High Sensitivity Bioluminescence Cell Tracking of Luciferase Positive Human iPSC-derived Cardiomyocytes

Background: Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) seem to be a promising cell source for cardiac repair purposes. However, there are biosafety concerns related to cell washout of cells with potentially teratogenic potential. We here developed and evaluated a biological system which allows for high sensitivity cell tracking using Bioluminescence Imaging (BLI).

Methods: To allow for BLI-based cell tracking, the hiPSCs were tagged with luciferase by a lentiviral knock-in. Luc+-hiPSC-CMs were differentiated using specific modulation of the Wnt/b-catenin- pathway and further used for hydrogel based engineered heart tissue (EHT) fabrication. To validate the sensitivity of BLI and the functionality of the enzyme, the CMs and EHTs were examined in 2D culture. To investigate the shielding of different organs and anatomical structures, CMs and EHTs with different cell concentrations were implanted in a rat model.

Results: After lentiviral knock-in, hiPSCs showed a strong bioluminescence signal. Genotyping of hiPSCs revealed genomic integration. FACS− and histological analysis of Luc+ hiPSCs presented a similar pluripotency profile compared with conventional hiPSCs. Both Luc− CMs and Luc+ CMs showed a comparable Troponin-T signal. Dilution series of hiPSC-CMs and EHTs revealed a linear correlation between cell number and luminescent signal intensity during BLI. In vitro analysis of Luc+ cells showed a detection limit of 10 cells. In-vivo bioluminescence imaging during chicken embryogenesis allowed for precise cell tracking during time course. In a small animal in-vivo model, implantation of EHTs and injection of hiPSC-CMs showed a strong luminescent signal with linear correlation between cell number and luminescent signal. Despite shielding of the animal’s body, a highly sensitive detection limit of 10,000 cells was observed.

Conclusion: This study successfully demonstrates the lentiviral transduction of hiPSCs with luciferase and their differentiation to Luc+ hiPSC-CMs. Knock-in of hiPSCs with luciferase did not reveal any negative effects on pluripotency and cardiomyocyte differentiation. BLI allowed for high-sensitivity cell tracking of Luc+ hiPSC-CMs.

NB: This abstract was presented in a similar form at a previous meeting.

Publication History

Article published online:
11 February 2025