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Establishment of a novel three‐dimensional primary culture model for hippocampal neurogenesis
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 s...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5492207/ https://www.ncbi.nlm.nih.gov/pubmed/28642339 http://dx.doi.org/10.14814/phy2.13318 |
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author | Usui, Tatsuya Sakurai, Masashi Kawasaki, Hideyoshi Ohama, Takashi Yamawaki, Hideyuki Sato, Koichi |
author_facet | Usui, Tatsuya Sakurai, Masashi Kawasaki, Hideyoshi Ohama, Takashi Yamawaki, Hideyuki Sato, Koichi |
author_sort | Usui, Tatsuya |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-5492207 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-54922072017-07-05 Establishment of a novel three‐dimensional primary culture model for hippocampal neurogenesis Usui, Tatsuya Sakurai, Masashi Kawasaki, Hideyoshi Ohama, Takashi Yamawaki, Hideyuki Sato, Koichi Physiol Rep Original Research 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. John Wiley and Sons Inc. 2017-06-22 /pmc/articles/PMC5492207/ /pubmed/28642339 http://dx.doi.org/10.14814/phy2.13318 Text en © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society. This is an open access article under the terms of the Creative Commons Attribution (http://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Original Research Usui, Tatsuya Sakurai, Masashi Kawasaki, Hideyoshi Ohama, Takashi Yamawaki, Hideyuki Sato, Koichi Establishment of a novel three‐dimensional primary culture model for hippocampal neurogenesis |
title | Establishment of a novel three‐dimensional primary culture model for hippocampal neurogenesis |
title_full | Establishment of a novel three‐dimensional primary culture model for hippocampal neurogenesis |
title_fullStr | Establishment of a novel three‐dimensional primary culture model for hippocampal neurogenesis |
title_full_unstemmed | Establishment of a novel three‐dimensional primary culture model for hippocampal neurogenesis |
title_short | Establishment of a novel three‐dimensional primary culture model for hippocampal neurogenesis |
title_sort | establishment of a novel three‐dimensional primary culture model for hippocampal neurogenesis |
topic | Original Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5492207/ https://www.ncbi.nlm.nih.gov/pubmed/28642339 http://dx.doi.org/10.14814/phy2.13318 |
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