{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T23:10:17Z","timestamp":1790291417093,"version":"4.1.0"},"posted":{"date-parts":[[2025,5,21]]},"group-title":"Neuroscience","reference-count":67,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,5,21]],"date-time":"2025-05-21T00:00:00Z","timestamp":1747785600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,5,21]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  The host retinal microglia and macrophage activation remains a major challenge for the integration of donor neurons following transplantation. Previously, we and others have shown that it is possible to increase donor retinal ganglion cell (RGC) survival by inhibiting the microglia-RGC interaction with Annexin V or through reprogramming microglia with the soluble Fas ligand. However, the exact mechanisms of the microglia\/macrophage activation and their heterogeneity following transplantation remain unknown. To address this question, the donor RGC were differentiated from Brn3b-Tdtomato-Thy1.2 human embryonic stem cells using a 3D protocol, followed by dissociation and RGC purification. RGC were delivered subretinally (1.5x10\n                  <jats:sup>4<\/jats:sup>\n                  viable cells\/eye) into 3-6-month-old\n                  <jats:italic>\n                    CX3CR1\n                    <jats:sup>GFP<\/jats:sup>\n                  <\/jats:italic>\n                  knock-in mice. Three days after transplantation retinas were dissociated into single-cell suspension and GFP-positive myeloid cells isolated using FACS. Of the sorted cells, up to 10,000 viable cells per sample were used for single-cell RNA library preparation and sequenced using the 10X Genomics Chromium platform. In addition, several retinas were fixed and stained for donor RGC (mCherry) and host microglia\/macrophages (Iba1). RNA Velocity was used to reconstruct the myeloid cell population and activation trajectory from scRNAseq data. We observed continuous bi-directional transition of microglia\/macrophages from a homeostatic to an activated state. We also observed that the response to the transplant falls into the classic disease-associated-microglia (DAM) activation paradigm with a decrease in expression of the homeostatic gene\n                  <jats:italic>Tmem119<\/jats:italic>\n                  and an increase in expression of disease-associated genes including\n                  <jats:italic>Apoe, Lgals3,<\/jats:italic>\n                  and\n                  <jats:italic>Spp1<\/jats:italic>\n                  . Our findings show that the host retinal myeloid cell population undergoes activation upon transplantation of stem-cell derived donor RGC, with a molecular profile of the activated cells similar to that of activated myeloid cells associated with neurodegenerative diseases of the brain and the eye. Advanced integrated transcriptomic analysis shows separate activated-to-homeostatic and homeostatic-to-activated trajectories suggesting the reversibility of this process.\n                <\/jats:p>","DOI":"10.1101\/2025.05.16.654622","type":"posted-content","created":{"date-parts":[[2025,5,21]],"date-time":"2025-05-21T17:55:12Z","timestamp":1747850112000},"source":"Crossref","is-referenced-by-count":5,"title":["Single-cell transcriptome of retinal myeloid cells in response to transplantation of human neurons reveals reversibility of microglial activation"],"prefix":"10.64898","author":[{"given":"Emil","family":"Kriukov","sequence":"first","affiliation":[{"name":"Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, MA, USA"},{"name":"Department of Ophthalmology, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anthony","family":"Mukwaya","sequence":"additional","affiliation":[{"name":"Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, MA, USA"},{"name":"Department of Ophthalmology, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul Francis","family":"Cullen","sequence":"additional","affiliation":[{"name":"Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, MA, USA"},{"name":"Department of Ophthalmology, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"George","family":"Baldwin","sequence":"additional","affiliation":[{"name":"Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, MA, USA"},{"name":"Department of Ophthalmology, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Volha V.","family":"Malechka","sequence":"additional","affiliation":[{"name":"Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, MA, USA"},{"name":"Department of Ophthalmology, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nasrin","family":"Refaian","sequence":"additional","affiliation":[{"name":"Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, MA, USA"},{"name":"Department of Ophthalmology, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nikita","family":"Bagaev","sequence":"additional","affiliation":[{"name":"Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, MA, USA"},{"name":"Department of Ophthalmology, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Everett","family":"Labrecque","sequence":"additional","affiliation":[{"name":"Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, MA, USA"},{"name":"Department of Ophthalmology, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sthavir","family":"Vinjamuri","sequence":"additional","affiliation":[{"name":"Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, MA, USA"},{"name":"Department of Ophthalmology, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Milica A.","family":"Margeta","sequence":"additional","affiliation":[{"name":"Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, MA, USA"},{"name":"Department of Ophthalmology, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Petr","family":"Baranov","sequence":"additional","affiliation":[{"name":"Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, MA, USA"},{"name":"Department of Ophthalmology, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026041122475722000_2025.05.16.654622v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/S0896-6273(02)00602-5"},{"key":"2026041122475722000_2025.05.16.654622v1.2","unstructured":"Soucy, J. R. et al. Retinal ganglion cell repopulation for vision restoration in optic neuropathy: a roadmap from the RReSTORe Consortium. Mol. Neurodegener. 18, 64 (2023)."},{"key":"2026041122475722000_2025.05.16.654622v1.3","doi-asserted-by":"crossref","unstructured":"Oswald, J. & Baranov, P . Regenerative medicine in the retina: from stem cells to cell replacement therapy. Ther Adv Ophthalmol 10, 2515841418774433 (2018).","DOI":"10.1177\/2515841418774433"},{"key":"2026041122475722000_2025.05.16.654622v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.omtm.2021.03.004"},{"key":"2026041122475722000_2025.05.16.654622v1.5","doi-asserted-by":"crossref","unstructured":"Murakami, Y. , Ishikawa, K. , Nakao, S. & Sonoda, K.-H . Innate immune response in retinal homeostasis and inflammatory disorders. Prog. Retin. Eye Res. 74, 100778 (2020).","DOI":"10.1016\/j.preteyeres.2019.100778"},{"key":"2026041122475722000_2025.05.16.654622v1.6","doi-asserted-by":"crossref","unstructured":"Tan, Z. et al. Microglia depletion exacerbates retinal ganglion cell loss in a mouse model of glaucoma. Exp Eye Res 225, 109273.","DOI":"10.1016\/j.exer.2022.109273"},{"key":"2026041122475722000_2025.05.16.654622v1.7","first-page":"4626","article-title":"Overexpression of Soluble Fas Ligand following Adeno-Associated Virus Gene Therapy Prevents Retinal Ganglion Cell Death in Chronic and Acute Murine Models of Glaucoma","volume":"197","year":"2016","journal-title":"Journal of immunology (Baltimore, Md.LJ: 1950)"},{"key":"2026041122475722000_2025.05.16.654622v1.8","doi-asserted-by":"crossref","unstructured":"Au, N. P. B. & Ma, C. H. E . Neuroinflammation, Microglia and Implications for Retinal Ganglion Cell Survival and Axon Regeneration in Traumatic Optic Neuropathy. Front Immunol 13, 860070 (2022).","DOI":"10.3389\/fimmu.2022.860070"},{"key":"2026041122475722000_2025.05.16.654622v1.9","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2022.07.014"},{"key":"2026041122475722000_2025.05.16.654622v1.10","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuroscience.2008.06.046"},{"key":"2026041122475722000_2025.05.16.654622v1.11","doi-asserted-by":"publisher","DOI":"10.1101\/2024.07.06.602307"},{"key":"2026041122475722000_2025.05.16.654622v1.12","doi-asserted-by":"publisher","DOI":"10.1021\/acsnano.8b00596"},{"key":"2026041122475722000_2025.05.16.654622v1.13","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2302089120"},{"key":"2026041122475722000_2025.05.16.654622v1.14","doi-asserted-by":"publisher","DOI":"10.1101\/2024.03.07.583961"},{"key":"2026041122475722000_2025.05.16.654622v1.15","doi-asserted-by":"publisher","DOI":"10.1093\/brain\/awt292"},{"key":"2026041122475722000_2025.05.16.654622v1.16","doi-asserted-by":"publisher","DOI":"10.1016\/j.stemcr.2020.12.001"},{"key":"2026041122475722000_2025.05.16.654622v1.17","doi-asserted-by":"publisher","DOI":"10.1016\/J.NEURON.2022.10.020"},{"key":"2026041122475722000_2025.05.16.654622v1.18","doi-asserted-by":"publisher","DOI":"10.1016\/S1350-9462(00)00027-6"},{"key":"2026041122475722000_2025.05.16.654622v1.19","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2017.08.008"},{"key":"2026041122475722000_2025.05.16.654622v1.20","doi-asserted-by":"publisher","DOI":"10.1016\/J.CELL.2021.04.048"},{"key":"2026041122475722000_2025.05.16.654622v1.21","doi-asserted-by":"publisher","DOI":"10.1016\/j.cels.2019.03.003"},{"key":"2026041122475722000_2025.05.16.654622v1.22","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-018-0414-6"},{"key":"2026041122475722000_2025.05.16.654622v1.23","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-020-0591-3"},{"key":"2026041122475722000_2025.05.16.654622v1.24","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-019-0619-0"},{"key":"2026041122475722000_2025.05.16.654622v1.25","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-018-0229-21"},{"key":"2026041122475722000_2025.05.16.654622v1.26","doi-asserted-by":"crossref","unstructured":"Faure, L. , Soldatov, R. , Kharchenko, P. V. & Adameyko, I . scFates: a scalable python package for advanced pseudotime and bifurcation analysis from single-cell data. Bioinformatics 39, btac746 (2022).","DOI":"10.1093\/bioinformatics\/btac746"},{"key":"2026041122475722000_2025.05.16.654622v1.27","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2019.05.031"},{"key":"2026041122475722000_2025.05.16.654622v1.28","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-21246-9"},{"key":"2026041122475722000_2025.05.16.654622v1.29","doi-asserted-by":"publisher","DOI":"10.1038\/s41596-024-01045-4"},{"key":"2026041122475722000_2025.05.16.654622v1.30","doi-asserted-by":"crossref","unstructured":"Borcherding, N. et al. Mapping the immune environment in clear cell renal carcinoma by single-cell genomics. Commun . Biol. 4, 122 (2021).","DOI":"10.1038\/s42003-020-01625-6"},{"key":"2026041122475722000_2025.05.16.654622v1.31","doi-asserted-by":"crossref","unstructured":"Luo, X.-G. & Chen, S.-D . The changing phenotype of microglia from homeostasis to disease. Transl. Neurodegener. 1, 9 (2012).","DOI":"10.1186\/2047-9158-1-9"},{"key":"2026041122475722000_2025.05.16.654622v1.32","doi-asserted-by":"crossref","unstructured":"Ruan, C. & Elyaman, W . A New Understanding of TMEM119 as a Marker of Microglia. Front. Cell. Neurosci. 16, 902372 (2022).","DOI":"10.3389\/fncel.2022.902372"},{"key":"2026041122475722000_2025.05.16.654622v1.33","doi-asserted-by":"publisher","DOI":"10.15252\/embj.2018100811"},{"key":"2026041122475722000_2025.05.16.654622v1.34","doi-asserted-by":"publisher","DOI":"10.1038\/s41433-023-02539-3"},{"key":"2026041122475722000_2025.05.16.654622v1.35","doi-asserted-by":"publisher","DOI":"10.1101\/2024.07.14.603469"},{"key":"2026041122475722000_2025.05.16.654622v1.36","doi-asserted-by":"publisher","DOI":"10.1101\/2024.04.23.589797"},{"key":"2026041122475722000_2025.05.16.654622v1.37","doi-asserted-by":"crossref","unstructured":"Pan, L. et al. IGFBPL1 is a master driver of microglia homeostasis and resolution of neuroinflammation in glaucoma and brain tauopathy. Cell Rep. 42, 112889 (2023).","DOI":"10.1016\/j.celrep.2023.112889"},{"key":"2026041122475722000_2025.05.16.654622v1.38","doi-asserted-by":"publisher","DOI":"10.1038\/s41593-024-01827-9"},{"key":"2026041122475722000_2025.05.16.654622v1.39","doi-asserted-by":"publisher","DOI":"10.1038\/nn.3599"},{"key":"2026041122475722000_2025.05.16.654622v1.40","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1525528113"},{"key":"2026041122475722000_2025.05.16.654622v1.41","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2017.05.018"},{"key":"2026041122475722000_2025.05.16.654622v1.42","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2019.02.007"},{"key":"2026041122475722000_2025.05.16.654622v1.43","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2019.04.062"},{"key":"2026041122475722000_2025.05.16.654622v1.44","doi-asserted-by":"publisher","DOI":"10.1002\/dvdy.1"},{"key":"2026041122475722000_2025.05.16.654622v1.45","doi-asserted-by":"crossref","unstructured":"Anderson, S. R. et al. Neuronal apoptosis drives remodeling states of microglia and shifts in survival pathway dependence. eLife 11, (2022).","DOI":"10.7554\/eLife.76564"},{"key":"2026041122475722000_2025.05.16.654622v1.46","doi-asserted-by":"crossref","unstructured":"Pitts, K. M. & Margeta, M. A . Myeloid masquerade: Microglial transcriptional signatures in retinal development and disease. Front. Cell. Neurosci. 17, 1106547 (2023).","DOI":"10.3389\/fncel.2023.1106547"},{"key":"2026041122475722000_2025.05.16.654622v1.47","first-page":"717","article-title":"The cGAS\u2013STING pathway in diabetic retinopathy and age-related macular degeneration. Futur","volume":"15","year":"2023","journal-title":"Med. Chem"},{"key":"2026041122475722000_2025.05.16.654622v1.48","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2409493121"},{"key":"2026041122475722000_2025.05.16.654622v1.49","doi-asserted-by":"publisher","DOI":"10.18632\/aging.205900"},{"key":"2026041122475722000_2025.05.16.654622v1.50","doi-asserted-by":"publisher","DOI":"10.1182\/blood.2020008911"},{"key":"2026041122475722000_2025.05.16.654622v1.51","doi-asserted-by":"publisher","DOI":"10.1038\/jid.2014.117"},{"key":"2026041122475722000_2025.05.16.654622v1.52","doi-asserted-by":"publisher","DOI":"10.1016\/S0024-3205(98)00107-6"},{"key":"2026041122475722000_2025.05.16.654622v1.53","doi-asserted-by":"publisher","DOI":"10.1002\/jmv.25578"},{"key":"2026041122475722000_2025.05.16.654622v1.54","doi-asserted-by":"publisher","DOI":"10.1016\/S0968-0004(02)02145-X"},{"key":"2026041122475722000_2025.05.16.654622v1.55","doi-asserted-by":"crossref","unstructured":"Rumford, J. E. et al. Forced MyD88 signaling in microglia impacts the production and survival of regenerated retinal neurons. Front. Cell Dev. Biol. 12, 1495586 (2024).","DOI":"10.3389\/fcell.2024.1495586"},{"key":"2026041122475722000_2025.05.16.654622v1.56","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(01)00295-1"},{"key":"2026041122475722000_2025.05.16.654622v1.57","doi-asserted-by":"crossref","unstructured":"Lalitha, S. et al. Pax6 modulates intra-retinal axon guidance and fasciculation of retinal ganglion cells during retinogenesis. Sci. Rep. 10, 16075 (2020).","DOI":"10.1038\/s41598-020-72828-4"},{"key":"2026041122475722000_2025.05.16.654622v1.58","doi-asserted-by":"publisher","DOI":"10.1101\/2024.10.16.618776"},{"key":"2026041122475722000_2025.05.16.654622v1.59","doi-asserted-by":"publisher","DOI":"10.1038\/ncb3493"},{"key":"2026041122475722000_2025.05.16.654622v1.60","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-021-01346-6"},{"key":"2026041122475722000_2025.05.16.654622v1.61","doi-asserted-by":"crossref","unstructured":"VandenBosch, L. S. et al. Developmental changes in the accessible chromatin, transcriptome and Ascl1-binding correlate with the loss in M\u00fcller Glial regenerative potential. Sci. Rep. 10, 13615 (2020).","DOI":"10.1038\/s41598-020-70334-1"},{"key":"2026041122475722000_2025.05.16.654622v1.62","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2020.01.007"},{"key":"2026041122475722000_2025.05.16.654622v1.63","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1510595112"},{"key":"2026041122475722000_2025.05.16.654622v1.64","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.13-12596"},{"key":"2026041122475722000_2025.05.16.654622v1.65","doi-asserted-by":"publisher","DOI":"10.1016\/j.expneurol.2003.10.021"},{"key":"2026041122475722000_2025.05.16.654622v1.66","doi-asserted-by":"publisher","DOI":"10.1016\/j.stemcr.2022.10.011"},{"key":"2026041122475722000_2025.05.16.654622v1.67","doi-asserted-by":"crossref","first-page":"131","DOI":"10.3233\/RNN-190941","article-title":"Cell transplantation of retinal ganglion cells derived from hESCs","volume":"38","year":"2020","journal-title":"Restorative neurology and neuroscience"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.05.16.654622","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,12]],"date-time":"2026-04-12T05:48:04Z","timestamp":1775972884000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.05.16.654622"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,5,21]]},"references-count":67,"URL":"https:\/\/doi.org\/10.1101\/2025.05.16.654622","relation":{},"subject":[],"published":{"date-parts":[[2025,5,21]]},"subtype":"preprint"}}