Cell culture

Human iPSCs were maintained on Matrigel-coated plates (Corning, Corning, New York, USA) in NutriStem XF medium (Sartorius, Göttingen, Germany) and sub-passaged every 2–3 days using Versene solution (Gibco, Waltham, MA, USA). Human leukemia-derived cells used in this study, including KG-1, U937 and K562 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultivated in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), L-glutamine and antibiotics (Gibco) in CO2 humidified atmosphere at 37 °C. Cells that were positive for mycoplasma contamination, as evaluated by MycoAlert PLUS Mycoplasma Detection Kit (Lonza, Basel, Switzerland), were discarded.

Viral production and transduction

For CAR-TIM3 transduction, the third-generation, lentiviral CAR construct (Creative Biolabs, Shirley, NY, USA), schematically depicted in Fig. 1a (upper), were inoculated in HEK293FT cells (Thermo Fisher Scientific, Waltham, MA, USA) in conjunction with VSV-G and dR8.2 plasmids (Addgene, #8454 and #8455) at a ratio of 10:1:4 using Lipofectamine 3000 (Thermo Fisher Scientific). Parental, single-cell clone iPS line, MUSIi-013-A, were dissociated into a single cell suspension using Accutase (Stemcell Technologies, Vancouver, BC, Canada) before seeding onto Matrigel-coated plate for 24 h, and transduced with concentrated lentiviral particles. Then, a single-cell clone dilution of transduced iPSCs was performed in a 96-well plate pre-seeding with irradiated human foreskin fibroblasts in the presence of SMC4 consisting PD0325901 (Sigma Aldrich, St. Louis, MO, USA), CHIR99021, Thiazovivin, and SB431542 (Stemcell Technologies). Emerging colonies were picked up for further iPSC characterization.

TIM3 overexpression in U937 cells was performed using pQCXIX-Myc-hTIM3 retroviral plasmid (Addgene, #110893), in which retroviral particles were produced in Plat-A packaging cells (Cell Biolabs, San Diego, CA). At 3-day post-transduction, U937 cells were expanded and sorted for TIM3-positive cells using FACS Aria Fusion cell sorter (BD Biosciences, San Jose, CA, USA).

Genomic DNA sequencing

Genomic DNA was isolated using a PureLink Genomic DNA Mini Kit (Invitrogen, Waltham, MA, USA). The target regions for DNA sequencing were amplified by PCR using Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) with specific primers (Fig. 1a, lower), and the resulting PCR products were purified by a GenepHlow Gel/PCR kit (Geneaid, New Taipei City, Taiwan). A total of 0.2 μg PCR product was then used for DNA sequencing using ABI PRISM BigDye™ Terminator Cycle Sequencing Kit v3.1 (1st BASE, Singapore).

Evaluation of CAR-TIM3 expression

CAR-TIM3 expression in iPSCs was determined by flow cytometry based on Fab fragments. Cells were incubated with FITC-conjugated anti-mouse-IgG, F(ab′)2 fragment antibody (F(ab′)2-FITC; Jackson ImmunoResearch, West Grove, PA, USA) for 30 min at 4 °C and analyzed using a BD FACS Canto (BD Biosciences). Western blotting of CD3ζ was used to confirm the successful introduction of CAR-TIM3.

CAR-TIM3 expression in the differentiated cells was evaluated by target antigen-based detection to ascertain its binding activity. Briefly, cells were incubated with 10 μg/mL rhTIM3 (23–200) protein with His tag to the C-terminus (Abcam, Cambridge, UK) for 1 h at 4 °C, followed by an incubation with FITC-conjugated anti-His tag antibody (His tag-FITC; Abcam) for 15 min at room temperature and flow cytometric analysis. Cells that were incubated with His tag-FITC, but not with rhTIM3, were used as a basal control. The percentage of cells that expressed CAR-TIM3 could be calculated from the subtraction of FITC-positive cells in the basal control from those with the target protein.

Immunofluorescence staining

Cells were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100/PBS for 10 min, and blocked with 3% bovine serum albumin (BSA)/PBS for 1 h. The cells were incubated with primary antibodies in 1% BSA/PBS overnight at 4 °C, followed by secondary antibodies at room temperature for 1 h. Nuclei were counterstained with Hoechst 33,342 (Thermo Fisher Scientific) for 30 min at room temperature and visualized under fluorescence microscope (Eclipse Ti-U, Nikon, Tokyo, Japan) with NIS-Elements D Software (version 4.30.00; Nikon).

Flow cytometry

For analysis of pluripotency markers, iPSCs were dissociated into single cells using TrypLE™ Select (Gibco), blocked with 10% human AB serum, stained with FITC-SSEA-3, PE-SSEA-4, Alexa Fluor 647-TRA-1-60, and PE-TRA-1-81 (BioLegend, San Diego, CA, USA), and analyzed by BD FACS Canto flow cytometer (BD Biosciences) with FlowJo software (V10.4.1).

For phenotypic analysis of HSPC induction and NK commitment, the cells were resuspended in FACS buffer (PBS with 2% BSA), incubated with antibody cocktail for 30 min at 4 °C in the dark, and analyzed by BD FACS Canto. The antibodies used in this study included PE-CD56, PerCP-CD45, APC-CD34 (BD Biosciences), FITC-CD16, PE/Cy7-CD43, FITC-Nkp46, PE-Nkp44, PerCP-CD94, APC-TIM3, PE/Cy7-KIR, Alexa Fluor 647-CD161 and BV605-Nkp30 (BioLegend).

qPCR analysis

Total RNA was isolated using TRI Reagent® (Molecular Research Center, Cincinnati, Ohio, USA) and converted to complementary DNA using the RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific). The qPCR reactions were performed on the CFX384 Touch Real-Time PCR detection system (Bio-Rad, Hercules, CA, USA) using SYBR™ Select Master Mix (Thermo Fisher Scientific). The cycle parameters started with an activation step at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min.

Karyotyping

The standard G-banded karyotyping was performed at the Department of Obstetrics and Gynecology, Faculty of Medicine Siriraj Hospital, Mahidol University. A total of 25 metaphases at a band resolution of 400–450 were analyzed.

STR analysis

STR analysis was performed at the Department of Forensic Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University. A total of 16 loci were tested.

Teratoma formation

The iPSCs were treated with 10 μM of Y-27632 for 1 h prior to harvesting. Cells were resuspended at 5 × 106 cells/100 μL of cold 30% (v/v) Matrigel® Matrix (Corning) in NutriStem medium, and then implanted intramuscularly into 4-week-old female SCID mice (Nomura Siam International Co., Ltd, Thailand). At 4 weeks after transplantation, the mice were sacrificed and the teratomas were removed and fixed in 10% formalin overnight before being embedded in paraffin wax. Samples were sectioned and examined by hematoxylin and eosin (H&E) staining. All animal experiments were approved by the Siriraj Animal Care and Use Committee (Si-ACUC) of the Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand (COA no. Si-ACUP 009/2561).

Spontaneous in vitro differentiation via EB formation

The iPSCs were harvested into small clumps using 1 mg/mL Dispase (Gibco) and cultured on low attachment dishes in knockout DMEM supplemented with 20% knockout serum replacement, 2 mM GlutaMAX™, 0.1 mM MEM non-essential amino acid, 0.1 mM β-mercaptoethanol, 1 × insulin-transferrin-selenium-ethanolamine, and 100 U/mL penicillin/streptomycin (Gibco). The medium was replaced every other day. On day 7, EBs were transferred onto a 0.1% gelatin-coated plate and cultured at 37 °C and 5% CO2 for another 3 weeks.

NK cell differentiation via spin EB formation

iPSCs were pre-treated with Rho kinase inhibitor (ROCKi, Stem Cell Technologies, Canada) for 1 h before single cell dissociation by Accutase. We have slightly modified the differentiation protocol from previous work by Lupo et al. [29]. Briefly, 5000 single cells were used to form EBs in a low-attachment round-bottom 96-well plate in hematopoietic differentiation medium comprising of 100 μL STEM diff APEL2 medium (Stem Cell Technologies) supplemented with 40 ng/mL SCF, 20 ng/mL each of VEGF and BMP4, and 10 μM ROCKi (for first 3 days). Plates were spin at 250 × g for 5 min to facilitate EB formation and placed in humidified normoxic CO2 incubator. Fresh medium was changed every 3 days by removal of 50 μL old medium and replacement with 100 μL fresh one. After 6 days, EBs were gently transferred onto Matrigel-coated plate in NK cell differentiation medium comprising of STEM Diff APEL2 medium, 20 ng/mL SCF, 20 ng/mL IL-7, 10 ng/mL IL-15, 10 ng/mL Flt-3 ligand, and 5 ng/mL IL-3 (only for first week). All cytokines used were obtained from R&D Systems (Minneapolis, MN, USA).

Cytotoxicity assay of CAR-NK cells against leukemia cells

Leukemia cells as the target cells were pre-stained with fluorescent PKH67 dye (Sigma Aldrich) according to manufacturer’s instructions. NK cells were harvested and pre-stimulated with 500 U/mL IL-2 and 25 ng/mL IL-15 overnight before use. Co-incubation of NK cells and target cells were performed at indicated E:T ratios for 4 h at 37 °C in a total cells of 2 × 104 cells/100 μL in a round-bottom 96-well plate. To determine the tumor killing activity of NK cells, cell mixtures were stained with PE-conjugated annexin-V and 7-AAD (BD Biosciences) in a binding buffer for 15 min at room temperature, and target cell death was evaluated only in PKH67-positive cells using BD FACS Canto (BD Biosciences).

Statistical analysis

Data were represented as mean ± SEM and statistical analysis were performed using GraphPad Prism software (San Jose, CA, USA). The comparison between WT and CAR-TIM3 groups were performed using unpaired t-tests. Differences with p-values less than 0.05 were considered statistically significant: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.