1Technical University of Munich / Klinikum rechts der Isar Internal Medicine Munich, Deutschland; 2Technical University of Munich / Klinikum rechts der Isar Nuclear Medicine Munich, Deutschland; 3Technical University of Munich School of Life Sciences Freising, Deutschland
Introduction and purpose
Cardiac progenitors have the capacity to adapt to environmental cues and to differentiate into multiple cardiac lineages, making them attractive candidates for cell-based treatment of heart failure. Recently, we demonstrated that the transplantation of human pluripotent stem cell (hPSC)-derived ventricular progenitors (HVPs) in injured pig hearts promoted remuscularization and prevented heart failure progression (Poch, C. M. et al. 2022) . Here, we engineered hiPSCs to express a novel reporter gene system (DTPA-R) that allows for quantitative positron emission tomography (PET) imaging of transplanted cells (Morath, V. et al. 2023). Tracking the grafted HVPs in vivo can provide useful dynamic information about their migration, viability, and proliferation within the heart. Importantly, the combination of PET and magnetic resonance imaging (MRI) would allow parallel assessment of heart function and its improvement upon HVP treatment. This multimodal imaging could help improve preclinical research as well as clinical studies of regenerative cell therapies.
Materials and methods
CRISPR/Cas9-based knock-in was used to introduce a PET reporter gene expression cassette in the AAVS1 safe-harbor locus of hiPSCs. The reporter gene cassette constantly expresses a membrane-anchored binding protein called DTPA-R, which is based on an extracellular Anticalin that can be targeted specifically with a Fluorine-18 [18F]F-DTPA radioligand and features a V5-epitope-tag which enables binding of anti-V5-tag antibodies. Cell surface expression and expression levels were assessed by flow cytometry and radiolabeling. Uptake of the reporter probe [18F]F-DTPA•natTb of induced cells was measured in cell culture and by ex vivo PET imaging of HVPs applied on cultured live pig heart slices.
Results
The engineered hiPSCs were created by nucleofection and antibiotic selection. We determined the correct gene knock-in, the absence of off-site mutations, and the absence of chromosomal abnormalities in the cells. Beating cardiomyocytes (CMs) emerged on day 9 of cardiac differentiation in vitro. The DTPA-R reporter was expressed at the cell surface at levels of about 2 million copies for hiPSCs. Expression of the receptor was confirmed in HVPs and CMs at the later time points. Radiolabeling of the hiPSCs-DTPA-R in vitro proved the expression of the receptor and significant difference in binding compared to the control cells (ratio:801 fold). HVPs survival, differentiation, and ability of radiolabeling was provend by ex vivo PET imaging of HVPs cultured on native pig heart slices at different concentrations: Obtained data confirmed the specific and efficient binding of the radioligand to the receptor (HVPs -DTPA-R): Slice 1 (1x106 cells, 0.7±0.1% ID/g); Slice 2 (2.5×106 cells, 13±2.2% ID/g); Slice 3 (3×106 cells, 1.7±0.2% ID/g), HVPs-control); Slice 4 (0 cells, 0.0±0% ID/g). Subsequent immunofluorescent stain of the V5 tag in the slices overlayed with the obtained PET signals.
Conclusions
This preparatory work will lay the foundation for tracking hiPSC derivatives in rodent and pig animal models of cardiac injury in vivo. Live monitoring of transplanted HVPs and their regenerative effect after myocardial infarction will be studied. Further investigation in animal models is essential to get a better understanding of the dynamic behavior of the cells in vivo and take a step forward to clinical translation.