Cell culture and in vitro differentiation of hiPSCs
The human iPSC line Ff-I14-s04 was kindly provided by the Center for iPS Cell Research and Application (CiRA) at the Kyoto University. The Ff-I14s04 line was established as an HLA homozygous iPSC line in a previous study [19]. The use of the iPSC line was approved by the Ethical Review Committee of the Shonan Health Innovation Park (Fujisawa, Kanagawa, Japan) and Kyoto University (#CiRA18-27). Cells were cultured and maintained on dishes coated by iMatrix-511 (Nippi, Tokyo, Japan) in StemFit AK03N (Ajinomoto, Tokyo, Japan) 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, Waltham, MA, USA), and subjected to differentiation experiments usually after over a 2-week running culture.
For differentiation culture to generate iPICs, we used 2D monolayer to static aggregate culture based on our previous report [15] and 3D stirred-floating aggregate culture [16]. The details of the typical 3D floating culture were as follows. A special note here is that 10 μM ALK5 inhibitor II (ALK5iII, Santa Cruz) at Stages 5–7 is replaced with ALK5 inhibitors, CDK8/19 inhibitors, and their combination, as alternatives to ALK5iII, depending on the purposes of Figs. 2, 3, 4 and 5.
Stage 1
Dissociated undifferentiated iPSCs were resuspended at a density of 2 × 105 cells/mL in the AK03N medium containing 10 μM Y-27632 (FUJIFILM Wako, Tokyo, Japan). The cells were cultured in a spinner type 30 mL bioreactor (ABLE Corporation & Biott Co., Ltd., Tokyo, Japan), or a vertical mixing 0.25 L bioreactor (SATAKE MultiMix Corporation, Saitama, Japan) throughout culturing. The next day, the aggregated cells were cultured in DMEM medium (Thermo Fisher Scientific) supplemented with 1% (v/v) penicillin/streptomycin (P/S, FUJIFILM Wako), 1× B27 (#17504001 or A1895601; Thermo Fisher Scientific), 1% Pluronic® F-68 (Sigma-Aldrich Co. LLC, Saint Louis, MO, USA) to reduce the fluid-induced mechanical damage, 5–10 ng/mL activin A (PeproTech, Cranbury, NJ, USA), 3 μM CHIR99021 (Axon Medchem, Reston, VA, USA), and 1% DMSO (FUJIFILM Wako). The following day, CHIR99021 was removed from the medium, and the culture was continued for another 2 days.
Stage 2
Cells were cultured in the MCDB 131 Medium (Thermo Fisher Scientific) supplemented with 1% P/S, 0.5× B27, 1% Pluronic® F-68, 50 ng/mL keratinocyte growth factor (KGF, R&D Systems, Minneapolis, MN, USA), 4.44 mM glucose (final concentration 10 mM, FUJIFILM Wako), 1.5 g/L NaHCO3 (FUJIFILM Wako), and 1% GlutaMAX (Thermo Fisher Scientific) for 4 days.
Stage 3
Cells were cultured in the 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, Toronto, Canada), 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, CA, USA) for 3 days.
Stage 4
Cells were cultured in 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, Vancouver, Canada), 0.1 μM TR05991851 (ROCK inhibitor, Takeda Pharmaceutical Company, Osaka, Japan), 0.5 μM PDBu (Merck Millipore, Billerica, MA, USA), and 5 ng/mL activin A for 4 days.
In the following stages 5–7, we used 10 μM ALK5iII and their alternatives (ALK5 inhibitors, CDK8/19 inhibitors, and their combination such as 3 μM SB 431542 and 0.3 μM senexin B, depending on the experimental purposes as mentioned above.
Stage 5
Cells were cultured in iMEM with 1% P/S, 0.5× B27, 1% Pluronic® F-68, 0.25 μM SANT-1 (Merck Millipore), 50 nM retinoic acid (Merck Millipore), 10 μM ALK5 inhibitor II (ALK5iII, Santa Cruz) or the alternative candidates, 100 nM LDN-193189 (MedChemExpress, Monmouth Junction, NJ, USA), 1 μM L-3,3′,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 in iMEM containing 1% P/S, 0.5× B27, 1% Pluronic® F-68, 1 μM RO4929097 (“GSI” in Fig. 1a, Chem Scene, Monmouth Junction, NJ, USA), 10 μM ALK5iII or the alternative candidates, 100 nM LDN-193189, and 1 μM T3 for 7 days. PD-166866 (1 μM, Merck Millipore) was added from day 4 of stage 6.
Stage 7
Stage 7 medium was based on a previous report [7] with our original modifications. Cells were cultured in the MCDB 131 medium with 1% P/S, 2% fat-free bovine serum albumin (FUJIFILM Wako), 20 mM glucose, 1.5 g/L NaHCO3, 1% GlutaMAX, 0.5% ITS-X (Thermo Fisher Scientific), 10 μM ALK5iII or the alternative candidates, 1 μM T3, 10 μM ZnSO4 (Merck Millipore), 1.4 IU/mL heparin sodium salt (Nacalai Tesque, Kyoto, Japan), 1 mM N-acetyl cysteine (Merck Millipore), 10 μM Trolox (FUJIFILM Wako), 2 μM R428 (Selleck Chemicals, Houston, TX, USA), 1 μM PD-166866, 3 μM TR06141363 (Multi-kinase inhibitor, Takeda Pharmaceutical Company), and 10 μM Y-27632, for 4 days. To generate iPICs for implantation, cells were dissociated and re-sized in an Elplasia 6-well microwell plate (Corning Incorporated, Corning, NY, USA) or a gas-permeable microwell culture bag (Toyo Seikan Group Holdings, Ltd., Yokohama, Japan) [20] and cultured in the stage 7 medium.
Ames mutagenicity assay
The Ames assay was performed to assess the mutagenicity of five compounds that displayed positive structural alerts in studies in silico (Fig. 1b). Salmonella typhimurium TA100, TA1535, TA98, and TA1537, and Escherichia coli WP2uvrA were used to detect either frameshift mutations (TA98 and TA1537) or base pair substitutions (TA100, TA1535, and WP2uvrA). These strains are widely used and recommended for use in ICH S2(R1) for the bacterial reversion assay.
In the current study, rat liver S9 fraction, an exogenous activator of post-mitochondrial supernatant, was not included because the mutagenicity of compounds should be assessed when simulating cell cultures but not in vivo after metabolism by liver enzymes. Briefly, each tester strain was mixed with seven concentrations (78.1–5000 μg/plate) of each compound and preincubated for 20 min in test tubes. Two plates per dose for the tested compounds and positive control group, and four plates per dose for the negative control group were used. Dimethyl sulfoxide (FUJIFILM Wako) was selected as solvent for the tested compounds and used as negative control. Strong mutagens served as positive controls: 9-aminoacridine hydrochloride monohydrate (9-AA, Sigma-Aldrich), and [6-Chloro-9-(3-[2-chloroethylamino]propylamino)-2-methoxyacridine] dihydrochloride (ICR 191, FUJIFILM Wako). Following preincubation, semisolid top agar was added to the tubes, and then, the mixtures were overlaid on minimal glucose agar plates. After the overlaid agar solidified, the plates were stored at 37 °C in an incubator for 48 h.
Cultures were examined for signs of compound precipitation and other abnormal conditions by eye. Subsequently, cytotoxicity (decreased background lawn) was assessed using a stereomicroscope. The number of revertant colonies on the plates was counted using an automatic colony analyzer (CA-11, System Science Co., Ltd., Tokyo, Japan) or manually when counting by the automatic colony analyzer was inappropriate, e.g., when precipitation was observed. A positive response was defined as the mean number of revertant colonies that was at least twofold greater than the mean negative control value in any test strain.
Flow cytometry analysis
The differentiation efficiency of β-cells (INS+NKX6.1+) and endocrine cells (CHGA+) was analyzed using immunostaining methods and flow cytometry on an LSRFortessa X20 instrument (BD Corporation, Franklin Lakes, NJ, USA), as described previously [5]. The primary antibodies used are detailed in Additional file 1: Table S1. Secondary antibodies were conjugated to Alexa Fluor 488, 546, 568, and 647 of the appropriate species (Thermo Fisher Scientific or Jackson ImmunoResearch, West Grove, PA, USA).
Study of iPIC implantation in type 1 diabetic mice
Male immunodeficient NOD.CB17-Prkdcscid/J (NOD-scid) mice were purchased from Charles River Laboratories Japan (Yokohama, Japan). They were fed commercial diet CE-2 (CLEA Japan, Tokyo, Japan) and received tap water ad libitum, together with appropriate weekly sanitation and enrichment. The care and use of the animals as well as the experimental protocols in this study were approved by the Institutional Animal Care and Use Committee of the Shonan Health Innovation Park (Shonan iPark), Takeda Pharmaceutical Company. Animals were excluded if monitored health condition was severe in many aspects like weight, intake, activity, fur condition, and so on. For euthanasia of diabetic mice, for example, we use 50% CO2 at a flow rate of 5.0 L/min to 9.0 L M-2 chamber (CLEA Japan, Tokyo, Japan) for over minimally 10 min. We can increase the flow rates once animals have lost consciousness.
The experimental unit is the individual animal (i.e., an implanted mouse) in this study. Male mice aged 8–9 weeks were intraperitoneally injected with five daily injections of streptozotocin (STZ, 50 mg/kg/day, Sigma-Aldrich) to induce type 1 diabetes and transferred to single housing to reduce fight, flight, freeze, or groom stress response that affect glucose levels. The hyperglycemic mice (n = 4–5 per group, total 14 mice for Fig. 5) were randomized based on blood glucose and body weight and were subcutaneously implanted with iPICs (3 × 106 cells/mouse, implanted by a well-trained operator using a standardized protocol) embedded in fibrin gel obtained by mixing 10 mg/mL fibrinogen (Merck Millipore) and 50 IU/mL thrombin solutions (Sigma-Aldrich). Non-fasted blood glucose and plasma human C-peptide levels were measured every 2 or 4 weeks using ACCU-CHEK Aviva (Roche Diagnostics, Basel, Switzerland) and Ultrasensitive human C-peptide ELISA (Mercodia AB, Uppsala, Sweden), respectively, according to the manufacturers’ instructions. An oral glucose tolerance test was conducted 21 weeks after iPIC implantation. The plasma samples were obtained before and after the glucose loading (2 g/kg) at the indicated time points for the measurements of plasma glucose (Glucose autokit, 439–90901, FUJIFILM Wako) and human C-peptide levels. These measurements are generally conducted in type 1 diabetes pre-clinical studies [5,6,7,8,9,10].
Immunostaining
For in vivo graft samples, the mice were dissected to obtain iPIC grafts for histological analysis over 6 months after the implantation. Paraformaldehyde (4%)-fixed grafts were dehydrated and embedded in paraffin. For in vitro aggregate samples, aggregates were frozen in Tissue-Tek O.C.T. compound (Sakura Finetek Japan, Tokyo, Japan) in a similar manner. Paraffin and frozen blocks were sectioned at 5–10 μm thickness, and they were used for immunofluorescence staining. After treatment with blocking solution (5% normal donkey serum/0.3% Triton X), the primary (Additional file 1: Table S1) and secondary antibodies (conjugated to Alexa Fluor 488 and 546 (Thermo Fisher Scientific)) were applied with appropriate wash steps. The sections were counterstained with Hoechst 33342 (1:200, Thermo Fisher Scientific) to label the nuclei. The sections were imaged using a fluorescent microscope (BZ-X700; Keyence, Osaka, Japan).
TR-FRET-based kinase profiling assays for tested compounds
TR-FRET-based competitive binding assays for ~ 350 kinases have been developed and conducted by Axcelead Drug Discovery Partners (Fujisawa, Japan) [21]. Briefly, BODIPY-FL- or Cy5-FL-conjugated staurosporine derivative that binds to a highly conserved ATP-binding pocket in kinases was used as a fluorescent probe and tested compounds were applied at 0.1 and 1 μM in competitive binding assays for ~ 350 recombinant protein kinases. TR-FRET fluorescent signals from terbium and BODIPY/Cy5-FL were measured using an EnVision plate reader (PerkinElmer, Waltham, MA, USA), and pIC50 values were extrapolated from the inhibition rate.
Single-cell RNA-sequencing and data processing
Single-cell RNA-seq libraries for iPICs were generated using the 10 × Genomics Chromium™ controller and Chromium Single Cell 3′ kits v3.1 (10 × Genomics, Pleasanton, CA, USA) according to the manufacturer’s instructions. Quality control and quantification of the obtained cDNA and library were conducted using high sensitivity DNA kits on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The libraries were subjected to next-generation sequencing using the HiSeq platform (Illumina, San Diego, CA, USA) 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 v5.0.1 (10 × Genomics). We imported UMI count matrices into R v3.6.3 software (Seurat v3.2.3 package) [22, 23] and performed normalization according to the package’s default setting. Cells with a percentage of mitochondrial gene counts over 20% were regarded as dead or damaged cells and removed from further analyses. UMI count matrices were scaled by regressing out the number of total UMI counts per cell and percentage of mitochondrial gene counts. Genes for dimensional reduction were selected based on 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 uniform manifold approximation and projection dimensional reduction [24, 25] were used to visualize the data. Analysis of DEGs in each cluster was performed using the Wilcoxon rank-sum test in Seurat. Subsets of DEGs with fold change > 2 at P < 0.05 were extracted and applied to the publicly available database, PanglaoDB (https://panglaodb.se/) to identify cell-type labels using clusterProfiler v3.14.3 package [26].
Statistical analysis
The Dunnett’s multiple comparison test was performed at a significance level of P < 0.05 to analyze the statistical significance of effects in iPIC differentiation studies. All statistical analyses, except for the Wilcoxon rank-sum test, were performed using Statistical Analysis System v9.3 (SAS Institute, Cary, NC, USA). The Wilcoxon rank-sum test in Additional file 1: Fig. 3 was performed using Seurat v3.2.3 package, as described above.