Establishment of a novel three-dimensional primary culture model for hippocampal neurogenesis

Physiol Rep. 2017 Jun;5(12):e13318. doi: 10.14814/phy2.13318.

Abstract

New neurons are generated in the adult hippocampus throughout life and contribute to the functions of learning and memory. Nevertheless, the mechanisms by which disrupted neurogenesis regulates central nervous system (CNS) disorders are not fully understood. Here, we established a novel 3D culture system of hippocampal neurogenesis using air liquid interface (ALI) culture and Matrigel culture from mouse hippocampus tissues. After isolated mouse hippocampus tissue fragments were seeded into ALI wells and cultured in stemness-stimulated media containing Wnt, EGF, Noggin and R-spondin for 7 days, small spheres gradually appeared in the tissues. To identify the cell components, immunohistochemical and immunofluorescence staining were performed. Expression of a mature neuronal cell marker, NeuN was observed in the tissues just after seeding. Expression of a neural stem cell marker, Nestin was observed in the tissues at day 7. To differentiate the Nestin-positive cells, they were passaged into Matrigel. Expression of Nestin but not an immature neuronal cell marker, doublecortin (DCX) was observed in the isolated cells. After 7 days of Matrigel culture, they showed the neurite outgrowth. Expression of Nestin was decreased compared with the one just after passaging, while DCX expression was increased. Western blotting analysis also showed Nestin expression was decreased, while expression of DCX, a neuronal cell marker, Tuj1 and a granule cell marker, Prox-1 was increased. Here, we establish the 3D culture of hippocampus tissues that might become a novel in vitro tool for monitoring the process of hippocampal neurogenesis. Our model might shed light into the mechanisms of pathogenesis of CNS disorders.

Keywords: 3D culture; differentiation; hippocampus; neural stem cell; neurogenesis.

MeSH terms

  • Animals
  • Cells, Cultured
  • Collagen / pharmacology
  • Drug Combinations
  • Hippocampus / cytology*
  • Hippocampus / growth & development
  • Laminin / pharmacology
  • Mice
  • Mice, Inbred C57BL
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism
  • Nestin / genetics
  • Nestin / metabolism
  • Neural Stem Cells / cytology*
  • Neural Stem Cells / drug effects
  • Neural Stem Cells / metabolism
  • Neurogenesis*
  • Neuropeptides / genetics
  • Neuropeptides / metabolism
  • Primary Cell Culture / methods*
  • Proteoglycans / pharmacology

Substances

  • Drug Combinations
  • Laminin
  • Microtubule-Associated Proteins
  • Nestin
  • Neuropeptides
  • Proteoglycans
  • doublecortin protein
  • matrigel
  • Collagen