Longitudinal monitoring of hiPSC-based cardiac grafts using bioluminescence imaging

Background Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair. However, monitoring of graft survival and cell distribution remains challenging. We developed a bioluminescence imaging (BLI)-based system to enable high-sensitivity in-vitro- as well as in-vivo monitoring of hiPSC-CMs. Methods Luciferase-tagged hiPSCs (Luc + -hiPSCs) were generated via lentiviral knock-in. Luc + -hiPSC-CMs were differentiated through Wnt/β-catenin pathway modulation and used to fabricate hydrogel-based engineered heart tissues (EHTs). BLI sensitivity and luciferase functionality were validated in 2D cultures. In vivo imaging was conducted using an ex ovo chicken embryogenesis model. Further, to assess signal shielding and graft survival, Luc + -hiPSC-CMs at varying cell concentrations were injected in a rat model, and longitudinally monitored. Results Luc + -hiPSCs exhibited strong bioluminescence signals post-knock-in, with stable genomic integration confirmed by genotyping. FACS and histological analyses demonstrated comparable pluripotency profiles between Luc + and conventional hiPSCs. Troponin-T expression was similar in Luc + and Luc - CMs. Electrophysiological measurements confirmed functionality of Luc + CMs. BLI revealed a linear correlation between cell number and luminescent signal intensity in dilution series, with a detection limit of 10 cells in vitro. In vivo BLI during chicken embryogenesis enabled precise, longitudinal cell tracking. Shielding experiments in a rat model demonstrated robust luminescent signals following EHT implantation or hiPSC-CM injection, with a linear signal-cell relationship and a detection limit of 10,000 cells. Longitudinal graft monitoring after hiPS-CM injection was feasible, with a reduction in photon emission signal over 7 days, allowing for assessment of graft survival. Conclusions This study successfully established luciferase knock-in in hiPSCs without compromising pluripotency or differentiation into functional cardiomyocytes. In contrast to previous approaches utilizing AAV to transduce mature hiPSC-CMs, our approach requires only a single lentiviral transduction, significantly saving time and resources. BLI enables monitoring of Luc + -hiPSC-CMs, to ensure in-vitro cell viability as a pre-transplant quality control, and to assess graft survival in a small animal in-vivo model