title: In vitro isolation of neural precursor cells from the adult pig subventricular zone.
abstract: In order to improve cell therapy techniques, we have characterized a multipotent neural precursor cell isolation technique from the subventricular zone of adult pig brain. The pig is a non-primate species that is immunologically closest to human. The proliferative zone of this neurogenic structure was first localized in situ in the pig brain by Ki-67 immunohistochemistry, as a ventral subfield of the Nissl-stained subventricular zone. For in vitro cultures, the striatal forebrain was sampled from deeply anaesthetized adult pigs and SVZ tissue explants were immediately microdissected out and dissociated in the appropriate medium. Primary cell culture in the presence of EGF and bFGF allowed growth of spherical masses that exhibited sustained growth and self-renewal capacity through six subsequent passages. Molecular characterization using reverse transcription-polymerase chain reaction (RT-PCR) showed that expanded pro-differentiating neurospheres expressed markers of proliferation, neural stem cells, and committed neural progenitors. After growth factor removal, the spheres became adherent and were shown to contain the three neural cell lineages by triple immunocytofluorescence and confocal microscopy. The present protocol therefore allowed for in vitro expansion of pig brain primary cells that display capacities for proliferation, self-renewal, and multipotency, i.e., the cardinal features of multipotent neural precursor cells.
authors.affiliation: Service de Chirurgie Maxillo-faciale et Plastique de face, HIA Laveran, BP 50, 13998 Marseille Armées, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Liard
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Segura
authors.forename: Aurélie
authors.initials: A
authors.lastname: Pascual
authors.forename: Pierrette
authors.initials: P
authors.lastname: Gaudreau
authors.forename: Thierry
authors.initials: T
authors.lastname: Fusai
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Moyse
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D004815:Epidermal Growth Factor / Q000378:metabolism; D016220:Fibroblast Growth Factor 1 / Q000378:metabolism; D007150:Immunohistochemistry; D019394:Ki-67 Antigen / Q000737:chemistry; D039902:Multipotent Stem Cells / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D013687:Telencephalon / Q000166:cytology*; D016707:Tissue Fixation
chemical_list: D015415:Biomarkers; D019394:Ki-67 Antigen; D012333:RNA, Messenger; D016220:Fibroblast Growth Factor 1; D004815:Epidermal Growth Factor