Accutase

The expression of CD73, CD90, CD105, CD34, CD45, and CD11b in GMSCs and CD11b, CD206, and CD86 in PBMC-derived macrophages were analyzed using the FACS Calibur (Becton Dickinson, USA) and the CellQuest software (Becton Dickinson). The adherent cells were washed with PBS and collected using Accutase (Nacalai Tesque) and resuspended in 50 μL staining buffer (BD Pharmingen, country). The harvested cells were blocked with 2 μL Human Trustain FcX (Fc receptor Blocking Solution; BioLegend) for 10 min at room temperature, and stained with 2 μL antibodies namely, FITC anti-human CD73, FITC anti-human CD90, Alexa Fluor® 488 anti-human CD105, FITC anti-human CD34, PE anti-human CD45, FITC anti-human CD11b, Alexa Fluor® 488 anti-human CD11b, PE anti-human CD206, APC anti-human CD206, PE anti-human CD86 and APC anti-human CD86 (BioLegend) in the dark for 30 min at 4 °C.

The CD45-negative cells used as cardiac myofibroblasts and cultured for 2 days on plastic plates were disassociated from plastic plates via accutase (Nacalai Tesque) treatment. The cells were fixed with 1% paraformaldehyde (PFA; Nacalai Tesque) in PBS for 10 min and were subsequently permeabilized with 0.5% saponin for 10 min. After the permeabilization, the cells were stained with Alexa488-conjugated α-SMA (1:200; Invitrogen; 53–9760–82) antibody and analyzed using FACSAria (BD Biosciences).

EL4 and Jurkat cells were harvested and washed twice with fluorescence-activated cell sorting (FACS) solution (2% FBS and 2 mM EDTA in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄)). Tumor-infiltrating cells, which were contained in the MC-38 cell mass, were collected from euthanized mice, along with MC-38 cells. Tumor cell masses were washed with Hanks’ Balanced Salt Solution (HBSS, Thermo Fisher Scientific) and chopped with sterile scissors in Accutase (Nacalai Tesque, Kyoto, Japan) to ensure a size no larger than 3 mm in diameter. All the chopped cell clumps were collected in plastic tubes, and Accutase was added to make a total of 5 ml/g of cell clumps. The tubes were agitated at 37°C for 30 min at 60 rotations per min. Then, the cells were collected through a 70 μm filter. The numbers of cells were counted and adjusted to a density of 1 × 10⁷/ml with FACS solution (PBS containing 2% FBS and 2 mM EDTA). After incubation with Fc blocker, cells were immunostained with antibodies shown in Supplementary Tables S3 and S4. After immunostaining, cells were washed and resuspended in FACS solution with 1/1000 of SYTOX Red dead cell stain (Thermo Fisher Scientific). Cells were analyzed using FACSAria IIIu (Becton, Dickinson and Company, NJ).

S516 was used for scRNA-seq. At day 80 of the myogenic differentiation, single cells were acquired upon incubation for 1 h with DMEM medium with Collagenase G (500 μg/ml) and H (500 μg/ml) (Meiji), followed by 10 min with Accutase (Nacalai) at 37°C. Then the cells were carefully detached by pipetting, filtered with a 50-nm mesh and washed twice with 1% BSA in HBSS (Gibco). The cells were resuspended in 1% BSA in HBSS to reach a concentration of 1,000 cells/μl. The cDNA library was prepared using the Next GEM Single Cell 3′ Gel Bead Kit v3.1 (1000129), Chromium Next GEM Chip G Single Cell Kit v3 (PN-1000127), Next GEM Single Cell 3′ GME Kit v3.1 (1000130), Next GEM Single Cell 3′ Library Kit v3.1 (1000158), and i7 Multiplex Kit (PN-120262) (10x Genomics) according to the 10x Genomics instructions. Then the cDNA library was run on an Illumina NextSeq 500 and HiSeq 4000.

All animal experiments were conducted at the Central Institute for Experimental Animals (CIEA). PLR327F-LD41 cells or MCC001F cells were dissociated with Accutase (Nacalai Tesque), suspended in StemPro hESC SFM medium with 50% Matrigel (Cat# 354234, Corning), and 1 × 10⁵ cells/100 µL were inoculated subcutaneously to both flanks of a female NOG mouse (CLEA Japan). Xenograft tumours were collected 35 or 48 days after injection of PLR327F-LD41 or MCC001F cells, respectively. Mice were euthanised by exsanguination under isoflurane anesthesia. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at CIEA and Forerunner Pharma Research Co., Ltd., and carried out in accordance with the relevant guidelines and regulations. The study was carried out in accordance with the ARRIVE guidelines.

Tet-mCherry or Tet-MyoD hiPSCs were cultured on Easy iMatrix-511 silk-coated plates (#892024, Nippi) in StemFit medium (AK02N, Ajinomoto) containing 100 μg/mL G418 (#938044, NacalaiTesque) or 0.5 μg/mL puromycin dihydrochloride (160-23151, Wako Chemicals). Cells were passaged every 7 days using Accutase (#12679-54, NacalaiTesque) and seeded on Easy iMatrix-511 silk-coated 6-well plates in the presence of 10 μM Y-27632 (NacalaiTesque) at a density of 1.5×104 cells/well for the first 2 days after plating. At 48 h after passaging, Y-27632 was removed and replaced with StemFit medium containing the appropriate antibiotic.

The expression of surface proteins within the collected cells was determined by using flow cytometry (FC). Cells were dissociated with Accutase (Nacalai Tesque) and stained with CD24 (1555427; BD Biosciences), CD44 (103012; BioLegend, 338820; BioLegend), CD44v5 (L MCA1729; Bio-Rad), CD44v6 (MCA1730; Bio-Rad), CD44v7 (MCA1731; Bio-Rad), CD44v9 (LKG-M003; Cosmo Bio), CD133 (372808; BioLegend), and 7-AAD (372808; BD Biosciences). The relative fluorescence intensities were measured by using an SH800 cell sorter (Sony Corporation, Tokyo, Japan). A two-dimensionality reduction step was performed using t-distributed stochastic neighbor embedding (t-SNE) to visualize high-dimensional cell surface marker expression data in a low-dimensional space. Data were analyzed by using the FlowJo software, Version 10.2 (FlowJo).