Elucidating the influence of microglia on retinal ganglion cells in a human pluripotent stem cell model

Abstract

Purpose : Glaucoma is the leading cause of irreversible blindness worldwide, affecting approximately 80 million people. In animal models of glaucoma, microglial activation has been associated with changes in morphology and proliferation, as well as the release of inflammatory factors, which contribute to the neurodegeneration of retinal ganglion cells (RGCs). Due to major differences identified between rodent microglia and RGCs compared to humans, there is a critical need for the development of novel human models that can be used to explore the cellular crosstalk between human microglia and RGCs and the role of these interactions in diseases such as glaucoma.

Methods : In the current study, we differentiated both microglia-like cells (MGLs), RGCs and astrocytes from human induced pluripotent stem cells (iPSCs). Microglial activation was then induced using Lipopolysaccharide (LPS) and confirmed using morphological analyses, immunostaining, and cytokine/chemokine profile screening. Healthy and LPS-activated microglia were then co-cultured with RGCs for up to 3 weeks and the effects of microglia upon RGCs were assessed using measurements of neurite complexity and functionally via multielectrode array. Finally, healthy and LPS-activated microglia and RGC co-cultures were also grown with astrocytes to assess both direct and indirect effects of activated microglia upon RGCs.

Results : Results indicated that following activation via treatment with LPS, MGLs exhibit more amoeboid morphological features and increased the expression of MHC-II, characteristic of activated microglia. MGLs also released significantly elevated levels of inflammatory cytokines including IL-6, IL-8, IL-1B and TNFa. When co-cultured with astrocytes, LPS-activated microglia promoted astrocyte reactivity. Additionally, co-cultures of MGLs and RGCs revealed that activated MGLs reduced RGC neurite complexity and decreased neuronal excitability, suggesting a contribution of microglia activation to RGC neurodegeneration. Finally, combined cultures of MGLs, astrocytes, and RGCs demonstrated complex interactions between cell types.

Conclusions : Taken together, the current study establishes the first known study to develop a human cellular model that allows for the examination of cellular interactions between human microglia, astrocytes and RGCs, including the study of microglial contributions to RGC neurodegeneration and neuroinflammation.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.