Animals

This study was approved by the University of Toronto’s Animal Care Committee (protocol #20012748). All procedures were conducted in accordance with the Canadian Council on Animal Care. Two-month-old female NOD SCID (n = 15) and CD-1 (n = 12) mice (Charles River Laboratories, Canada) weighing between 20 and 25 g were utilized. The reporting of animal experiments adheres to the ARRIVE guidelines (https://arriveguidelines.org/arrive-guidelines).

Human embryonic stem cell (hESC) cell culture and maintenance

ESI-017 hESCs (ESI BIO, SKU: ES-700; Alameda, CA, USA) [13] were cultured on Matrigel™ hESC-qualified Matrix (Corning; Corning, NY, USA) coated tissue culture plates using mTeSR™ Plus medium (StemCell Technologies; Vancouver, BC, Canada) in an incubator set at 37 °C and 5% CO2. Cells were maintained in an undifferentiated state as colonies and passaged using ReLeSR™ (StemCell Technologies).

Generation of ferritin-overexpressing hESCs

A non-viral CRISPR/Cas9 gene editing system was used to integrate the human ferritin transgene into the safe-harbor locus AAVS1 of ESI-017 hESCs as previously described [12]. Transfected cells were sorted for eGFP expression before expansion of single colonies to produce clonal cell lines. Clones were selected based on the ferritin expression level determined by Western blot.

Cardiac differentiation and flow cytometry

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were differentiated as previously described [14, 15]. In brief, hESCs were cultured as single cells in mTeSR™ One medium (StemCell Technologies) with 10 µM ROCK inhibitor Y27632 (StemCell Technologies) until ready for embryoid bodies (EBs) generation. EBs were formed by culturing cells at a density of 1 × 106 cells/ml on an orbital shaker (45 rpm) overnight in a low oxygen environment (5% CO2, 5% O2, 90% N2) with aggregation medium. The aggregation medium contained STEMPRO 34 medium (Thermo Fisher Scientific; Waltham, MA USA), L-glutamine (2 mM, Thermo Fisher Scientific), transferrin (150 μg/mL, Roche; Basel, Switzerland), L-ascorbic acid (50 μg/mL, Sigma-Aldrich; St. Louis, MO, USA), monothioglycerol (MTG, Sigma-Aldrich), bone morphogenetic protein-4 (BMP4, 1 ng/mL, R&D Systems; Minneapolis, MN, US), and ROCK inhibitor Y-27632 (10 μM, StemCell Technologies). EBs were then treated with a growth factor based three-stage induction using BMP4 (10 ng/ml, R&D Systems), fibroblast growth factor basic (bFGF, 5 ng/ml, R&D Systems), and activin A (6 ng/ml, R&D Systems) for 2 days, then Wnt inhibitor IWP2 (2 μM, Tocris; Bristol, UK) and vascular endothelial growth factor (VEGF, 10 ng/mL, R&D Systems) for 3 days. The aggregates were dissociated into single cells on Day 6 of differentiation and plated into Growth Factor Reduced Matrigel (Corning)-coated flasks at a density of 80,000 cm2 in presence of VEGF (5 ng/ml) for another 6 days. Starting Day 12 of differentiation, cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) with B-27 supplement (Thermo Fisher Scientific) until Day 17 when the cells were cryopreserved. hESC-CM populations were assessed using a LSRII/Fortessa flow cytometer (BD Bioscience; San Jose, CA, USA) with primary antibodies against cardiac troponin T (cTnT) and myosin light chain 2 (MLC2v).

Western blot

Cells were washed with phosphate buffer saline (PBS) and lysed on ice with lysis buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor). Lysates were collected and centrifuged at 15,000 g for 15 min at 4 °C. Supernatants were collected, and protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). For each sample, an equal amount of protein was mixed with 4 × Laemmili Sample Buffer (BioRad; Hercules, CA, USA), 10 mM dithiothreitol (DTT, Sigma-Aldrich), and boiled at 95 °C for 5 min. Proteins were resolved by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) + 0.05% Tween-20 for 1 h at room temperature, incubated overnight at 4 °C with primary antibodies (anti-ferritin and anti-β-actin antibodies, Abcam; Cambridge, United Kingdom), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam) for 1 h at room temperature. The chemiluminescent signals were developed by Clarity Western ECL Substrate (Bio-Rad) and imaged on a ChemiDoc™ imaging system (Bio-Rad).

Cellular toxicity assays

To investigate cellular toxicity associated with ferritin overexpression and Mn supplementation, we assessed cells for viability, proliferation, and metabolic activity. Wild type and ferritin-overexpressing hESCs were seeded on 24-well and 96-well plates at a density of 40,000 cells/cm2. Twenty-four hours after seeding, cells were dosed with MnCl2 for 24 h. After dosing, cells were washed twice with normal culture medium. LIVE/DEAD™ Viability/Cytotoxicity Kit (Invitrogen; Waltham, MA, USA) was used to qualitatively assessed cell viability. Calcium AM, ethidium homodimer-1, and Hoechst 33342 (Invitrogen) were added to the cells to label live cells, dead cells, and cell nuclei, respectively. Representative images were captured using a Leica DMi8 inverted epifluorescence microscope. Cell proliferation and metabolic activity were assayed using CyQUANT™ NF Cell Proliferation Assay (Invitrogen) and WST-1 reagent (ROCHE; Basel, Switzerland), respectively. The reagents were added to cells according to the manufacturer instructions. The absorbance or the fluorescence intensity was measured using a PerkinElmer Envision 2104 Multilabel Plate Reader.

Immunofluorescence microscopy

To evaluate pluripotency markers in hESCs and cardiac markers in hESC-CMs, cells were cultured on Matrigel-coated glass coverslips in 24-well plates for immunofluorescence imaging. Cells were fixed in 4% paraformaldehyde for 15 min, permeated in 0.1% Triton for 5 min, blocked with 3% BSA at 37 °C for 20 min, and incubated with primary antibodies (anti-OCT4, anti-SSEA4, anti-cTnT, anti-sarcomeric-α-actinin antibodies, Abcam; Cambridge, United Kingdom) at room temperature for 1 h. Cells were then blocked with 10% donkey serum for 30 min and incubated with Alexa Fluor conjugated secondary antibodies (Thermo Fisher Scientific) at room temperature for 1 h. Cell nuclei were stained with Hoechst 33342 (Invitrogen) for 5 min. The coverslips were mounted and imaged using an Olympus FLUOVIEW FV3000 laser scanning confocal microscope.

Inductively coupled plasma atomic emission spectroscopy (ICP-AES) quantification of Mn content

Cells with or without MnCl2 supplementation were washed with PBS before collection. The cell pellets were digested with 35% HNO3 with sonication at 60 °C for 3 h. Samples were topped up with distilled deionized water (ddH2O) to a final concentration of 2% HNO3 and filtered through 0.22 µm membranes. Samples were stored at 4 °C before analysis on an Optima 7300 DV ICP-AES spectrometer at the Department of Chemistry, University of Toronto (Toronto, Canada).

In vitro MRI

Cells with or without MnCl2 supplementation were washed with PBS before collection. Cells were then dissociated, centrifuged at 300 g for 5 min, washed with PBS, and transferred into wells in a pre-cut 96-well PCR microplate. The microplate was centrifuged at 300 g for 5 min and the supernatant was removed. Cell pellets were kept on ice until MRI on a preclinical 3 T MR scanner (MR Solutions; Guildford, United Kingdom).

MRI was performed by placing the microplate in the center of a mouse whole-body coil. A 2D T1-weighted spin echo (SE) sequence was run with the following parameters: repetition time (TR) = 500 ms, echo time (TE) = 11 ms, matrix = 256 × 256, field-of-view (FOV) = 50 × 50 mm, slice thickness = 1.0 mm, voxel size = 0.1953 × 0.1953 × 1.0 mm3, number of signal averages (NSA) = 4. T1 mapping was performed using a variable flip angle approach [16] with a 3D spoiled gradient echo (SPGR) sequence: TR = 11 ms, TE = min, flip angle (FA) = [2°, 3°, 10°, 20°], matrix = 256 × 128 × 12, FOV = 50 × 50 mm, slice thickness = 1.0 mm, voxel size = 0.195 × 0.390 × 1.0 mm3, NSA = 4.

In vivo MRI

On the day of cell transplantation, hESCs were dissociated from 10 cm dishes using ReLeSR, washed with PBS, centrifuged at 300 g for 5 min, and kept on ice until ready for cell injection. Cells were manually counted with a hemocytometer. For each leg in each animal, 3 × 106 cells in a volume of 50 µl were injected into the gastrocnemius muscle using a 1 ml syringe and 27 G needle. After the baseline MRI scan, 0.4 mmol/kg of Mn was administrated subcutaneously (for a mouse weighing 25 g, 100 µl of 0.1 M MnCl2 solution was injected).

Mice were induced on 5% isoflurane in 100% oxygen (1.0 L/min), maintained at 1.5–2.0% isoflurane during the entire imaging session, and placed head-first in a prone position inside the mouse whole-body coil. Body temperature was maintained at 37 °C using an air-heating system built into the mouse holder. A pneumatic respiratory pillow was placed on the abdomen to monitor respiration throughout imaging and to provide respiratory gating. A high-resolution 2D fat-suppressed T1-weighted fast spin echo (FSE) sequence was run with the following parameters: TR = 743 ms, TE = 11 ms, echo train length (ETL) = 4, matrix = 256 × 248, FOV = 60 × 45 mm, slice thickness = 1.0 mm, voxel size = 0.234 × 0.181 × 1.0 mm3, and NSA = 2. T1 mapping was performed using a variable flip angle approach [16] with a 3D SGPR sequence: TR = 11 ms, TE = “MIN”, FA = [2°, 10°, 20°], matrix = 256 × 128 × 24, FOV = 60 × 45 mm, slice thickness = 1.0 mm, voxel size = 0.234 × 0.351 × 1.0 mm3, and NSA = 4. A high-resolution 2D T2-weighted FSE sequence was also run to exclude fluid contributions either from the injectate or from inflammation: TR = 3658 ms, TE = 68 ms, ETL = 8, matrix = 256 × 240, FOV = 60 × 45 mm, slice thickness = 1.0 mm, voxel size = 0.234 × 0.187 × 1.0 mm3, and NSA = 2. MRI was performed daily during the first five days post-cell transplantation and thereafter every other week (Day 1–5, Week 2, Week 4, and Week 6, etc.). After the last imaging session, NOD SCID mice were euthanized by cardiac arrest with 10% potassium chloride injection into the right ventricle under anesthesia with 5% isoflurane in 100% oxygen. CD-1 mice were euthanized by cardiac puncture under anesthesia with 5% isoflurane in 100% oxygen.

MRI data analysis

Quantitative T1 data analysis was performed in-house using Matlab (v2022, MathWorks, Natick, MA, USA). Regions of interest (ROIs) were drawn manually around the enhancing region on T1-weighted FSE. T1 relaxation times were averaged across the ROI to arrive at a mean value.

Histological analysis

The leg muscles were fixed with 4% paraformaldehyde, processed, and paraffin-embedded for histological analysis. Sections 5 µm thick were stained using hematoxylin and eosin (H&E, Newcomer Supply; Middleton, WI, USA), immunostained using anti-Ku80 antibody (Cell Signalling Technology; Danvers, MA, USA), followed by HRP-conjugated secondary antibody (Abcam), developed with SignalStain DAB Substrate Kit (Cell Signalling), and counterstained with hematoxylin.

Statistical analysis

Data are presented as mean ± SEM, except for in vitro T1 values, which are presented as mean ± SD. For in vitro studies, two-sample comparisons were made using a Welch's t-test. For in vivo studies, data were analyzed in Matlab using a three-way analysis of variance (ANOVA), where ferritin/wild type, teratoma formation, and time post-cell transplantation were independent variables. Fisher’s post-hoc analysis was performed for multiple comparisons. In all cases, significance was reported at a p-value of 5%.