Lentiviral Transduction of Human-Induced Pluripotent Stem Cells (hiPSCs) for High Sensitivity Bioluminescence Cell Tracking

Background: Human induced pluripotent stem cells (hiPSCs) are a promising cell source for cardiac repair. In studies, two different application techniques are used; intramyocardial injection of hiPSC-derived cardiomyocytes (CM) and implantation of three-dimensional engineered heart tissue (EHT). However, there are biosafety concerns related to cell distribution of cells with teratogenic potential. Limited data are available for cell distribution after implantation. We here generated luciferase positive hiPSCs-derived cardiomyocytes, to allow for in vivo bioluminescence cell tracking.

Method: Transduction of hiPSCs with a tissue unspecific promoter was performed using a lentiviral vector. The Luc+-hiPSCs were identified by puromycin selection. Functional enzyme testing was performed using BLI measurement. Both, the Luc+ and Luc− hiPSCs were tested for pluripotency by FACS analysis and confocal microscopy. The Luc+ hiPSCs were differentiated into hiPSC-derived cardiomyocytes using specific inhibition and activation of the Wnt/β-catenin-signaling pathway. The CMs were further used for hydrogel based EHT fabrication. To evaluate the sensitivity of BLI for cell tracking, both Luc+ CMs and Luc+ EHTs were studied at different cell concentrations in vitro and in vivo. Furthermore, shielding of different organ systems and anatomical structures was investigated in a rat model.

Results: After knock-in hiPSCs showed a positive bioluminescence signal. Genotyping of hiPSCs revealed genomic integration of the gene. FACS analysis and histological analysis of Luc+ hiPSCs (78.88 ± 3.85, n = 6) presented a similar pluripotency character in comparison to Luc− hiPSCs (87.8% ± 2.46, n = 6). Both Luc− CMs (70.1% ± 4.96, n = 6) and Luc+ CMs (72% ± 7.36, n = 6) showed a positive signal for troponin-T. Dilution series of hiPSC-CMs (R ² = 0.97) and EHTs (R ² = 0.99) revealed a strong correlation between cell number and luminescent signal intensity. In vitro analysis of Luc+ cells showed the detection limit of 10 cells (6.3 × 104 [p/s/cm²/sr]) compared with signals without cells (5.2 × 104 [p/s/cm²/sr]). Dilution series of hiPSC-CMs implanted in a rat model presented different shielding for each organ (heart = 1.15 × 106 [p/s/cm²/sr], lungs = 1.1 × 106 [p/s/cm²/sr], liver = 1.75 × 106 [p/s/cm²/sr], kidney = 2.42 × 106 [p/s/cm²/sr]) and a detection limit of 10,000 cells.

Conclusion: This study demonstrates that BLI of lentiviral transduced hiPSCs is suitable for tracking living cells—even small cell numbers—with high sensitivity. Knock-in of hiPSCs with luciferase did not reveal any negative effects on pluripotency character and differentiation potential.

Publication History

Article published online:
28 January 2023