Bright Ferritin for Non-Invasive MRI Monitoring of the Fate of Transplanted hPSC-Cardiomyocytes in the Infarcted Rat Heart.

Figures

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  FIGURE 1. Generation of ferritin‐overexpressing hPSCs and hPSC‐derived cardiomyocytes. (A) Schematic of the plasmid vector to insert human ferritin gene into the AAVS1 locus in the genome of hPSC using a non‐viral CRISPR/Cas9 system. Adapted with permission from [Szulc DA, Lee XA, Cheng HM, Cheng HM. Bright Ferritin‐a Reporter Gene Platform for On‐Demand, Longitudinal Cell Tracking on MRI. iScience. 2020;23 (8):101350], copyright 2020. (B) Representative immunofluorescence images of wild type (WT) and ferritin‐overexpressing (ferritin) hPSCs showing the expression of pluripotency marker OCT4 (orange) (n = 3). Gene insertion was confirmed by eGFP (green). Cell nuclei were stained by Hoechst 33342 (blue). Scale bar = 50 μm. (C) Representative immunofluorescence images of WT and ferritin hPSC‐CMs showing the expression of cardiac marker cardiac troponin T (cTnT) (red) (n = 3). Scale bar = 50 μm. (D) Representative flow cytometry data of WT and ferritin hPSC‐CMs demonstrating the percentages of cTnT positive cells after in vitro differentiation. (E) Plot showing mean percentages of cTnT positive cells in WT and ferritin hPSC‐CMs (92.8% ± 3.7% vs. 92.4% ± 2.3%), with no significant difference…

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  FIGURE 2. Bright contrast efficiency of ferritin hPSC‐CMs supplemented with MnCl2. WT and ferritin hPSC‐CMs were dosed with 0.1 mM MnCl2 for 2, 6, and 24 h, showing that adverse effects on viability and phenotype emerged at 24 h. (A) Representative images of WT and ferritin hPSC‐CMs assessed by LIVE/DEAD assay (n = 3). Calcium AM (green), ethidium homodimer‐1 (red), and Hoechst 33342 (blue) were added to label live cells, dead cells, and cell nuclei, respectively. Scale bar = 200 μm. (B) Total live cells quantified by LIVE/DEAD assay (n = 3). *p < 0.1, analyzed using two‐way ANOVA with Dunnett post hoc tests. (C) Representative immunofluorescence staining of cardiac contractile proteins cTnT (white) and sarcomeric α‐actinin (red) in WT and ferritin hPSC‐CMs (n = 3). Cell nuclei were stained by Hoechst 33342 (blue). Scale bar = 20 μm. (D) Average sarcomere length measured in individual cells (n = 10). **p < 0.01, analyzed using two‐way ANOVA with Dunnett post hoc tests. (E) T 1‐weighted fast spin echo (gray‐scale) and T 1 map (pseudo‐color) of cell pellets imaged by in vitro MRI. (F) Quantitative T 1 relaxation times. (G) Intracellular Mn content quantified by ICP‐AES (n = 3). *p < 0.05,…

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  FIGURE 3. Electrophysiological characterizations of hPSC‐CMs with MnCl2. (A) Representative action potential and calcium transient traces of WT and ferritin hPSC‐CMs paced at 1.5 Hz. Cells were cultured in normal cell media (Baseline), 0.1 mM MnCl2 for 6 h (6 h Mn), or 0.1 mM MnCl2 for 6 h followed by 24 h recovery in normal cell media (Recovery). Functional parameters of WT and ferritin hPSC‐CMs measured at 1.5 Hz, including action potential duration to 90% (B) and 30% (C) repolarization (APD90, APD30), APD30/APD90 ratio (D), calcium transient amplitude (E), calcium transient time to peak (F), time to 50% calcium transient decay (G), and contraction displacement (at spontaneous beating) (H). Functional parameters measured at 1.0 and 2.0 Hz were shown in Figure S3. No significant difference between WT and ferritin hPSC‐CMs at Baseline; **p < 0.01, ***p < 0.001, difference vs. Baseline, analyzed using two‐way ANOVA with Dunnett post hoc tests; Contraction Displacement, 6 h Mn WT vs. 6 h Mn Ferritin, p < 0.05, analyzed using two‐way ANOVA with Tukey's post hoc tests. All data are presented as mean ± SEM (n = 4).

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  FIGURE 4. In vivo long‐term tracking of hPSC‐CMs in healthy rat hearts using bright ferritin MRI. (A) Representative short‐axis end‐diastolic CINE cardiac MRI of healthy rat heart at various time points after intramyocardial transplantation of WT and ferritin hPSC‐CMs, followed by subcutaneous MnCl2 injection (post‐Mn). White arrows indicate bright contrast originating from transplanted hPSC‐CMs. Scale bar = 2 mm. (B) Quantitative post‐Mn T 1‐weighted MRI signal calculated by selecting region of interest (ROI) from WT and ferritin hPSC‐CMs. Signal intensity from dorsal muscle was used as internal reference. Data are presented as mean ± SEM (WT, n = 2; Ferritin, n = 3). *p < 0.05, **p < 0.01, ferritin vs. WT, analyzed using two‐way ANOVA with Tukey post hoc tests. (C) Cardiac graft (black dotted lines) confirmed by immunohistochemistry staining using a human‐specific antibody against the nuclear antigen Ku80 (brown) and an antibody against myosin heavy chain (pink). Scale bar = 2 mm. (D) Graft size quantified by histology in rats 8 weeks after receiving WT and ferritin hPSC‐CMs. Data are presented as mean ± SEM (WT, n = 3; Ferritin, n = 4). RV: Right ventricle; LV: Left ventricle.

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  FIGURE 5. Histology‐MRI slice correspondence for hPSC‐CMs distribution. MRI findings from a representative rat who received ferritin hPSC‐CMs transplantation were corroborated against histological analysis. Multi‐slice CINE MRI in the short‐axis view was acquired from base to apex to cover the LV. Bright contrast signal (white arrows) were observed within the myocardium in slices between mid‐ventricle to apex. The rat was sacrificed after MRI and the heart was sectioned and matched to the corresponding MRI slices. H&E and IHC staining confirmed cell graft (open arrows). Scale bar = 2 mm. Myosin heavy chain staining are presented in Figure S4.

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  FIGURE 6. Transplantation of ferritin hPSC‐CMs in rats with myocardial infarction. (A) Schematic of the study design for hPSC‐CMs tracking in infarcted rat hearts using bright ferritin MRI. Rats with myocardial infarcts received intracardial transplantation of ferritin hPSC‐CMs (n = 6), WT hPSC‐CMs (n = 3), or saline (n = 3). Cardiac MRI for cardiac functional assessment was performed on Day −8, −4, and 1. Cardiac MRI with subcutaneous MnCl2 supplementation for longitudinal cell tracking was performed at Week 1, 2, 4, and 6. (B) Quantitative T 1‐weighted MRI signal calculated by selecting ROI at the cell injection site. Signal intensity from dorsal muscle was used for internal reference. Data are presented as mean ± SEM (ferritin, n = 4; WT, n = 2; saline, n = 2). *p < 0.05, ferritin vs. WT and saline, analyzed using two‐way ANOVA with Tukey post hoc tests. (C) Graft size quantified by histology in rats who received WT (n = 3) and ferritin hPSC‐CMs (n = 6). (D) Infarct size quantified by histology in rats who received WT hPSC‐CMs (n = 3), ferritin hPSC‐CMs (n = 6), and saline (n = 3). (E) Change in LVEF between Day −4 and Week 6 in rats who received WT hPSC‐CMs (n = 2), ferritin hPSC‐CMs…

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  FIGURE 7. Monitoring the retention of hPSC‐CMs in the infarcted heart using bright ferritin MRI. (A) Representative in vivo short‐axis end‐diastolic CINE MRI of the infarcted rat heart at various time points after intramyocardial transplantation of ferritin hPSC‐CMs, followed by subcutaneous MnCl2 administration (post‐Mn). Two rats (Rat 1 and Rat 2) showed different cell retention as indicated by bright contrast signal on MRI (yellow arrows). (B) Histological analysis confirmed the presence of cell graft in Rat 1 and minimal cell graft in Rat 2 at Week 6 (yellow dotted lines). (C) In vivo and ex vivo MRI of the same rat heart with ferritin hPSC‐CMs, showing bright contrast signal on T 1‐weighted images with corresponding low T 1's on T 1 map (yellow arrows). (D) Histological analysis of the rat heart in (C) confirmed the existence of cell graft (yellow dotted lines).