Study population
The Institutional Review Board (IRB) of the Catholic University of Korea, Seoul St. Mary’s Hospital, approved this study (IRB number: KC16TISI0774). We recruited pre-KT patients (n = 20) who had never been treated with anti-diabetes medications and banked their PBMCs for further experiments. Of these, five patients were diagnosed with DM 1 year after KT; we selected matched patients with DM (n = 4) and non-DM individuals (n = 4) on the basis of the clinical index (Tables S1 and S2).
iPSC differentiation
iPSCs from four patients with DM and four non-DM individuals were generated using PBMCs, as previously described [10]. Briefly, the PBMCs obtained from each group were cultured for 4 days) at 37°C in an incubator with 5% CO2 in StemSpan medium (09650; STEMCELL Technologies, Vancouver, Canada), which includes StemSpan CC100 (02690; STEMCELL Technologies), to expand CD34-positive cells. The expanded PBMCs were transfected using the CytoTune-iPS Sendai Reprogramming Kit (A16517; Life Technologies, Carlsbad, CA, USA), which includes the Yamanaka factors (Oct4, Sox2, KLF4, and c-Myc). PBMCs were induced to form iPSCs via centrifugation, and the resultant attached cells were expanded and purified by colony picking.
PP cell differentiation
Human iPSCs were subcultured in dishes coated with Matrigel (354277; Corning Life Sciences, Bedford, MA, USA) at 37°C in an incubator with 5% CO2. Fresh mTeSR1 medium (05850; STEMCELL Technologies), which was replaced once per day, was used as the culture medium. iPSCs were split using trypsin– ethylenediaminetetraacetic acid (TE) (15400054; Life Technologies) at 70% confluence, and 10 µM of a rho-associated kinase (ROCK) inhibitor (1254; TOCRIS Bioscience, Bristol, UK) was added to the newly passaged cells. The STEMdiff pancreatic progenitor kit (05120; STEMCELL Technologies) was used as the culture medium for differentiation into PP cells.
Cell counting kit (CCK)-8 assay
iPSC-derived PP cells were differentiated in 96-well microplates for the CCK-8 assay. After differentiation, the cells were subjected to various Tac treatments for specified durations. CCK-8 solution (CK04-01; Dojindo Molecular Technologies, Kumamoto, Japan) was added to each well for 2 h. Subsequently, absorbance was measured at 450 nm using a VersaMax ELISA Reader (Molecular Devices, Sunnyvale, CA, USA).
Quantitative real-time (qRT)-polymerase chain reaction (PCR)
RNA was extracted from iPSCs or PP cells using RNA-Bee (CD-105B; Tel-Test, Friendswood, TX, USA), as per the manufacturer’s instructions. First-strand cDNA was synthesized and subjected to qRT-PCR performed using SYBR Green Master Mix (DYRT1200; Dyne Bio Inc, Seongnam-si, South Korea) in a LightCycler 480 system (Roche, Basel, Switzerland). Target gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression using the change-in-threshold method. Primer sequences are listed in Table S3.
Flow cytometry
iPSCs or iPSC-derived PP cells were dissociated using TE (15400054; Life Technologies). The cells were washed twice with FACS buffer (phosphate-buffered saline [PBS] containing 1% bovine serum albumin and 10 mM sodium azide), permeabilized for 30 min using flow cytometry fixation and permeabilization solution (554714; BD Biosciences, San Jose, CA, USA), washed with wash buffer, stained with anti-OCT3/4 (60093AD.1; STEMCELL Technologies) and anti-insulin (565689; BD Biosciences) antibodies for 1 h each, and then washed with FACS buffer. Subsequent analysis was performed using a BD LSRFortessa cell analyzer (BD Biosciences). Next, the data obtained were analyzed using the FlowJo V10 Single Cell Analysis Software (TreeStar Inc., OR, USA).
Suspension culture of PP cells
For further maturation of PP cells, suspension culture of PP cells was performed as previously described [11]. The PP cells were treated with 5 mg/mL dispase (07913; STEMCELL Technologies) for 5 min, followed by gentle pipetting to obtain cell clumps (< 100 µm). The cell clusters were transferred into a polystyrene 125 mL Spinner Flask (3152; Corning Life Sciences) and spun at 80–100 rpm overnight in suspension with DMEM-HG (10-017-CV; Corning Life Science) supplemented with 1 µmol/L ALK5 inhibitor II (ALX-270-445-M005; Enzo Life Sciences, Farmingdale, NY, USA), 100 ng/mL Noggin (6057-NG-100; R&D Systems, Minneapolis, MN, USA), and 1% B27 (17504077; Life Technologies).
Immunofluorescence staining
Cell clusters were obtained in 1.5 cc tubes after suspension culture for insulin staining. The cell clusters were then incubated with 4% paraformaldehyde for 15 min at 4’C and washed thrice in PBS in RT (room temperature). Subsequently, they were incubated with 0.1% Triton X-100 for 10 min and with 10% normal donkey serum for 1 h at RT. The primary antibodies, that is, anti-insulin (18–0067; Invitrogen, Camarillo, CA, USA), anti-OCT3/4 (5279; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-SOX2 (365823; Santa Cruz Biotechnology), and anti-SSEA4 (MAB4304; Millipore Sigma, Burlington, MA, USA) antibodies, were incubated at 4°C overnight. On the next day, they were incubated with a secondary Cyanine3 (Cy3; Jackson ImmunoResearch, West Grove, PA, USA)-conjugated antibody for 2 h at RT. Subsequently, they were stained with 4′,6-diamidine-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA) for nucleic acid staining. Images were obtained using a Zeiss LSM700 confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany).
Electron microscopy (EM)
After the iPSCs differentiated into PP cells, the PP cells were fixed in 2.5% glutaraldehyde, 0.1 M phosphate buffer, and 1% OSO4 and then embedded in Epon 812. Ultrathin sections were cut, stained with uranyl acetate/lead citrate, and photographed under a JEM-1200EX transmission electron microscope (JEOL Ltd., Tokyo, Japan). The sections were randomly scanned at 20 different spots per sample at 5000× magnification.
Library preparation and sequencing
For control and test RNAs, library construction was performed using the QuantSeq 3’ mRNA-Seq Library Prep Kit (Lexogen, Inc., Austria), according to the manufacturer’s instructions. In brief, total RNA samples (500 ng each) were prepared, an oligo-dT primer containing an Illumina-compatible sequence at its 5′ end was hybridized to the RNA, and reverse transcription was performed. After RNA template degradation, second-strand synthesis was initiated by a random primer containing an Illumina-compatible linker sequence at its 5′ end. The double-stranded library was purified using magnetic beads to remove all reaction components. The library was amplified to add the complete adapter sequences required for cluster generation. The final library was purified from the PCR components. High-throughput sequencing was performed as single-end 75-bp sequencing using a NextSeq 500 system (Illumina, Inc., USA).
Data analysis
QuantSeq 3 mRNA-Seq reads were aligned using Bowtie2 [12]. Bowtie2 indices were either generated from the genome assembly sequence or the representative transcript sequences for alignment to the genome and transcriptome. The alignment file was used for assembling transcripts, estimating their abundance, and detecting differential gene expression. Differentially expressed genes (DEGs) were identified on the basis of counts from unique and multiple alignments by using coverage in BEDtools [13]. The read count (RC) data were processed on the basis of the TMM + CPM normalization method by utilizing EdgeR within R (R development Core Team, 2020) using Bioconductor [14]. Gene classification was based on searches performed using the DAVID (http://david.abcc.ncifcrf.gov/) and Medline databases (https://www.ncbi.nlm.nih.gov/).
Statistical analyses
Data have been expressed in terms of mean ± standard error (SE) from at least three independent experiments. Multiple comparisons between groups were performed by one-way analysis of variance with the Bonferroni post hoc test using the Prism software (version 7.03 for Windows; GraphPad Software, La Jolla, CA, USA). Statistical significance was set at P < 0.05.