Complement regulatory advantages of primary human amniotic epithelial cells following splenic intraportal transplantation into mouse liver
Ayumi Yamazaki1, Millei Kaga1, Chika Takano2,3, Isamu Taiko1, Kazunori Kanemaru1, Toshio Miki1.
1Department of Physiology, Nihon University School of Medicine, Tokyo, Japan; 2Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, Tokyo, Japan; 3 Department of Pediatrics and Child Health, Nihon University School of Medicine, Tokyo, Japan
Background: Human amniotic epithelial cells (hAECs), a type of placenta-derived stem cell, are considered suitable for clinical application due to their low immunogenicity and lack of tumorigenicity. In murine models of inherited metabolic disorders, the therapeutic efficacy of hAECs has been demonstrated following their transplantation via the portal vein. Previously, we successfully established a non-invasive in vivo cell tracking system using immortalized hAECs (iAECs) labeled with luciferase and green fluorescent protein (Luc2/GFP) . Our results showed that iAECs transplanted via the spleen localized exclusively to the liver without affecting the recipient’s respiratory condition, supporting this route as a safe method of cell delivery. In the present study, we aimed to further evaluate the immunological advantages of hAECs by comparing them with other cell types.
Materials and Methods: Primary hAECs (pAECs), iAECs, and human dermal fibroblasts (HDFs) were labeled with Luc2/GFP using a lentiviral vector. Each cell type (1 × 10⁶ cells) was injected into the spleen of recipient mice. Subsequently, TakeOni (AkaLumine-HCl), a luciferin analog emitting near-infrared bioluminescence (λmax = 675 nm), was administered intraperitoneally. Bioluminescent signals were captured using the IVIS® Kinetic imaging system approximately 10 minutes after substrate administration and monitored over time. Temporal changes in photon flux were statistically analyzed to compare among the different cell types. After imaging, the mice were euthanized, and the liver was harvested for molecular and histological analyses to assess engraftment.
In addition, the expression of complement regulatory proteins (CD46, CD55, and CD59) on each cell type was analyzed by flow cytometry.
Results: Stable expression of both luciferase and GFP was confirmed in all cell types. Following intrasplenic transplantation, near-infrared bioluminescent signals were immediately detected in the spleen and subsequently localized to the liver. The bioluminescence signal gradually declined over time. No signal was detected in the lungs or intestines for any of the transplanted cell types.
Flow cytometry revealed that pAECs exhibited high expression of all tested complement regulatory proteins: CD46, CD55, and CD59. In contrast, iAECs showed comparable expression of CD59 but lower expression of CD46 and CD55. HDFs demonstrated lower expression of all three complement regulatory proteins compared to both pAECs and iAECs.
Conclusion: Compared to iAECs and HDFs, pAECs exhibited higher expression of complement regulatory proteins, which may underlie their superior immunological profile by attenuating complement-mediated rejection and cytotoxic injury in both xenogeneic and allogeneic settings.