title: A neural extracellular matrix-based method for in vitro hippocampal neuron culture and dopaminergic differentiation of neural stem cells.
abstract: BACKGROUND
  The ability to recreate an optimal cellular microenvironment is critical to understand neuronal behavior and functionality in vitro. An organized neural extracellular matrix (nECM) promotes neural cell adhesion, proliferation and differentiation. Here, we expanded previous observations on the ability of nECM to support in vitro neuronal differentiation, with the following goals: (i) to recreate complex neuronal networks of embryonic rat hippocampal cells, and (ii) to achieve improved levels of dopaminergic differentiation of subventricular zone (SVZ) neural progenitor cells.
  
  
  METHODS
  Hippocampal cells from E18 rat embryos were seeded on PLL- and nECM-coated substrates. Neurosphere cultures were prepared from the SVZ of P4-P7 rat pups, and differentiation of neurospheres assayed on PLL- and nECM-coated substrates.
  
  
  RESULTS
  When seeded on nECM-coated substrates, both hippocampal cells and SVZ progenitor cells showed neural expression patterns that were similar to their poly-L-lysine-seeded counterparts. However, nECM-based cultures of both hippocampal neurons and SVZ progenitor cells could be maintained for longer times as compared to poly-L-lysine-based cultures. As a result, nECM-based cultures gave rise to a more branched neurite arborization of hippocampal neurons. Interestingly, the prolonged differentiation time of SVZ progenitor cells in nECM allowed us to obtain a purer population of dopaminergic neurons.
  
  
  CONCLUSIONS
  We conclude that nECM-based coating is an efficient substrate to culture neural cells at different stages of differentiation. In addition, neural ECM-coated substrates increased neuronal survival and neuronal differentiation efficiency as compared to cationic polymers such as poly-L-lysine.
authors.affiliation: Tissue Engineering Laboratory, Department of Bioengineering, Instituto Biodonostia, Hospital Universitario Donostia, San Sebastian, 20014, Spain. patricia.garcia@biodonostia.org
authors.forename: Patricia
authors.initials: P
authors.lastname: García-Parra
authors.forename: Marcos
authors.initials: M
authors.lastname: Maroto
authors.forename: Fabio
authors.initials: F
authors.lastname: Cavaliere
authors.forename: Neia
authors.initials: N
authors.lastname: Naldaiz-Gastesi
authors.forename: José Iñaki
authors.initials: JI
authors.lastname: Álava
authors.forename: Antonio G
authors.initials: AG
authors.lastname: García
authors.forename: Adolfo
authors.initials: A
authors.lastname: López de Munain
authors.forename: Ander
authors.initials: A
authors.lastname: Izeta
mesh_terms: D016229:Amyloid beta-Peptides / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation / Q000502:physiology*; D002470:Cell Survival; D002478:Cells, Cultured; D002552:Cerebral Ventricles / Q000166:cytology; D002809:Chondroitin Sulfates / Q000494:pharmacology; D059290:Dopaminergic Neurons / Q000502:physiology* / Q000648:ultrastructure; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000502:physiology*; D005260:Female; D006624:Hippocampus / Q000166:cytology*; D066298:In Vitro Techniques; D008297:Male; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D009419:Nerve Tissue Proteins / Q000378:metabolism; D058953:Neural Stem Cells / Q000502:physiology*; D009840:Oligomycins / Q000494:pharmacology; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D012402:Rotenone / Q000494:pharmacology; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D009840:Oligomycins; D012402:Rotenone; D002809:Chondroitin Sulfates; D014446:Tyrosine 3-Monooxygenase