title: A method for generating high-yield enriched neuronal cultures from P19 embryonal carcinoma cells.
abstract: P19 embryonal carcinoma (EC) cells are an invaluable tool for approximating the mechanisms that govern neuronal differentiation but with an enormous degree of simplification and have primarily been used to model the early stages of neurogenesis. However, they are often cultured under conditions that promote unrestricted non-neuronal growth that compromises neuronal viability. In this study we report an improved method to differentiate P19 EC cells that gives rise to high yields of functionally and morphologically mature neurons while significantly reducing the over-growth of non-neuronal cells in the cultures. In this protocol, P19 EC cells are induced in Minimum Essential Medium alpha supplemented with all-trans retinoic acid (RA) and 2.5% serum, and cultured as a monolayer. After RA-induction, cells are cultured on Matrigel coated-plates using defined media comprised of Neurobasal-A medium temporally supplemented with N2 and then B-27 for the remaining culture period. By treating the culture with Cytosine β-d-arabinofuranoside and 2'-Deoxycytidine for five days, the cultures are reliably promoted toward the neuronal differentiation vs non-neuronal differentiation, this accounting for a progressive neuronal enrichment of the cultures reaching 56% after 20 days of culture. P19-derived neural progenitor cells progressively expressed neuronal markers such as NeuN, Calretinin, Calbindin and Synapsin I in close resemblance to that occurring in vivo in the central nervous system (CNS). Furthermore, RA-induced P19 EC cells progressively acquired functional neuronal traits and after approximately 3 weeks in culture revealed mature neurophysiological properties, characteristics of CNS neurons. This protocol allows for a more specific assessment of the neuronal differentiation processes in vitro.
authors.affiliation: Department of Anatomy with Radiology, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand; Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand.
authors.forename: Hector J
authors.initials: HJ
authors.lastname: Monzo
authors.affiliation: Department of Pharmacology, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand; Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand.
authors.forename: Thomas I H
authors.initials: TIH
authors.lastname: Park
authors.affiliation: Department of Physiology, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand; Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand.
authors.forename: Johanna M
authors.initials: JM
authors.lastname: Montgomery
authors.affiliation: Department of Anatomy with Radiology, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand; Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand.
authors.forename: Richard L M
authors.initials: RLM
authors.lastname: Faull
authors.affiliation: Department of Pharmacology, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand; Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand.
authors.forename: Mike
authors.initials: M
authors.lastname: Dragunow
authors.affiliation: Department of Anatomy with Radiology, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand; Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand. Electronic address: m.curtis@auckland.ac.nz.
authors.forename: Maurice A
authors.initials: MA
authors.lastname: Curtis
mesh_terms: D000818:Animals; D018236:Carcinoma, Embryonal / Q000473:pathology* / Q000503:physiopathology*; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D051379:Mice; D055495:Neurogenesis / Q000502:physiology*; D009474:Neurons / Q000166:cytology* / Q000502:physiology*; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D014212:Tretinoin