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Increasing evidence implicates microglia, the resident immune cells of the central nervous system, as key modulators of RGC health and disease progression. However, the precise mechanisms by which microglia influence RGCs remain poorly understood, particularly in the human context. In this study, we established human pluripotent stem cell (hPSC)-derived co-culture systems incorporating microglia, astrocytes, and RGCs to explore how microglia shape RGC growth and maturation under physiological conditions. We first examined the impact of homeostatic microglia on RGCs in both co-culture and tri-culture systems, revealing distinct influences of cell types in co-culture compared to when they were grown individually. We then modeled inflammatory states by activating microglia with lipopolysaccharide (LPS) and evaluated their effects on RGCs both directly and in the context of astrocyte co-culture. This stepwise, reductionist approach enabled us to dissect the cellular interactions driving RGC vulnerability in inflammatory conditions relevant to optic neuropathies. Our findings provide new insight into the complex neuroimmune landscape that underlies RGC degeneration and identify key pathways that may serve as therapeutic targets across a range of optic nerve diseases.<\/jats:p>","DOI":"10.1101\/2025.07.01.662573","type":"posted-content","created":{"date-parts":[[2025,7,4]],"date-time":"2025-07-04T17:40:40Z","timestamp":1751650840000},"source":"Crossref","is-referenced-by-count":3,"title":["A human pluripotent stem cell tri-culture platform to elucidate microglial regulation of retinal ganglion cells in neuroinflammation"],"prefix":"10.64898","author":[{"given":"Jade","family":"Harkin","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"C\u00e1tia","family":"Gomes","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaylee D.","family":"Tutrow","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Reham","family":"Afify","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shruti V.","family":"Patil","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kiersten H.","family":"Pe\u00f1a","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aaron","family":"Baker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sailee S.","family":"Lavekar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kang-Chieh","family":"Huang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jason S.","family":"Meyer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025070714403272000_2025.07.01.662573v1.1","doi-asserted-by":"publisher","DOI":"10.1002\/glia.22408"},{"key":"2025070714403272000_2025.07.01.662573v1.2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-immunol-051116-052358"},{"key":"2025070714403272000_2025.07.01.662573v1.3","doi-asserted-by":"publisher","DOI":"10.1124\/jpet.120.265058"},{"key":"2025070714403272000_2025.07.01.662573v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.conb.2015.12.004"},{"key":"2025070714403272000_2025.07.01.662573v1.5","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.1711-16.2016"},{"key":"2025070714403272000_2025.07.01.662573v1.6","doi-asserted-by":"crossref","unstructured":"L. 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