Four hiPSC lines [KUXP (WTSIi008-A), QOLG (WTSIi004-B), ROBP (WTSIi018-A), and KOLF (WTSIi090-A)] were obtained from the European Bank for Induced Pluripotent Stem Cells (EBiSC) and maintained in feeder-free, serum-free 2D culture at 37 °C and 5% CO2. For CM differentiation, hiPSCs were grown in E8 Flex medium (Thermofisher, A2858501) on recombinant human vitronectin (Thermofisher, A14700)-coated plates for 3–4 days until reaching approximately ~ 85% confluency. Differentiation into cardiomyocytes was initiated using a protocol adapted from Burridge et al. [4] Briefly, hiPSCs were incubated in CDM3 medium; RPMI 1640 supplemented with 75 mg/mL recombinant human albumin (Merck, A7931) and 64 mg/mL L-ascorbic acid 2-phosphate (Merck, 49752) with the addition of 6 μM CHIR99021 (Cambridge Bioscience, CAY13122) for 48 h. On day 2, the medium was replaced with CDM3 containing 2 μM Wnt-C59 (Selleckchem, S7037), and from day 4 onwards, cells were maintained in CDM3 alone, with media refreshed every two days. Alternatively, where indicated, hiPSC-CMs were generated using a commercially available Cardiomyocyte Differentiation Kit (Thermofisher, A2921201), following the manufacturer’s instructions.

Adult male Sprague–Dawley rats (8–10 weeks) were purchased from Charles River UK and underwent either MI induction via left coronary artery (LCA) ligation or a sham surgical procedure, as described previously [20]. For MI, the LCA was ligated at the level of the lower edge of the left atrial appendage via left thoracotomy under isoflurane anaesthesia and mechanical ventilation. Successful induction of ischaemia was confirmed by regional epicardial colour change and the presence of dyskinesia. Following chest and skin closure, animals were extubated and allowed to recover in their cage. Sham-operated animals underwent an open-chest procedure without LCA ligation. All animals were sacrificed 24 h post-surgery. The left-ventricular (LV) wall was then harvested, either freshly homogenised or snap-frozen in liquid nitrogen and stored at –80 °C for later use. For each individual experiment, one LV was used per homogenate.

Tissue homogenisation was performed PBS supplemented with protease inhibitors (cOmplete™, EDTA-free Protease Inhibitor Cocktail; Sigma-Aldrich, 11,873,580,001) using a Precellys 24 tissue homogeniser. Homogenates were centrifuged at 6000 × g for 15 min, and the supernatant was collected for immediate use or stored at –80 °C for subsequent experiments.

All animal procedures were approved by the institutional ethics committee at Queen Mary University of London and the UK Home Office. Experiments conformed to the Principles of Laboratory Animal Care (National Society for Medical Research) and the Guide for the Care and Use of Laboratory Animals (U.S. National Institutes of Health, 1996).

Human iPSC-CMs were seeded at a density of ≥ 2.5 × 105 cells/cm2 to form a confluent monolayer. To promote cell attachment, hiPSC-CMs were incubated overnight with 10 μM Y-27632 (Rock inhibitor; Cambridge BioScience, HY-10071-5 mg). The following day, a scratch wound was generated across the hiPSC-CM monolayer using a sterile P1000 tip. After washing with PBS to remove detached cells, hiPSC-CMs were incubated for up to 24 h with either cell media alone or medium supplemented with LV homogenate from MI or sham-operated rat hearts (diluted as specified).

Cell migration was assessed by capturing images at defined time points using either a Keyence BZ-X810 microscope or an EVOS XL Core Cell Imaging System. Quantitative analysis was performed using ImageJ software. Percentage wound closure was calculated as follows:

$$\begin &\frac}^\right)-initial\, wound\, area \,\left(}^\right)}}^\right) } \\ &\quad \times 100\end$$

Cell front migration was calculated as follows;

$$\begin\frac}^\right)-initial\, wound\, area\, \left(}^\right)}\right)},\end$$

in which the height of the image represents the y-axis, to obtain the difference in horizontal distance (x-axis), i.e., the cell front migration (μm). This number is then divided by the incubation time to obtain the cell front migration speed (μm/h). For the TGF-β inhibition experiment (Fig. 4), 10 μM SB431542 (Tocris, 1614/1) was supplemented to the media.

All TWM assays were performed in 24-well plates. Transwell inserts with 8 μM pore size (Starstedt, 83.3932.800) were pre-coated on both sides with ~ 22 μg/cm2 Geltrex Ready to Use (Thermofisher, A1569601) for 1 h at 37 °C. After aspiration of the residual coating, 1 × 105 hiPSC-CMs per insert were seeded into the upper chamber in cardiomyocyte support medium supplemented with 10 μM Y-27632 and incubated overnight to allow cell attachment. The next day, the medium in the lower chamber was replaced with cell medium alone or medium supplemented with LV homogenate from MI or sham-operated rat hearts at the indicated dilution. After 24 h of incubation, cells were fixed in 4% paraformaldehyde for 15 min at room temperature (RT) followed by a PBS wash and incubation with DAPI for 15 min at RT. To ensure that only migrated cells on the lower surface were visualised, the upper surface of the insert was gently swabbed with a cotton applicator to remove non-migrated cells. The membranes were then carefully excised and mounted onto glass microscope slides. Images were acquired on a Keyence BZ-X180 or Zeiss LSM800 microscope. Quantification of migrated cells was performed using the ImageJ software. For the TGF-β inhibition experiments, 10 μM SB431542 (Tocris, 1614/1) was supplemented to the upper insert 10 min prior to their incubation with medium and LV homogenate. For the TGF-β neutralisation studies, we used an anti-TGF-β−1,2,3 antibody (1D11.16.8, Thermofisher, 16–9243-85) or an IgG isotype control at a concentration of 20 μg/mL.

TGF-β1 levels were measured in homogenates from sham and MI tissues using a TGF-beta 1 Quantikine ELISA kit (R and D, Cat #DB100C). To activate latent TGF-β1, tissue supernatants were incubated with 1 M HCl for 10 min, followed by neutralisation with 1.2 M NaOH/0.5 M HEPES. Activated samples or standards were added to wells pre-coated with TGF-β1 capture antibody, along with the assay diluent. After a 2 h incubation at RT, plates were washed four times before addition of the TGF-β1 conjugate. Plates were then incubated for a further 2 h, followed by four washes. Substrate solution was then added and incubated for 30 min at RT in the dark. The reaction was stopped by the addition of Stop Solution and absorbance was measured at 450 nm with wavelength correction at 540 nm using a microplate reader.

Human iPSCs were seeded onto Transwell inserts at day 15 of CM differentiation, as previously described. After 24 h of incubation with a 1:10 dilution of left-ventricular homogenate from an MI rat heart in the lower compartment, culture media was aspirated and the inserts washed in PBS. To isolate migrating and non-migrating cells, the upper and lower surfaces of the inserts were swabbed separately.

RNA was extracted by lysing the cells directly on the inserts using RLT lysis buffer from the Qiagen RNeasy mini kit (Qiagen, 74,104), following the manufacturer’s instructions. Samples with RNA integrity numbers (RIN) ≥ 9.4 and a minimum input of 100 ng of total RNA were used for bulk RNA-seq (20 million reads per samples). Library preparation and paired-end 150 bp sequencing (PE150) were performed by Novogene Ltd, who also conducted downstream data analysis.

Human iPSCs were harvested by incubation with EDTA for 3–5 min, followed by gentle detachment with culture media. Cells were seeded onto 16 mm coverslips in 12 well plates pre-coated with recombinant human vitronectin and cultured for 2–4 days before fixation with 4% paraformaldehyde in PBS for 15 min at 37 °C.

Ahead of immunostaining, hiPSC-CMs were replated overnight using the CM dissociation kit (StemCell Technologies, 05025). Briefly, wells were washed twice with PBS before incubation with CM dissociation medium (StemCell Technologies, 05026) at 37 °C for 10–15 min. Detached cells were collected using a 10 mL serological pipette and transferred into tubes containing CM support medium (StemCell Technologies, 05027). After centrifugation at 300 g for 5 min, the supernatant was removed, and the cell pellet was resuspended in CM support medium supplemented with 5 µM Y-27632. Cells were seeded into a new vitronectin-coated plate at the desired density (≥ 2.5 × 105 cells/cm2) and allowed to attach overnight. The following day, cells were fixed with 4% paraformaldehyde in PBS for 15 min at 37 °C.

After fixation, hiPSCs and hiPSC-CMs were washed and permeabilised with 0.1% Triton-X-100 in PBS for 10 min at RT, then blocked using 10% donkey serum/PBS for 1 h at RT. Primary antibodies were applied overnight at 4 °C in the dark. After washing, cells were incubated with appropriate secondary antibodies for 30 min at 37 °C. DAPI was used as a nuclear counterstain. All wash steps were performed with 0.1% Tween in PBS. Imaging was conducted using a Zeiss LSM 800 or Keyence BZ-X810 microscope. The antibodies used for immunostaining can be found in Supplementary Tables S4 and S5.

For Fig. 4B, Transwell inserts were fixed, washed, permeabilised, blocked, and stained according to the above steps within the 24-well plates, and staining was performed with Phospho-SMAD2/3 (Thermofisher, PA5-110,155) at a 1:200 dilution.

Human iPSCs were harvested by incubation with EDTA for 3–5 min followed by aspiration, followed by gentle detachment with culture media. Cells were then transferred to 15 mL tubes and centrifuged at 200 g for 5 min. The supernatant was aspirated, and cells were resuspended into single cells in filtered FACS buffer (PBS, 4% w/v bovine serum albumin (BSA), 10% v/v penicillin–streptomycin (Pen-Strep), 4% v/v EDTA). After repeating the last step, the cells were stained for 30 min at RT. The antibodies used can be found in Supplementary Tables S4–5.

HiPSC-CMs were dissociated with the Cardiomyocyte Dissociation Kit (see above) and stained with the Inside Stain Kit (Miltenyi Biotech, 130-090-477) according to the manufacturer’s protocol. Briefly, dissociated cells are counted with Trypan Blue and up to 1 million cells are washed by adding FACS buffer. After centrifugation at 200 g for 10 min, the cells are resuspended in 1:1 FACS buffer/Inside Fix buffer. After 20 min of incubation in the dark at RT, the cells are washed with FACS buffer and centrifuged at 200 g for 5 min. The washing step is repeated in Inside Perm. The cells were directly stained in Inside Perm buffer for 10–30 min at RT, or indirectly for 1 h at RT and with secondary antibodies for 30 min at RT in the dark. The antibodies used can be found in Supplementary Tables S4–5.

HiPSCs/hiPSC-CMs were subsequently washed and resuspended in incubation medium and were kept on ice until analysis. Data were acquired on a BD LSRFortessa instrument and analysed using FlowJo software.

Total RNA isolation was performed using the RNeasy mini kit (Qiagen) according to the manufacturer’s protocol. Briefly, harvested cells or heart homogenates were lysed in RLT lysis buffer for 5 min at RT. The resulting cell lysate was transferred to a QIAShredder column and was centrifuged at full speed for 3 min. Next, 70% ethanol was added to the lysate, which was transferred to an RNeasy silica column, followed by centrifugation at 1.2 × 104 rpm for 15 s. This was followed by a washing step in RW1 wash buffer and a DNase I incubation for 15 min at RT, followed by another wash step with RW1 and twice with RPE, followed by dry centrifugation of the column at 1.2 × 104 rpm for 1 min. The RNA was eluted in RNase free ddH2O for 2 min at RT followed by centrifugation at 1.2 × 104 rpm for 1 min. Eluted RNA was kept on ice until further use or stored at −80 °C for future use. RNA was subsequently reverse transcribed into cDNA with the High-Capacity Reverse Transcription Kit (Thermofisher, 4,368,814) with the following thermal cycler settings: 10 min at 25 °C, 120 min at 37 °C, and 5 min at 85 °C. Gene expression was quantified by real-time PCR of 10 ng cDNA of each sample with the PowerUp SYBR Green supermix (Thermofisher, A257420). β-actin and GAPDH were used as housekeeping genes. Primers for each gene are given in Table S6.

The plate was transferred to a QuantStudio 7.0 Real-Time PCR machine (Applied Biosystems) and the following parameters were applied: 5 min 95 °C, 40 cycles of 15 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C, followed by a melt curve analysis from 55 to 95 °C.

All data were analysed using Graphpad Prism 9.0 software (Graphpad, San Diego, CA). Data are represented as mean ± standard deviation (SD). Statistical analysis was performed on data with a replicate number of 3 and above. Technical replicates are indicated with ‘n’ and biological replication with ‘N’, as detailed in the figure legends. For the comparison between two groups, two-tailed unpaired Student’s t tests were performed. Multiple group comparisons were performed with one-way or two-way analysis of variance (ANOVA) with Tukey’s post hoc test correction (3 groups) or Dunnett’s correction (> 3 groups). If the number of replicates was not equal between groups, a mixed-effects analysis was performed instead of an ANOVA, as the latter cannot deal with missing values. Statistical significance was accepted at p < 0.05.