Abstract P1066: Engineering Of Ferritin Overexpressing Human Induced Pluripotent Stem Cells For In Vivo Tracking Of Grafts Using Magnetic Resonance Imaging
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Medientyp:
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Titel:
Abstract P1066: Engineering Of Ferritin Overexpressing Human Induced Pluripotent Stem Cells For In Vivo Tracking Of Grafts Using Magnetic Resonance Imaging
Beschreibung:
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<jats:bold>Introduction:</jats:bold>
Transplantation of human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) emerges as a promising approach for the treatment of heart diseases. However, it is an unmet challenge to precisely localize the hiPSC-CMs upon transplantation and quantify the graft size in the live large animals. To address this issue, we established a new approach to track grafted CMs in large animals using magnetic resonance imaging detectable reporter molecules, which allows the assessment of graft size in live animals at different time points.
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<jats:bold>Methods:</jats:bold>
We employed a CRISPR/Cas9 system as a virus-free approach to overexpress ferritin in hiPSCs.
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<jats:bold>Results:</jats:bold>
hiPSC cells were transfected with ferritin heavy chain CRISPR activation plasmid for 24 hours. Before antibiotic selection, we established a kill curve to determine the appropriate antibiotic concentration. Based on the kill curve, we conducted antibiotic selection using puromycin for 7 days and isolated transfected colonies for further culture. Next, we treated various concentrations of iron chloride (0-100 μM) to the FHC transfected hiPSC for 4 hours to examine a proper concentration of iron loading for MRI detection. There was no significant difference in cell viability between 0 and 25 μM of iron chloride. In addition, we confirmed transfection efficiency with higher ferritin expression in transfected cells compared to controls (non-transfected wild-type hiPSC). Following, we plan to differentiate ferritin overexpressing hiPSC into cardiomyocytes, inject the cells into porcine hearts, and conduct magnetic resonance imaging to detect the engrafted transgenic cells in live animals.
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<jats:bold>Conclusion:</jats:bold>
The ferritin-expressing hiPSC line has the potential to provide a reliable measurement of the graft size in large animals.
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