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
- 37 Downloads
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.
KeywordsCluster of differentiation 9 Ependymal cells Tanycytes Third ventricle
We would like to thank Editage (www.editage.jp) for English language editing.
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.
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.
- Coskun V, Wu H, Blanchi B, Tsao S, Kim K, Zhao J, Biancotti JC, Hutnick L, Krueger RC Jr, Fan G, de Vellis J, Sun YE (2008) CD133+ neural stem cells in the ependyma of mammalian postnatal forebrain. Proc Natl Acad Sci U S A 105(3):1026–1031. https://doi.org/10.1073/pnas.0710000105 CrossRefPubMedPubMedCentralGoogle Scholar
- Fujiwara K, Maekawa F, Kikuchi M, Takigami S, Yada T, Yashiro T (2007) Expression of retinaldehyde dehydrogenase (RALDH)2 and RALDH3 but not RALDH1 in the developing anterior pituitary glands of rats. Cell Tissue Res 328(1):129–135. https://doi.org/10.1007/s00441-006-0345-7 CrossRefPubMedGoogle Scholar
- Haan N, Goodman T, Najdi-Samiei A, Stratford CM, Rice R, El Agha E, Bellusci S, Hajihosseini MK (2013) Fgf10-expressing tanycytes add new neurons to the appetite/energy-balance regulating centers of the postnatal and adult hypothalamus. J Neurosci 3(14):6170–80. https://doi.org/10.1523/JNEUROSCI.2437-12.2013. CrossRefGoogle Scholar
- Hamilton LK, Truong MK, Bednarczyk MR, Aumont A, Fernandes KJ (2009) Cellular organization of the central canal ependymal zone, a niche of latent neural stem cells in the adult mammalian spinal cord. Neuroscience 164(3):1044–1056. https://doi.org/10.1016/j.neuroscience.2009.09.006 CrossRefPubMedGoogle Scholar
- Hemler ME (2003) Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain. Annu Rev Cell Dev Biol 19:397–422. https://doi.org/10.1146/annurev.cellbio.19.111301.153609 CrossRefPubMedGoogle Scholar
- Horiguchi K, Nakakura T, Yoshida S, Tsukada T, Kanno N, Hasegawa R, Takigami S, Ohsako S, Kato T, Kato Y (2016) Identification of THY1 as a novel thyrotrope marker and THY1 antibody-mediated thyrotrope isolation in the rat anterior pituitary gland. Biochem Biophys Res Commun 480(2):273–279. https://doi.org/10.1016/j.bbrc.2016.10.049 CrossRefPubMedGoogle Scholar
- Horiguchi K, Fujiwara K, Yoshida S, Nakakura T, Arae K, Tsukada T, Hasegawa R, Takigami S, Ohsako S, Yashiro T, Kato T, Kato Y (2018) Isolation and characterisation of CD9-positive pituitary adult stem/progenitor cells in rats. Sci Rep 8(1):5533. https://doi.org/10.1038/s41598-018-23923-0 CrossRefPubMedPubMedCentralGoogle Scholar
- Jin Y, Takeda Y, Kondo Y, Tripathi LP, Kang S, Takeshita H, Kuhara H, Maeda Y, Higashiguchi M, Miyake K, Morimura O, Koba T, Hayama Y, Koyama S, Nakanishi K, Iwasaki T, Tetsumoto S, Tsujino K, Kuroyama M, Iwahori K, Hirata H, Takimoto T, Suzuki M, Nagatomo I, Sugimoto K, Fujii Y, Kida H, Mizuguchi K, Ito M, Kijima T, Rakugi H, Mekada E, Tachibana I, Kumanogoh A (2018) Double deletion of tetraspanins CD9 and CD81 in mice leads to a syndrome resembling accelerated aging. Sci Rep 8(1):5145. https://doi.org/10.1038/s41598-018-23338-x CrossRefPubMedPubMedCentralGoogle Scholar
- Lee DA, Bedont JL, Pak T, Wang H, Song J, Miranda-Angulo A, Takiar V, Charubhumi V, Balordi F, Takebayashi H, Aja S, Ford E, Fishell G, Blackshaw S (2012) Tanycytes of the hypothalamic median eminence form a diet-responsive neurogenic niche. Nat Neurosci 15(5):700–702. https://doi.org/10.1038/nn.3079 CrossRefPubMedPubMedCentralGoogle Scholar
- Millhouse OE (1971) A Golgi study of third ventricle tanycytes in the adult rodent brain. Zeitschrift fur Zellforschung und mikroskopische Anatomie (Vienna, Austria : 1948) 121 (1):1–13Google Scholar
- Peddibhotla SS, Brinkmann BF, Kummer D, Tuncay H, Nakayama M, Adams RH, Gerke V, Ebnet K (2013) Tetraspanin CD9 links junctional adhesion molecule-A to alphavbeta3 integrin to mediate basic fibroblast growth factor-specific angiogenic signaling. Mol Biol Cell 24(7):933–944. https://doi.org/10.1091/mbc.E12-06-0481 CrossRefPubMedPubMedCentralGoogle Scholar
- Pfenninger CV, Roschupkina T, Hertwig F, Kottwitz D, Englund E, Bengzon J, Jacobsen SE, Nuber UA (2007) CD133 is not present on neurogenic astrocytes in the adult subventricular zone, but on embryonic neural stem cells, ependymal cells, and glioblastoma cells. Cancer Res 67(12):5727–5736. https://doi.org/10.1158/0008-5472.can-07-0183 CrossRefPubMedGoogle Scholar
- Pfenninger CV, Steinhoff C, Hertwig F, Nuber UA (2011) Prospectively isolated CD133/CD24-positive ependymal cells from the adult spinal cord and lateral ventricle wall differ in their long-term in vitro self-renewal and in vivo gene expression. Glia 59(1):68–81. https://doi.org/10.1002/glia.21077 CrossRefPubMedGoogle Scholar
- Robins SC, Stewart I, McNay DE, Taylor V, Giachino C, Goetz M, Ninkovic J, Briancon N, Maratos-Flier E, Flier JS, Kokoeva MV, Placzek M (2013) alpha-Tanycytes of the adult hypothalamic third ventricle include distinct populations of FGF-responsive neural progenitors. Nature communications 4:2049. doi: https://doi.org/10.1038/ncomms3049