Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Berditchevski F (2001) Complexes of tetraspanins with integrins: more than meets the eye. J Cell Sci 114(Pt 23):4143–4151
Boucheix C, Rubinstein E (2001) Tetraspanins. Cellular and molecular life sciences : CMLS 58(9):1189–1205. https://doi.org/10.1007/pl00000933
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
Del Bigio MR (2010) Ependymal cells: biology and pathology. Acta Neuropathol 119(1):55–73. https://doi.org/10.1007/s00401-009-0624-y
Feng R, Wen J (2015) Overview of the roles of Sox2 in stem cell and development. Biol Chem 396(8):883–891. https://doi.org/10.1515/hsz-2014-0317
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
Goodman T, Hajihosseini MK (2015) Hypothalamic tanycytes-masters and servants of metabolic, neuroendocrine, and neurogenic functions. Front Neurosci 9:387. https://doi.org/10.3389/fnins.2015.00387
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.
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
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
Hemler ME (2005) Tetraspanin functions and associated microdomains. Nat Rev Mol Cell Biol 6(10):801–811. https://doi.org/10.1038/nrm1736
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
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
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
Le Naour F, Rubinstein E, Jasmin C, Prenant M, Boucheix C (2000) Severely reduced female fertility in CD9-deficient mice. Science (New York, NY) 287(5451):319–321
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
Maecker HT, Todd SC, Levy S (1997) The tetraspanin superfamily: molecular facilitators. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 11(6):428–442
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):1–13
Ohata S, Alvarez-Buylla A (2016) Planar organization of multiciliated ependymal (E1) cells in the brain ventricular epithelium. Trends Neurosci 39(8):543–551. https://doi.org/10.1016/j.tins.2016.05.004
Pastrana E, Silva-Vargas V, Doetsch F (2011) Eyes wide open: a critical review of sphere-formation as an assay for stem cells. Cell Stem Cell 8(5):486–498. https://doi.org/10.1016/j.stem.2011.04.007
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
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
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
Pierzchalski A, Mittag A, Bocsi J, Tarnok A (2013) An innovative cascade system for simultaneous separation of multiple cell types. PLoS One 8(9):e74745. https://doi.org/10.1371/journal.pone.0074745
Rizzoti K, Lovell-Badge R (2017) Pivotal role of median eminence tanycytes for hypothalamic function and neurogenesis. Mol Cell Endocrinol 445:7–13. https://doi.org/10.1016/j.mce.2016.08.020
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
Rodriguez EM, Blazquez JL, Pastor FE, Pelaez B, Pena P, Peruzzo B, Amat P (2005) Hypothalamic tanycytes: a key component of brain-endocrine interaction. Int Rev Cytol 247:89–164. https://doi.org/10.1016/s0074-7696(05)47003-5
Silva-Vargas V, Crouch EE, Doetsch F (2013) Adult neural stem cells and their niche: a dynamic duo during homeostasis, regeneration, and aging. Curr Opin Neurobiol 23(6):935–942. https://doi.org/10.1016/j.conb.2013.09.004
Yoshida S, Nishimura N, Ueharu H, Kanno N, Higuchi M, Horiguchi K, Kato T, Kato Y (2016) Isolation of adult pituitary stem/progenitor cell clusters located in the parenchyma of the rat anterior lobe. Stem Cell Res 17(2):318–329. https://doi.org/10.1016/j.scr.2016.08.016
Acknowledgments
We would like to thank Editage (www.editage.jp) for English language editing.
Funding
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).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
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.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Horiguchi, K., Yoshida, S., Hasegawa, R. et al. 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. Cell Tissue Res 379, 497–509 (2020). https://doi.org/10.1007/s00441-019-03132-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00441-019-03132-5