Functional consequences of the suppression of MHC-II expression on human stem cell derived retinal pigment epithelium (hES-RPE)

Abstract

Purpose: Subretinal implantation of human stem cell derived retinal pigment epithelium cells (hES-RPE) is an emerging method to replace degenerated RPE in age related macular degeneration. But, immune reaction to the implanted cells, that necessitates potentially harmful immunosuppressive agents, is a major obstacle for the success of hESC-RPE implantation. Expression of Major Histocompatibility Complex-II (MHC-II) molecules on RPE is the key event for the recognition of allogeneic antigens of these cells by the host immune system. We hypothesize that the suppression of Class II Transactivator (CIITA), an essential regulator of MHC-II expression, through RNA interference will down regulate MHC-II expression on hESC-RPE cells, and thus, will reduce immune recognition of these cells by host immune system.

Methods: The H9 human embryonic stem cell line was used for derivation of RPE cells. hESC-RPE monolayer polarization (trans epithelial resistance above 300 Ohm/cm2) was achieved with long-term growth on permeable inserts. A pool of constructs consisting of three siRNA targeting CIITA were used to suppress CIITA expression. Transfected cells and controls treated with scrambled RNA were stimulated with interferon gamma to induce MHC-II expression. Stimulated expression of CIITA and MHC-II was quantified by qPCR and western blot techniques. Transfected cells were co-cultured with naïve lymphocytes and reactive and regulatory T cell differentiation was analyzed by flow cytometry.

Results: Polarized hES-RPE were successfully transfected with anti-CIITA siRNA. Interferon gamma-stimulated CIITA and MHC-II expression was knocked down with more than 95% efficiency. CIITA-knocked down RPE's ability to stimulate reactive T-lymphocyte differentiation was limited.

Conclusions: RNA modulation of CIITA expression in polarized hESC-RPE results in significant suppression of MHC-II expression upon immune stimulation. Stable suppression of MHC-II expression in differentiated hESC-RPE cells can render these cells less immunogenic, potentially leading to improved survival of implanted cells. Such treatment would have a major clinical impact if the need for systemic use of immunosuppressive agents was eliminated.

Keywords: 412 age-related macular degeneration • 555 immunomodulation/immunoregulation • 721 stem cells