Introduction: 

Hepatocyte transplantation has been applied clinically as a therapeutic approach for congenital metabolic liver diseases, offering the potential to partially compensate for liver function without the need for whole-organ replacement. However, its application is limited by a critical shortage of donor hepatocytes. As an alternative cell source, human amniotic epithelial cells (hAECs), derived from the placenta, have attracted attention due to their multipotency, low immunogenicity, and non-tumorigenic nature. The therapeutic efficacy of hAEC transplantation has been demonstrated in small animal disease models. In this study, aiming toward future clinical application, we performed allogeneic transplantation of porcine AECs in a large animal model and investigated the feasibility and safety of clinically relevant cell transplantation procedures.

Methods:

Porcine amniotic epithelial cells (pAECs) were aseptically isolated from late-gestation porcine placentas and cryopreserved. Some of the pAECs were subjected to comprehensive gene expression analysis with RNA-seq. The pAECs from male fetuses were selected and then labeled with eGFP and Luc2 reporter genes by a lentiviral vector. These labeled cells were transplanted via the portal vein into the liver of 10-week-old female piglets. One month after transplantation, recipient pigs were euthanized, and liver, spleen, and lung tissues were harvested. Immediately after liver excision, a luciferin analog (AkaLumine-HCl) was administered via the portal vein, and Luc2-positive cells in the liver were visualized using an in vivo imaging system (IVIS®). Tissues were subjected to hematoxylin and eosin staining, immunofluorescence staining, and fluorescence in situ hybridization (FISH) using probes specific to the porcine Y chromosome. 

Results:

pAECs were successfully isolated from porcine placentas with minimal contamination by other cell types and showed CD49f expression similar to hAECs. Lentiviral labeling with eGFP/Luc2 was successful, allowing detection via FACS and near-infrared luciferase imaging. Using a catheter-based intraportal transplantation method, eGFP/Luc2-labeled male-derived pAECs were transplanted into four female pigs without major perioperative complications. All animals survived for one month without any complications. IVIS imaging of the excised tissues revealed bioluminescence in some regions of the liver. 

Conclusion:

This study provides important insights into AEC transplantation in a large animal model as a preliminary step toward clinical application. Intraportal transplantation of allogeneic AECs was successfully and safely performed. IVIS imaging revealed bioluminescence in the excised liver, strongly suggesting successful engraftment of the transplanted cells. Further analyses will be conducted to confirm the engraftment of the male donor cells and their distribution within the liver.