Sunao Sugita1, Kenichi Makabe1, Shota Fujii1,2, Masayo Takahashi1. 1. Laboratory for Retinal Regeneration, Center for Biosystems Dynamics Research, RIKEN, Kobe, Hyogo, Japan. 2. Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
Abstract
Purpose: To determine whether human induced pluripotent stem (iPS) cell-derived retinal pigment epithelial (RPE) cells (iPS-RPE) can express complement factors. Methods: To confirm expression of complement factors in human iPS-RPE cells, we performed flow cytometry, immunohistochemistry, ELISA, and quantitative RT-PCR for the following: C3, C5, CFB (Factor B), C5b-9 (membrane attack complex [MAC]), CFH (Factor H), CFI (Factor I), CD46, CD55, CD59, clusterin, and vitronectin. We also prepared iPS-RPE cells in the presence of recombinant IFN-γ, recombinant TNF-α, lipopolysaccharide, supernatants of naïve T cells, and T helper 1 (Th1) cells. For the transplantation, after preparation of iPS-RPE cells from cynomolgus monkeys, the iPS-RPE cells (allografts) were transplanted into the subretinal space in monkeys. After surgery, monkeys were euthanized for IHC evaluation of the retinal section and determination of complement factors (C3, C5, CFB, MAC, and C1q), cytokines, and immunoglobulin G (IgG). Results: Human iPS-RPE cells expressed complement activators and inhibitors. iPS-RPE cells highly expressed complement factors during inflammatory conditions, especially IFN-γ exposure including Th1 cell supernatants. In immune attack eyes after allogeneic iPS-RPE cell transplantation, complement activators such as C3, CFB, C5, and MAC were detected around the host RPE layer, grafted RPE cells, inflammatory retinal lesions, and transplanted subretinal space. In addition, we observed a large number of C1q and IgG double positive and IFN-γ positive inflammatory cells in the retinal sections. Conclusions: iPS-derived RPE cells greatly expressed complement factors. Thus, RPE cells might be activated and produce complement factors after exposure to infiltrating inflammatory cells in the eye.
Purpose: To determine whether human induced pluripotent stem (iPS) cell-derived retinal pigment epithelial (RPE) cells (iPS-RPE) can express complement factors. Methods: To confirm expression of complement factors in humaniPS-RPE cells, we performed flow cytometry, immunohistochemistry, ELISA, and quantitative RT-PCR for the following: C3, C5, CFB (Factor B), C5b-9 (membrane attack complex [MAC]), CFH (Factor H), CFI (Factor I), CD46, CD55, CD59, clusterin, and vitronectin. We also prepared iPS-RPE cells in the presence of recombinant IFN-γ, recombinant TNF-α, lipopolysaccharide, supernatants of naïve T cells, and T helper 1 (Th1) cells. For the transplantation, after preparation of iPS-RPE cells from cynomolgus monkeys, the iPS-RPE cells (allografts) were transplanted into the subretinal space in monkeys. After surgery, monkeys were euthanized for IHC evaluation of the retinal section and determination of complement factors (C3, C5, CFB, MAC, and C1q), cytokines, and immunoglobulin G (IgG). Results:HumaniPS-RPE cells expressed complement activators and inhibitors. iPS-RPE cells highly expressed complement factors during inflammatory conditions, especially IFN-γ exposure including Th1 cell supernatants. In immune attack eyes after allogeneic iPS-RPE cell transplantation, complement activators such as C3, CFB, C5, and MAC were detected around the host RPE layer, grafted RPE cells, inflammatory retinal lesions, and transplanted subretinal space. In addition, we observed a large number of C1q and IgG double positive and IFN-γ positive inflammatory cells in the retinal sections. Conclusions: iPS-derived RPE cells greatly expressed complement factors. Thus, RPE cells might be activated and produce complement factors after exposure to infiltrating inflammatory cells in the eye.
Authors: Kapil Bharti; Anneke I den Hollander; Aparna Lakkaraju; Debasish Sinha; David S Williams; Silvia C Finnemann; Catherine Bowes-Rickman; Goldis Malek; Patricia A D'Amore Journal: Exp Eye Res Date: 2022-07-11 Impact factor: 3.770