Introduction: Human amniotic epithelial cells (hAECs), one of the placental stem cells, are highly regarded for their multipotency, low immunogenicity, and non-tumorigenic characteristics, making them suitable for clinical application. Our previous study demonstrated that hAECs acquire spontaneous differentiation into functional hepatocytes upon transplantation into the liver through the portal vein. Additionally, we have successfully demonstrated the therapeutic efficacy of intraportal transplantation of hAECs in several murine models of congenital liver metabolic disorders. Despite some of the disease model mice being immunocompetent, comparable therapeutic efficacy of hAECs was demonstrated under the xenotransplantation setting. Here, this study employed a noninvasive in vivo live cell imaging system to investigate the feasibility of hAEC engraftment into immunocompetent mice and aimed to elucidate the in vivo cell kinetics following intraportal transplantation of hAECs.
Method: Previously we have established a mouse model with a subcutaneous splenic injection port, which enables cell transplantation into the portal vein with a minimally invasive procedure. In this study, we utilized this mouse model and immortalized hAECs (ihAECs), which were further labeled with luciferase 2 and green fluorescent protein (Luc2/GFP). The spleens of immunocompetent mice (BALB/c) were translocated underneath the skin while maintaining intact vascular pedicles. One week after the surgery, 1 x 10⁶ Luc2/GFP ihAECs were injected into the spleen followed by TakeOni (AkaLumine-HCl, 300 nmol, intraperitoneally), a luciferin analog that exhibits near-infrared emission (λmax= 675 nm). Subsequently, we assessed the photon flux around 10 minutes after the injection of the substrate using the IVIS^® Kinetic system. The mice were euthanized on day 7 and subjected to histological analysis. 
Results: The Luc2/GFP ihAECs stably expressed luciferase and GFP. The near-infrared signal was immediately detected in the spleen and migrated to the liver after about 3 min. When measured every 24 hours, the signal was also confirmed in the liver 7 days after transplantation despite xenotransplantation without immunosuppression. No signals were detected in the lungs or intestinal tract. After 7 days, the recipient mice were euthanized, and histological analysis revealed that the Luc2/GFP ihAECs remained in the liver as aggregated cell clusters.
Conclusion: In this study, we successfully established a cell tracking system and observed in vivo cell kinetics using Luc2/GFP-labeled ihAECs and IVIS^® imaging. The cell kinetics data indicated that the injected ihAECs via portal vein are retained in the liver and spleen only, thus it is a safe cell transplantation route that does not affect the recipient's respiratory status. Further studies including a comparison to other cell types and lymphocyte subset analysis related to activation of effector T cells are required to elucidate the immunological benefits of hAECs.