{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T12:55:54Z","timestamp":1773924954537,"version":"3.50.1"},"reference-count":37,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T00:00:00Z","timestamp":1773878400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Joint Special Funds for the Department of Science and Technology of Yunnan Province\u2013Kunming Medical University","award":["202501AY070001-027"],"award-info":[{"award-number":["202501AY070001-027"]}]},{"name":"Yunnan Revitalization Talent Support Program","award":["XDYC-QNRC-2023-0177"],"award-info":[{"award-number":["XDYC-QNRC-2023-0177"]}]},{"name":"First-Class Discipline Team of Kunming Medical University","award":["2024XKTDPY16"],"award-info":[{"award-number":["2024XKTDPY16"]}]},{"name":"College Students\u2019 Innovation and Entrepreneurship Training Program of Kunming Medical University","award":["202510678052"],"award-info":[{"award-number":["202510678052"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Dogs represent a promising animal model for analyzing human neurodegenerative diseases, owing to their similarities to humans in nervous system architecture and behavioral phenotypes. Neural stem cells (NSCs) serve as a highly valuable in vitro experimental model for investigating neurogenesis, neurodegenerative disease pathogenesis, and neural molecular biology; however, studies on immortalized canine neural stem cell lines remain scarce. Herein, we successfully established an immortalized canine hippocampal neural stem cell line that can be continuously passaged in vitro via SV40 large T antigen (SV40LT) viral infection and subsequent cellular transformation. Both the immortalized NSCs and their normal parental counterparts differentiated into neuronal and glial lineages under induced differentiation conditions. Normal canine hippocampal NSCs can be passaged for no more than 10 generations, whereas the immortalized line can be passaged indefinitely while maintaining a normal karyotype. This immortalized canine hippocampal NSC line can act as a critical experimental tool for future research into neural differentiation mechanisms and stem cell-derived therapeutic strategies for neurological disorders in dogs.<\/jats:p>","DOI":"10.3390\/cells15060543","type":"journal-article","created":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T08:54:37Z","timestamp":1773910477000},"page":"543","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Establishment of an Immortalized Canine Hippocampal Neural Stem Cell Line via SV40LT Retroviral Transduction"],"prefix":"10.3390","volume":"15","author":[{"given":"Yankun","family":"Ke","sequence":"first","affiliation":[{"name":"Institute of Biomedical Engineering, Kunming Medical University, Kunming 650500, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zixin","family":"Li","sequence":"additional","affiliation":[{"name":"Faculty of First Clinical Medical, Kunming Medical University, Kunming 650500, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Huaiyu","family":"Wang","sequence":"additional","affiliation":[{"name":"Institute of Biomedical Engineering, Kunming Medical University, Kunming 650500, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yixuan","family":"Zhang","sequence":"additional","affiliation":[{"name":"Institute of Biomedical Engineering, Kunming Medical University, Kunming 650500, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shiyu","family":"Xu","sequence":"additional","affiliation":[{"name":"Institute of Biomedical Engineering, Kunming Medical University, Kunming 650500, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0259-9024","authenticated-orcid":false,"given":"Longlong","family":"Zhang","sequence":"additional","affiliation":[{"name":"Institute of Biomedical Engineering, Kunming Medical University, Kunming 650500, China"},{"name":"Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650201, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e2414890","DOI":"10.1002\/advs.202414890","article-title":"Unraveling Neurodevelopment: Synergistic Effects of Intrinsic Genetic Programs and Extrinsic Environmental Cues","volume":"12","author":"Wang","year":"2025","journal-title":"Adv. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1197","DOI":"10.1186\/s12967-025-07266-9","article-title":"Exploration of biomaterial and stem cell-based strategies for promoting neuronal regeneration and creating engineered 3D in-vitro disease models","volume":"23","author":"Khodve","year":"2025","journal-title":"J. Transl. Med."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Zholudeva, L.V., Bourbeau, D., Hall, A., Spruance, V., Ogbolu, V., Qiang, L., Sakiyama-Elbert, S., and Lane, M.A. (2026). Beyond Transplantation: Engineering Neural Cell Therapies and Combination Strategies for Spinal Cord Repair. Brain Sci., 16.","DOI":"10.3390\/brainsci16010113"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1433","DOI":"10.1126\/science.287.5457.1433","article-title":"Mammalian neural stem cells","volume":"287","author":"Gage","year":"2000","journal-title":"Science"},{"key":"ref_5","first-page":"1321","article-title":"Specific ablation of Nampt in adult neural stem cells recapitulates their functional defects during aging","volume":"33","author":"Stein","year":"2014","journal-title":"EMBO J."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"176","DOI":"10.18632\/aging.100446","article-title":"Age-related cognitive decline: Can neural stem cells help us?","volume":"4","author":"Artegiani","year":"2012","journal-title":"Aging"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"897","DOI":"10.1016\/j.cell.2016.10.021","article-title":"Adult Neurogenesis in the Hippocampus: From Stem Cells to Behavior","volume":"167","author":"Schafer","year":"2016","journal-title":"Cell"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2312","DOI":"10.1634\/stemcells.2007-0040","article-title":"A novel, immortal, and multipotent human neural stem cell line generating functional neurons and oligodendrocytes","volume":"25","author":"Lamorte","year":"2007","journal-title":"Stem Cells"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"559","DOI":"10.1016\/j.yexcr.2003.11.025","article-title":"Long-term molecular and cellular stability of human neural stem cell lines","volume":"294","author":"Villa","year":"2004","journal-title":"Exp. Cell Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1038\/35036093","article-title":"Hayflick, his limit, and cellular ageing","volume":"1","author":"Shay","year":"2000","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2815","DOI":"10.1038\/nprot.2007.402","article-title":"Retrovirally mediated telomerase immortalization of neural progenitor cells","volume":"2","author":"Roy","year":"2007","journal-title":"Nat. Protoc."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3255","DOI":"10.1073\/pnas.85.9.3255","article-title":"Immortalization of mouse neural precursor cells by the c-myc oncogene","volume":"85","author":"Bartlett","year":"1988","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"271","DOI":"10.3727\/096368916X692852","article-title":"Establishment and Characterization of Immortalized Minipig Neural Stem Cell Line","volume":"26","author":"Choi","year":"2017","journal-title":"Cell Transplant."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1186\/scrt220","article-title":"Conditionally immortalized stem cell lines from human spinal cord retain regional identity and generate functional V2a interneurons and motorneurons","volume":"4","author":"Cocks","year":"2013","journal-title":"Stem Cell Res. Ther."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1204","DOI":"10.1634\/stemcells.2006-0409","article-title":"Brain transplantation of immortalized human neural stem cells promotes functional recovery in mouse intracerebral hemorrhage stroke model","volume":"25","author":"Lee","year":"2007","journal-title":"Stem Cells"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"nwac147","DOI":"10.1093\/nsr\/nwac147","article-title":"A single-nucleus transcriptomic atlas of the dog hippocampus reveals the potential relationship between specific cell types and domestication","volume":"9","author":"Zhou","year":"2022","journal-title":"Natl. Sci. Rev."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Zhang, L., Ye, M., Zhu, L., Cha, J., Li, C., Yao, Y.G., and Mao, B. (2020). Loss of ZC4H2 and RNF220 Inhibits Neural Stem Cell Proliferation and Promotes Neuronal Differentiation. Cells, 9.","DOI":"10.3390\/cells9071600"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"653","DOI":"10.1016\/j.stem.2016.03.020","article-title":"Pharmacological Reprogramming of Fibroblasts into Neural Stem Cells by Signaling-Directed Transcriptional Activation","volume":"18","author":"Zhang","year":"2016","journal-title":"Cell Stem Cell"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Nunes, B.T.D., de Mendon\u00e7a, M.H.R., Simith, D.B., Moraes, A.F., Cardoso, C.C., Prazeres, I.T.E., de Aquino, A.A., Santos, A., Queiroz, A.L.N., and Rodrigues, D.S.G. (2019). Development of RT-qPCR and semi-nested RT-PCR assays for molecular diagnosis of hantavirus pulmonary syndrome. PLoS Neglected Trop. Dis., 13.","DOI":"10.1371\/journal.pntd.0007884"},{"key":"ref_20","first-page":"475","article-title":"gfap and nestin reporter lines reveal characteristics of neural progenitors in the adult zebrafish brain","volume":"238","author":"Lam","year":"2009","journal-title":"Dev. Dyn. Off. Publ. Am. Assoc. Anat."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"10217","DOI":"10.1523\/JNEUROSCI.1469-15.2015","article-title":"FGF Signaling Is Necessary for Neurogenesis in Young Mice and Sufficient to Reverse Its Decline in Old Mice","volume":"35","author":"Kang","year":"2015","journal-title":"J. Neurosci. Off. J. Soc. Neurosci."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Hall, C.M., Lasli, S., Serwinski, B., Djordjevic, B., Sheridan, G.K., and Moeendarbary, E. (2023). Hippocampus of the APP(NL-G-F) mouse model of Alzheimer\u2019s disease exhibits region-specific tissue softening concomitant with elevated astrogliosis. Front. Aging Neurosci., 15.","DOI":"10.3389\/fnagi.2023.1212212"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1016\/j.neulet.2016.12.068","article-title":"Analysis of gene expression in Ca2+-dependent activator protein for secretion 2 (Cadps2) knockout cerebellum using GeneChip and KEGG pathways","volume":"639","author":"Sadakata","year":"2017","journal-title":"Neurosci. Lett."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"735","DOI":"10.1002\/cne.20947","article-title":"Differential distributions of the Ca2+-dependent activator protein for secretion family proteins (CAPS2 and CAPS1) in the mouse brain","volume":"495","author":"Sadakata","year":"2006","journal-title":"J. Comp. Neurol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1017","DOI":"10.1002\/hipo.22287","article-title":"Age-related differences in oligodendrogenesis across the dorsal-ventral axis of the mouse hippocampus","volume":"24","author":"Yamada","year":"2014","journal-title":"Hippocampus"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"102600","DOI":"10.1016\/j.stemcr.2025.102600","article-title":"Adult hippocampal neurogenesis: New avenues for treatment of brain disorders","volume":"20","author":"Chen","year":"2025","journal-title":"Stem Cell Rep."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1586\/14789450.5.2.175","article-title":"Proteomics of neural stem cells","volume":"5","author":"Skalnikova","year":"2008","journal-title":"Expert Rev. Proteom."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1548","DOI":"10.1091\/mbc.e06-11-1013","article-title":"Immortalization of neural precursors when telomerase is overexpressed in embryonal carcinomas and stem cells","volume":"19","author":"Schwob","year":"2008","journal-title":"Mol. Biol. Cell"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2802","DOI":"10.1101\/gad.207142.112","article-title":"Neural-specific Sox2 input and differential Gli-binding affinity provide context and positional information in Shh-directed neural patterning","volume":"26","author":"Peterson","year":"2012","journal-title":"Genes Dev."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.pnpbp.2013.04.002","article-title":"Brain structural abnormalities in Doberman pinschers with canine compulsive disorder","volume":"45","author":"Ogata","year":"2013","journal-title":"Prog. Neuro-Psychopharmacol. Biol. Psychiatry"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1016\/j.tics.2022.01.003","article-title":"Comparing wolves and dogs: Current status and implications for human \u2018self-domestication\u2019","volume":"26","author":"Range","year":"2022","journal-title":"Trends Cogn. Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"7748","DOI":"10.1523\/JNEUROSCI.0303-19.2019","article-title":"Significant Neuroanatomical Variation Among Domestic Dog Breeds","volume":"39","author":"Hecht","year":"2019","journal-title":"J. Neurosci. Off. J. Soc. Neurosci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1706","DOI":"10.1101\/gr.3736605","article-title":"The canine genome","volume":"15","author":"Ostrander","year":"2005","journal-title":"Genome Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"375","DOI":"10.1177\/0271678X19827446","article-title":"Large animals in neurointerventional research: A systematic review on models, techniques and their application in endovascular procedures for stroke, aneurysms and vascular malformations","volume":"39","author":"Herrmann","year":"2019","journal-title":"J. Cereb. Blood Flow Metab. Off. J. Int. Soc. Cereb. Blood Flow Metab."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Hernandez, J., Rhimi, S., Kriaa, A., Mariaule, V., Boudaya, H., Drut, A., Jablaoui, A., Mkaouar, H., Saidi, A., and Biourge, V. (2022). Domestic Environment and Gut Microbiota: Lessons from Pet Dogs. Microorganisms, 10.","DOI":"10.3390\/microorganisms10050949"},{"key":"ref_36","first-page":"ajvr.25.02.0053","article-title":"Recent advances in diagnostic and therapeutic strategies for canine cognitive dysfunction","volume":"86","author":"Kim","year":"2025","journal-title":"Am. J. Vet. Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"S42","DOI":"10.1038\/nm1064","article-title":"Stem cell therapy for human neurodegenerative disorders-how to make it work","volume":"10","author":"Lindvall","year":"2004","journal-title":"Nat. Med."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/6\/543\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T09:11:19Z","timestamp":1773911479000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/6\/543"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,19]]},"references-count":37,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2026,3]]}},"alternative-id":["cells15060543"],"URL":"https:\/\/doi.org\/10.3390\/cells15060543","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,19]]}}}