK5 inhibitor II (Santa Cruz Biotechnology), 100 nM LDN-193189 (MedChemExpress), 1 μM L-3,3ʹ,5-triiodothyronine (T3, Merck Millipore), 50 ng/ml basic fibroblast growth factor (bFGF, PeproTech), 1 μM XAV939 (Merck Millipore), and 10 μM Y-27632 for 2 days.
Stage 6
Cells were cultured with iMEM containing 1% P/S, 0.5 × B27, 1% Pluronic® F-68, 1 μM γ-secretase inhibitor, RO4929097 (“GSI” in Supplementary Fig. 1, Chem Scene), 10 μM ALK5 inhibitor II, 100 nM LDN-193189, and 1 μM T3 for 4 days. To generate s6-iPIC-B, cells were treated with the same medium for another 7 days. In the case of s6-iPIC-A and iPIC, 1 μM PD-166866 (Merck Millipore) was added starting on the fourth day. In Fig. 4c–h, 3 μM GSK 461364 (Cayman), CFI-400945 (Apexbio), 1 μM TR06141363 (Takeda original multi-kinase inhibitor), and 2-deoxy-D-glucose (2-DG) were added as alternatives to PD-166866 in the s6-iPIC-A protocol. To generate iPIC, cells were dissociated on the seventh day at stage 6, seeded into Elplasia multi-well plates (4440, Corning) and cultured for the remaining 4 days in Stage 7 medium.
Stage 7
Stage 7 medium is based on Rezania et al. with some modifications. Cells were exposed to MCDB 131 medium with 1% P/S, 2% fat-free BSA (FUJIFILM Wako), 14.44 mM glucose (to generate a final concentration of 20 mM), 1.5 g/L NaHCO3, 1% GlutaMAX, 0.5% ITS-X (Thermo Fisher Scientific), 10 μM ALK5 inhibitor II, 1 μM T3, 10 μM ZnSO4 (Merck Millipore), 1.4 IU/ml heparin sodium salt (Nacalai Tesque), 1 mM N-acetyl cysteine (Merck Millipore), 10 μM Trolox (FUJIFILM Wako), 2 μM R428 (Selleck), 1 μM PD-166866, 3 μM TR06141363, and 10 μM Y-27632, for 4 days.
Flow cytometry
Differentiation efficacy and quality at individual stages based on the developmental markers were analyzed with immunostaining methods and LSRFortessa X20 flow cytometry equipment (BD), as described previously10. Data were processed with FlowJo software. The primary antibodies are listed in Supplementary Table 1. Secondary antibodies of the appropriate species were conjugated to AlexaFluor 488, 546, 568 and 647 of appropriate species (Thermo Fisher Scientific or Jackson).
Type 1 diabetes mouse model
NOD.CB17-Prkdc-scid/J (NOD-scid) mice were obtained from Charles River. Male mice between the ages of 8 and 9 weeks were intraperitoneally injected with multiple low doses of streptozotocin (STZ, 50 mg/kg/day for 5 days, Sigma). Mice that became hyperglycemic within 2–3 weeks after STZ injection were subjected to implantation experiments as a type 1 diabetes mouse model (STZ-NOD-scid mice). All animal studies were conducted at Shonan iPark, one of the AAALAC international accreditation facilities, and approved by the iPark institutional animal care and use committee. All experiments were performed in accordance with the relevant guidelines and regulations, including the ARRIVE guidelines.
Implantation and in vivo assessment
Differentiated iPIC aggregates were mixed with 100 μL of fibrinogen/50 μL of thrombin solution, incubated at 37 °C for 5 min and then implanted in the subcutaneous space of anaesthetized STZ-NOD-scid mice (3–4 × 106 cells/mouse). Fibrinogen from human plasma (Merck Millipore) and thrombin (Sigma) were reconstituted in iMEM and in PBS to make 10 mg/mL and 50 IU/mL solutions, respectively, and stored at − 80 °C until use. For the kidney capsule implantation study (Supplementary Fig. 7), s6-iPIC-A (1.4 × 106 cells/mouse) or s6-iPIC-B (3.6 × 106 cells/mouse) was implanted directly into the kidney capsule without fibrin gel. We monitored the blood glucose levels of implanted animals using an Accu-Chek Aviva system (Roche DC Japan) and collected plasma samples from the tail vein on the indicated days. For the oral glucose tolerance test, the mice were fasted overnight and orally injected with a 2 g/kg glucose solution (Otsuka), and plasma samples were collected from the tail vein before and 15, 30, 60 and 120 min after injection.
Plasma glucose and hormone measurements
Plasma glucose, human C-peptide and mouse C-peptide levels were measured using the glucose test C-II Wako (Fujifilm Wako), Mercodia Ultrasensitive C-peptide ELISA (Mercodia) and mouse C-peptide measurement kit (Morinaga) according to the manufacturer’s instructions.
Tissue processing and immunostaining
Implanted grafts were collected and fixed with 4% paraformaldehyde (FUJIFILM Wako) for over 24 h at 4 °C and embedded in paraffin or frozen in OCT compound. Paraffin blocks were sectioned at 5 μm and used for hematoxylin and eosin staining, Masson trichrome staining and immunostaining. Frozen blocks were sectioned at 10 μm and used for immunofluorescence staining. The primary antibodies are listed in Supplementary Table 1. Secondary antibodies were conjugated to AlexaFluor 488, 546 or 568 (Thermo Fisher Scientific or Jackson). Frozen sections were also counterstained with Hoechst (Thermo Fisher Scientific) to label the nucleus.
Single-cell RNA sequencing library preparation, sequencing and data processing
A total of 6 samples (1 sample of iPIC, 3 samples of s6-iPIC-A, 1 sample of s6-iPIC-B, and 1 sample of reference human islet) underwent scRNA-seq. Human islets were purchased from PLODO. All 5 samples of three versions of iPICs were prepared from Ff-I14s04 line according to the inducers of Supplementary Fig. 1. iPICs were cultured from stage 1 with 3D stirred-floating culture with shear stress using bio-reactor (Biott). As an exception, only 1 of 3 samples of s6-iPIC-A was cultured in 2D monolayer from stages 1 to 4, and then in static aggregate culture without shear stress using non-adhesive V bottom 96 well plate (SUMITOMO BAKELITE) in accordance with our methods10. Single-cell RNA-seq libraries were generated using the 10 × Genomics Chromium™ controller and Chromium Single Cell 3′ kits v2 (10 × Genomics) according to the manufacturer’s instructions. Successful cDNA amplification and library construction were ensured with High Sensitivity DNA kits on an Agilent 2100 Bioanalyzer (Agilent). The obtained libraries were sequenced using Hi-seq (Illumina) with 150 bp paired-end reads at a depth of > 100,000 reads per cell. Sequencing reads were aligned to the human GRCh38 genome reference, and gene counts were quantified as UMIs using Cell Ranger v2.0.1 (10 × Genomics). We imported UMI count matrices into the R v3.3.1 software Seurat v2.0.1 package46,47, where normalization was performed according to the package’s default setting. Cells with mitochondrial gene counts over 10% were regarded as dead or damaged cells and removed for further analyses. UMI count matrices were scaled by regressing out the number of total UMI counts per cell and the percentage of mitochondrial gene counts. Genes for dimensional reduction were selected by the average expression and dispersion of each gene, and principal component analysis was performed. Principal components were used for Seurat’s shared nearest neighbor graph clustering and t-distributed stochastic neighbor embedding (t-SNE) dimensional reduction to create a visualization of data. The cell cycle was evaluated and scored using the expression of genes known as S-phase, G1, and G2M markers. To estimate cell types and similarities within iPICs with reference to human organs, we performed reference component analysis (RCA) using the RCA v1.0.0 package31. Differential gene expression analysis of each cluster compared with the others was performed using the likelihood-ratio test for single-cell gene expression in Seurat. For trajectory analysis, processed UMI count matrices were imported to a single-cell dataset for the Monocle v2.6.3 package28,29,30. We selected the genes for ordering cells with ‘dpFeature’ in monocle and contracted the single-cell trajectories via the ‘DDRTree’ algorithm.
Measurement of inhibitory effects on FGFR and PLK isoforms
The percent (%) inhibition of FGFR and PLK isoforms by PD-166866, TR06141363, GSK 461364, and CFI-400945 was evaluated with a time-resolved fluorescence resonance energy transfer (TR-FRET)-based competitive binding assay36, performed using 1536-well, white, flat-bottomed plates (Greiner Bio-One). Assay buffer comprised 50 mmol/L HEPES, 10 mmol/L MgCl2, 1 mmol/L EGTA, 0.1 mmol/L DTT, and 0.01% Brij-35. Test compounds (1 and 0.1 µmol/L) were mixed with recombinant kinase proteins with an epitope tag, a terbium-labelled anti GST-tag antibody, and fluorescent labelled ligands. After 1 h incubation at room temperature, TR-FRET signals were measured with EnVision (PerkinElmer). DMSO and 5 µmol/L control inhibitor (Staurosporine or other multi kinase inhibitors) were used as 0 and 100% controls, respectively. The percent (%) inhibition was calculated based on the signals of the 0% and 100% inhibition samples in the absence and presence of control inhibitors, respectively.
Analysis of mRNA expression by quantitative real-time PCR
After iPIC differentiation, cDNA samples were synthesized from lysates using TaqMan Gene Expression Cells-to-Ct kits (Thermo Fisher Scientific) according to the manufacturer’s instructions, followed by quantitative real-time polymerase chain reaction analysis using a Prism 7900HT sequence detector (Thermo Fisher Scientific). The thermal cycling parameters were 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The mRNA levels were analyzed with the comparative Ct method (2-ΔΔCt) using RPLP0 as the housekeeping gene. The TaqMan Gene Expression assays (Thermo Fisher Scientific) used herein were as follows: Hs00170586_m1 (FGB), Hs00356521_m1 (AGR2), and Hs00420895_gH (RPLP0).
Statistical analysis
Dunnett’s multiple-comparison test was performed at a significance level of P < 0.05 to determine statistical significance in Figs. 3c,d, 4c,e. Additionally, the Aspin-Welch test or Student’s t-test was performed at a significance level of P < 0.05 to determine statistical significance between two groups in Fig. 3c,d. The dose–response relationships in Fig. 4f,h were tested using Williams or Shirley-Williams tests with a one-tailed significance level of P < 0.025 based on the results of the homogeneity of variance test (Bartlett’s test). All statistical analyses were performed using Statistical Analysis System version 9.3 (SAS Institute, NC, USA).
