Saponin

iPSC-derived myogenic cells on day 3 were collected and incubated with 0.2% saponin (Nacalai Tesque) and 4% paraformaldehyde (Nacalai Tesque) on ice for 5 min for permeabilization and fixation. Then, the cells were labeled with antibodies against cleaved caspase-3 (1:20; #9602S; CST) or MyoG (1:20; #NBP2-33056; Novus Biologicals). The following isotype controls were used: rabbit IgG Isotype Control (Alexa Fluor 647 Conjugate) (1:20; #3452S; CST) and mouse (MOPC-21) mAb IgG₁ Isotype Control (Alexa Fluor 647 Conjugate) (1:20; #4843S; CST). Antibodies were incubated with cells for 90 min at room temperature. The labeled cells were analyzed with BD FACSAria (BD Biosciences), and the results were analyzed and processed with FlowJo software (Tree Star Inc.).

To investigate the expression of Arg-1 in human monocytes and macrophages, human MNCs from tumor, spleen, and PB of HSC-NOG-hIL-6 Tg mice or HSC-NOG non-Tg mice were fixed in 2% formaldehyde (Nacalai Tesque, Kyoto, Japan) for 15 min at room temperature, and subsequently stained with anti-human CD68-PE-Cy7 (Y1/82A) and anti-human Arg-1-APC (Clone # 658922; R&D Systems) or APC-conjugated mouse IgG2b isotype control (MPC-11; BioLegend) in the presence of 0.5% saponin (Nacalai Tesque) for permeabilization. Stained samples were analyzed using a BD FACSAriaII™ flow cytometer (BD Biosciences).

Human iPSC colonies were dissociated into single cells and cultured on coverslips (24 × 60 mm, Matsunami) for a few days. Cells on the coverslips were washed twice with phosphate-buffered saline (PBS) for 3 min, fixed in 4% paraformaldehyde (PFA) in 0.3 × PBS for 10 min and washed again in PBS. For permeabilisation, cells were treated with 0.5% saponin (Nacalai Tesque) and 0.5% Triton X-100 in PBS for 20 min, incubated in 20% glycerol (Merck Millipore) in PBS for at least 30 min and subjected to repeated freeze–thaw cycles in liquid nitrogen five times. After washing in PBS for 2 min, cells were incubated in 0.1 N HCl for 10 min, washed in PBS for 5 min, treated in 0.02% pepsin in 0.01 N HCl at 37 °C for 4 min and washed with 0.05 M MgCl₂ in PBS for 5 min. Next, cells were postfixed with 1% PFA in PBS for 10 min, and washed in PBS for 5 min and then in 2 × SSC for 5 min. Cells on coverslips were stored at 4 °C in 50% formamide (Wako) in 2 × SSC until hybridisation.

Flow cytometry was conducted in accordance with our previous study with modifications.¹ Differentiated cardiovascular cells and cardiac tissue sheets were dissociated by incubation with Accutase and stained with one or a combination of the following surface markers: anti-PDGFRβ conjugated with phycoerythrin (PE), clone 28d4, 1:100 (BD, Franklin Lakes, NJ, USA) for MCs, and anti-VE-cadherin conjugated with phycoerythrin (PE), clone 55-7h1, 1:100 (BD) for ECs. To eliminate dead cells, cells were stained with the LIVE/DEAD fixable Aqua dead cell staining kit (Thermo Fisher). For cell surface markers, staining was carried out in PBS with 5% FBS. For intracellular proteins, staining was carried out in cells fixed with 4% paraformaldehyde (PFA) in PBS. Cells were stained with the anti-cardiac isoform of troponin T (cTnT) (clone 13-11) (Thermo Fisher) labelled with APC using Zenon technology (Thermo Fisher) (1:50) for CMs. The staining was performed in PBS with 5% FBS and 0.75% saponin (Nacalai Tesque). The stained cells were analyzed by CytoFLEX S (Beckman Coulter, Brea, CA, USA). Data was collected from at least 10,000 events. Data was analyzed with CytExpert software (Beckman Coulter). The percentage of CMs, ECs and MCs in cardiac tissue sheets used in the present study was as follows: CM, 35.53±10.47 %, EC, 1.86±0.93 %, MC, 37.43±7.07 % (n=6).

For histological analysis, 0.5 cm distal colon was fixed in Zinc Formalin (Polyscience Inc.) for 3 hours and then embedded in paraffin blocks. We then prepared 5 μm paraffin cross-sections, which were used for hematoxylin and eosin (H&E) staining (Mayer’s Hematoxylin solution, 1% Eosin Solution, Wako) following standard procedures. The histological images were captured using the BX51-P Polarizing microscope (Olympus) and processed with the Olympus D.P. Controller 2002 software.
For immunohistochemistry analysis, 0.5 cm distal colon was fixed overnight in 4% paraformaldehyde (PFA) (Wako) at 4°C and mounted in embedding medium Tissue-Tek O.C.T Compound (Sakura). The tissues were cut into 8 μm sections and permeabilized with 0.2% saponin (Nacalai Tesque) in PBS. The sections were then blocked with 5% goat serum (Wako) for Mucin 2 detection. Subsequently, the sections were stained with anti-Mucin2 (1:200, rabbit, clone: H-300, Santa Cruz Biotechnology) at 4°C overnight. For the second antibody, Alexa Fluor 488 conjugated donkey anti-rabbit IgG antibody (1:400, Thermo Fisher Scientific) was used with DAPI (1:1000, Dojindo). The sections were assessed using the Leica TCS SP8 (Leica Microsystems).