Abstract
SOX2 is one of the key transcription factors involved in maintenance of neural progenitor identity. However, its function during the process of neural differentiation, including phases of lineage-specification and terminal differentiation, is still poorly understood. Considering growing evidence indicating that SOX2 expression level must be tightly controlled for proper neural development, the aim of this research was to analyze the effects of constitutive SOX2 overexpression on outcome of retinoic acid-induced neural differentiation of pluripotent NT2/D1 cells. We demonstrated that in spite of constitutive SOX2 overexpression, NT2/D1 cells were able to reach final phases of neural differentiation yielding both neuronal and glial cells. However, SOX2 overexpression reduced the number of mature MAP2-positive neurons while no difference in the number of GFAP-positive astrocytes was detected. In-depth analysis at single-cell level showed that SOX2 downregulation was in correlation with both neuronal and glial phenotype acquisitions. Interestingly, while in mature neurons SOX2 was completely downregulated, astrocytes with low level of SOX2 expression were detected. Nevertheless, cells with high level of SOX2 expression were incapable of entering in either of two differentiation pathways, neurogenesis or gliogenesis. Accordingly, our results indicate that fine balance between undifferentiated state and neural differentiation depends on SOX2 expression level. Unlike neurons, astrocytes could maintain low level of SOX2 expression after they acquired glial fate. Further studies are needed to determine whether differences in the level of SOX2 expression in GFAP-positive astrocytes are in correlation with their self-renewal capacity, differentiation status, and/or their phenotypic characteristics.
Similar content being viewed by others
Abbreviations
- RA:
-
retinoic acid
References
Bylund, M., Andersson, E., Novitch, B. G., and Muhr, J. (2003) Vertebrate neurogenesis is counteracted by Sox1-3 activity, Nat. Neurosci., 6, 1162–1168.
Cavallaro, M., Mariani, J., Lancini, C., Latorre, E., Caccia, R., Gullo, F., Valotta, M., DeBiasi, S., Spinardi, L., Ronchi, A., Wanke, E., Brunelli, S., Favaro, R., Ottolenghi, S., and Nicolis, S. K. (2008) Impaired generation of mature neurons by neural stem cells from hypomorphic Sox2 mutants, Development, 135, 541–557.
Ferri, A. L., Cavallaro, M., Braida, D., Di Cristofano, A., Canta, A., Vezzani, A., Ottolenghi, S., Pandolfi, P. P., Sala, M., DeBiasi, S., and Nicolis, S. K. (2004) Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain, Development, 131, 3805–3819.
Graham, V., Khudyakov, J., Ellis, P., and Pevny, L. (2003) SOX2 functions to maintain neural progenitor identity, Neuron, 39, 749–765.
Miyagi, S., Masui, S., Niwa, H., Saito, T., Shimazaki, T., Okano, H., Nishimoto, M., Muramatsu, M., Iwama, A., and Okuda, A. (2008) Consequence of the loss of Sox2 in the developing brain of the mouse, FEBS Lett., 582, 2811–2815.
Rizzino, A. (2008) Transcription factors that behave as master regulators during mammalian embryogenesis function as molecular rheostats, Biochem. J., 411, e5–7.
Thomson, M., Liu, S. J., Zou, L. N., Smith, Z., Meissner, A., and Ramanathan, S. (2011) Pluripotency factors in embryonic stem cells regulate differentiation into germ layers, Cell, 145, 875–889.
Boyer, L. A., Lee, T. I., Cole, M. F., Johnstone, S. E., Levine, S. S., Zucker, J. P., Guenther, M. G., Kumar, R. M., Murray, H. L., Jenner, R. G., Gifford, D. K., Melton, D. A., Jaenisch, R., and Young, R. A. (2005) Core transcriptional regulatory circuitry in human embryonic stem cells, Cell, 122, 947–956.
Macarthur, B. D., Ma’ayan, A., and Lemischka, I. R. (2009) Systems biology of stem cell fate and cellular reprogramming, Nat. Rev. Mol. Cell Biol., 10, 672–681.
Wegner, M., and Stolt, C. C. (2005) From stem cells to neurons and glia: a Soxist’s view of neural development, Trends Neurosci., 28, 583–588.
Avilion, A. A., Nicolis, S. K., Pevny, L. H., Perez, L., Vivian, N., and Lovell-Badge, R. (2003) Multipotent cell lineages in early mouse development depend on SOX2 function, Genes Dev., 17, 126–140.
Bani-Yaghoub, M., Tremblay, R. G., Lei, J. X., Zhang, D., Zurakowski, B., Sandhu, J. K., Smith, B., Ribecco-Lutkiewicz, M., Kennedy, J., Walker, P. R., and Sikorska, M. (2006) Role of Sox2 in the development of the mouse neocortex, Dev. Biol., 295, 52–66.
Ellis, P., Fagan, B. M., Magness, S. T., Hutton, S., Taranova, O., Hayashi, S., McMahon, A., Rao, M., and Pevny, L. (2004) SOX2, a persistent marker for multipotential neural stem cells derived from embryonic stem cells, the embryo or the adult, Dev. Neurosci., 26, 148–165.
Taranova, O. V., Magness, S. T., Fagan, B. M., Wu, Y., Surzenko, N., Hutton, S. R., and Pevny, L. H. (2006) SOX2 is a dose-dependent regulator of retinal neural progenitor competence, Genes Dev., 20, 1187–1202.
Hutton, S. R., and Pevny, L. H. (2011) SOX2 expression levels distinguish between neural progenitor populations of the developing dorsal telencephalon, Dev. Biol., 352, 40–47.
Favaro, R., Valotta, M., Ferri, A. L., Latorre, E., Mariani, J., Giachino, C., Lancini, C., Tosetti, V., Ottolenghi, S., Taylor, V., and Nicolis, S. K. (2009) Hippocampal development and neural stem cell maintenance require Sox2-dependent regulation of Shh, Nat. Neurosci., 12, 1248–1256.
Andrews, P. W. (1984) Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro, Dev. Biol., 103, 285–293.
Coyle, D. E., Li, J., and Baccei, M. (2011) Regional differentiation of retinoic acid-induced human pluripotent embryonic carcinoma stem cell neurons, PLoS One, 6, e16174.
Pleasure, S. J., Page, C., and Lee, V. M. (1992) Pure, postmitotic, polarized human neurons derived from NTera 2 cells provide a system for expressing exogenous proteins in terminally differentiated neurons, J. Neurosci., 12, 1802–1815.
Xu, Y. X., Hirose, Y., Zhou, X. Z., Lu, K. P., and Manley, J. L. (2003) Pin1 modulates the structure and function of human RNA polymerase II, Genes Dev., 17, 2765–2776.
Drakulic, D., Krstic, A., and Stevanovic, M. (2012) Establishment and initial characterization of SOX2-overexpressing NT2/D1 cell clones, Genet. Mol. Res., 11, 1385–1400.
Rutka, J. T., Murakami, M., Dirks, P. B., Hubbard, S. L., Becker, L. E., Fukuyama, K., Jung, S., Tsugu, A., and Matsuzawa, K. (1997) Role of glial filaments in cells and tumors of glial origin: a review, J. Neurosurg., 87, 420–430.
Salaun, C., James, D. J., Greaves, J., and Chamberlain, L. H. (2004) Plasma membrane targeting of exocytic SNARE proteins, Biochim. Biophys. Acta, 1693, 81–89.
Herzog, W., and Weber, K. (1978) Microtubule formation by pure brain tubulin in vitro. The influence of dextran and poly(ethylene glycol), Eur. J. Biochem., 91, 249–254.
Maddodi, N., Bhat, K. M., Devi, S., Zhang, S. C., and Setaluri, V. (2010) Oncogenic BRAFV600E induces expression of neuronal differentiation marker MAP2 in melanoma cells by promoter demethylation and down-regulation of transcription repressor HES1, J. Biol. Chem., 285, 242–254.
Podrygajlo, G., Tegenge, M. A., Gierse, A., Paquet-Durand, F., Tan, S., Bicker, G., and Stern, M. (2009) Cellular phenotypes of human model neurons (NT2) after differentiation in aggregate culture, Cell Tissue Res., 336, 439–452.
Popovic, J., Stanisavljevic, D., Schwirtlich, M., Klajn, A., Marjanovic, J., and Stevanovic, M. (2014) Expression analysis of SOX14 during retinoic acid induced neural differentiation of embryonal carcinoma cells and assessment of the effect of its ectopic expression on SOXB members in HeLa cells, PLoS One, 9, e91852.
Sandhu, J. K., Sikorska, M., and Walker, P. R. (2002) Characterization of astrocytes derived from human NTera-2/D1 embryonal carcinoma cells, J. Neurosci. Res., 68, 604–614.
Goodfellow, C. E., Graham, S. E., Dragunow, M., and Glass, M. (2011) Characterization of NTera2/D1 cells as a model system for the investigation of cannabinoid function in human neurons and astrocytes, J. Neurosci. Res., 89, 1685–1697.
Guillemain, I., Alonso, G., Patey, G., Privat, A., and Chaudieu, I. (2000) Human NT2 neurons express a large variety of neurotransmission phenotypes in vitro, J. Comp. Neurol., 422, 380–395.
Hartley, R. S., Margulis, M., Fishman, P. S., Lee, V. M., and Tang, C. M. (1999) Functional synapses are formed between human NTera2 (NT2N, hNT) neurons grown on astrocytes, J. Comp. Neurol., 407, 1–10.
Gasque, P., Jones, J., Singhrao, S. K., and Morgan, B. (1998) Identification of an astrocyte cell population from human brain that expresses perforin, a cytotoxic protein implicated in immune defense, J. Exp. Med., 187, 451–460.
Mulder, S. D., Nielsen, H. M., Blankenstein, M. A., Eikelenboom, P., and Veerhuis, R. (2014) Apolipoproteins E and J interfere with amyloid-beta uptake by primary human astrocytes and microglia in vitro, Glia, 62, 493–503.
Trotti, D., Aoki, M., Pasinelli, P., Berger, U. V., Danbolt, N. C., Brown, R. H., Jr., and Hediger, M. A. (2001) Amyotrophic lateral sclerosis-linked glutamate transporter mutant has impaired glutamate clearance capacity, J. Biol. Chem., 276, 576–582.
Trotti, D., Danbolt, N. C., and Volterra, A. (1998) Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration? Trends Pharmacol. Sci., 19, 328–334.
Trotti, D., Rolfs, A., Danbolt, N. C., Brown, R. H., Jr., and Hediger, M. A. (1999) SOD1 mutants linked to amyotrophic lateral sclerosis selectively inactivate a glial glutamate transporter, Nat. Neurosci., 2, 848.
Seki, Y., Feustel, P. J., Keller, R. W., Jr., Tranmer, B. I., and Kimelberg, H. K. (1999) Inhibition of ischemia-induced glutamate release in rat striatum by dihydrokinate and an anion channel blocker, Stroke, 30, 433–440.
Crunelli, V., Carmignoto, G., and Steinhauser, C. (2014) Astrocytic targets provide new avenues for the therapeutic treatment of epilepsy, Neuroscientist, March 21 [E-pub ahead of print].
Rothstein, J. D., Dykes-Hoberg, M., Pardo, C. A., Bristol, L. A., Jin, L., Kuncl, R. W., Kanai, Y., Hediger, M. A., Wang, Y., Schielke, J. P., and Welty, D. F. (1996) Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate, Neuron, 16, 675–686.
Tanaka, K., Watase, K., Manabe, T., Yamada, K., Watanabe, M., Takahashi, K., Iwama, H., Nishikawa, T., Ichihara, N., Kikuchi, T., Okuyama, S., Kawashima, N., Hori, S., Takimoto, M., and Wada, K. (1997) Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1, Science, 276, 1699–1702.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published in Russian in Biokhimiya, 2014, Vol. 79, No. 11, pp. 1442–1451.
Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM14-118, September 7, 2014.
Rights and permissions
About this article
Cite this article
Klajn, A., Drakulic, D., Tosic, M. et al. SOX2 overexpression affects neural differentiation of human pluripotent NT2/D1 cells. Biochemistry Moscow 79, 1172–1182 (2014). https://doi.org/10.1134/S0006297914110042
Received:
Revised:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S0006297914110042