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RGC degeneration in injury and\/or disease also occurs in a compartmentalized manner, with distinct injury responses in axonal and somatodendritic compartments. Thus, the goal of this study was to establish a novel microfluidic-based platform for the analysis of RGC compartmentalization in health and disease states. Human pluripotent stem cell (hPSC)-derived RGCs were seeded into microfluidics, enabling the recruitment and isolation of axons apart from the somatodendritic compartment. Initial studies explored axonal outgrowth and compartmentalization of axons and dendrites. We then compared the differential response of RGCs differentiated from hPSCs carrying the OPTN(E50K) glaucoma mutation with isogenic control RGCs in their respective axonal and somatodendritic compartments, followed by analysis of axonal transport. Further, we explored the axonal transcriptome via RNA-seq, focusing on disease-related axonal differences. Finally, we established models to uniquely orient astrocytes along the axonal compartment combined with modulation of astrocyte reactivity as a pathological feature of neurodegeneration. Overall, RGC culture within microfluidic chips allowed enhanced cell growth and maturation, including long-distance axonal projections and proper compartmentalization, while patient-specific RGCs exhibited axonal outgrowth deficits as well as decreased rate of axonal transport. Finally, the induction of astrocyte reactivity uniquely along the proximal region of RGC axons led to the onset of neurodegenerative phenotypes in RGCs. These results represent the first study to effectively recapitulate the highly compartmentalized properties of hPSC-derived RGCs in healthy and disease states, providing a more physiologically relevant in vitro model for neuronal development and degeneration.<\/jats:p>","DOI":"10.1101\/2025.05.02.651934","type":"posted-content","created":{"date-parts":[[2025,5,8]],"date-time":"2025-05-08T12:50:18Z","timestamp":1746708618000},"source":"Crossref","is-referenced-by-count":1,"title":["Modeling human retinal ganglion cell axonal outgrowth, development, and pathology using pluripotent stem cell-based microfluidic platforms"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8491-6822","authenticated-orcid":false,"given":"C\u00e1tia","family":"Gomes","sequence":"first","affiliation":[{"name":"Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202"},{"name":"Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kang-Chieh","family":"Huang","sequence":"additional","affiliation":[{"name":"Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202"},{"name":"Department of Biology, Indiana University Purdue University Indianapolis, Indianapolis, IN 46202"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sailee S.","family":"Lavekar","sequence":"additional","affiliation":[{"name":"Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202"},{"name":"Department of Biology, Indiana University Purdue University Indianapolis, Indianapolis, IN 46202"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jade","family":"Harkin","sequence":"additional","affiliation":[{"name":"Department of Biology, Indiana University Purdue University Indianapolis, Indianapolis, IN 46202"},{"name":"Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carson","family":"Prosser","sequence":"additional","affiliation":[{"name":"Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202"},{"name":"Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yue","family":"Fang","sequence":"additional","affiliation":[{"name":"Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claire","family":"Kalem","sequence":"additional","affiliation":[{"name":"Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202"},{"name":"Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adrian","family":"Oblak","sequence":"additional","affiliation":[{"name":"Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202"},{"name":"Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chi","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9037-6033","authenticated-orcid":false,"given":"Jason S.","family":"Meyer","sequence":"additional","affiliation":[{"name":"Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202"},{"name":"Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202"},{"name":"Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202"},{"name":"Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, IN 46202"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026041121073680000_2025.05.02.651934v1.1","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.1711-16.2016"},{"key":"2026041121073680000_2025.05.02.651934v1.2","doi-asserted-by":"crossref","unstructured":"L. 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