Isolation and characterization of cluster of differentiation 9-positive ependymal cells as potential adult neural stem/progenitor cells in the third ventricle of adult rats

  • Kotaro HoriguchiEmail author
  • Saishu Yoshida
  • Rumi Hasegawa
  • Shu Takigami
  • Shunji Ohsako
  • Takako Kato
  • Yukio KatoEmail author
Regular Article


Ependymal cells located above the ventricular zone of the lateral, third, and fourth ventricles and the spinal cord are thought to form part of the adult neurogenic niche. Many studies have focused on ependymal cells as potential adult neural stem/progenitor cells. To investigate the functions of ependymal cells, a simple method to isolate subtypes is needed. Accordingly, in this study, we evaluated the expression of cluster of differentiation (CD) 9 in ependymal cells by in situ hybridization and immunohistochemistry. Our results showed that CD9-positive ependymal cells were also immunopositive for SRY-box 2, a stem/progenitor cell marker. We then isolated CD9-positive ependymal cells from the third ventricle using the pluriBead-cascade cell isolation system based on antibody-mediated binding of cells to beads of different sizes and their isolation with sieves of different mesh sizes. As a result, we succeeded in isolating CD9-positive populations with 86% purity of ependymal cells from the third ventricle. We next assayed whether isolated CD9-positive ependymal cells had neurospherogenic potential. Neurospheres were generated from CD9-positive ependymal cells of adult rats and were immunopositve for neuron, astrocyte, and oligodendrocyte markers after cultivation. Thus, based on these findings, we suggest that the isolated CD9-positive ependymal cells from the third ventricle included tanycytes, which are special ependymal cells in the ventricular zone of the third ventricle that form part of the adult neurogenic and gliogenic niche. These current findings improve our understanding of tanycytes in the adult third ventricle in vitro.


Cluster of differentiation 9 Ependymal cells Tanycytes Third ventricle 



We would like to thank Editage ( for English language editing.

Funding information

This work was supported by the JSPS KAKENHI (grant nos. 16K08475 to K.H., 21380184 to Y.K., and 24580435 to T.K.), by a MEXT-supported Program for the Strategic Research Foundation at Private Universities (2014–2018), and by the Meiji University International Institute for BioResource Research (MUIIR).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

The current study was approved by the Committee on Animal Experiments of the School of Agriculture, Meiji University, and Kyorin University based on the NIH Guidelines for the Care and Use of Laboratory Animals. This article does not contain any studies with human participants.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Kotaro Horiguchi
    • 1
    • 2
    Email author
  • Saishu Yoshida
    • 2
    • 3
  • Rumi Hasegawa
    • 1
  • Shu Takigami
    • 1
  • Shunji Ohsako
    • 1
  • Takako Kato
    • 2
  • Yukio Kato
    • 2
    Email author
  1. 1.Laboratory of Anatomy and Cell Biology, Department of Health SciencesKyorin UniversityTokyoJapan
  2. 2.Institute of EndocrinologyMeiji UniversityKawasakiJapan
  3. 3.Department of BiochemistryThe Jikei University School of MedicineTokyoJapan

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