Isolation and characterization of neural progenitor cells from adult canine brains

Objective: To isolate and characterize neural stem and progenitor cell populations in the brain of adult dogs. Animals: 7 healthy adult dogs. Procedures: Dogs (age, 10 to 60 months) were euthanized for reasons unrelated to the study. The subventricular zone (SVZ) adjacent to the lateral ventricles and subgranular zone (SGZ) of the hippocampus were isolated and used to generate single cell suspensions for nonadherent culture. The resulting primary neurospheres were serially passaged to assess self-renewal capacity. Neurospheres were differentiated by the withdrawal of growth factors and the addition of serum. Differentiated and undifferentiated neurospheres were analyzed via reverse transcriptase PCR assay or immunocytochemical staining for markers of pluripotency and neural lineage. Results: Neurospheres were generated from the SVZ and SGZ in all dogs. The SVZ generated more primary neurospheres than did the SGZ. Serial passage was successful, although few neurospheres could be generated after the fifth passage. Undifferentiated neurospheres were positive for SOX2, nestin, and glial fibrillary acidic protein (GFAP) and negative for OCT4 and NANOG. After differentiation, GFAP, neuronal class III β-tubulin, and 2′, 3′-cyclic nucleotide 3′-phosphodiesterase–positive progeny were noted migrating out of the neurospheres. Conclusions and Clinical Relevance: Results suggested the persistence of SOX2-positive, nestin-positive, GFAP-positive, OCT4-negative, and NANOG-negative neural progenitor cells in the SVZ and SGZ regions of mature canine brains, which are capable of producing multiple cell lineages. This study may serve as a basis for future studies investigating the role of these cells in various disease processes, such as neoplasia, or for regenerative purposes.