Stem Cell Reviews and Reports

, Volume 8, Issue 4, pp 1129–1137 | Cite as

Role of miRNAs in Neuronal Differentiation from Human Embryonic Stem Cell—Derived Neural Stem Cells

  • Jing Liu
  • Jackline Githinji
  • Bridget Mclaughlin
  • Kasia Wilczek
  • Jan Nolta


microRNAs (miRNAs) are important modulators in regulating gene expression at the post-transcriptional level and are therefore emerging as strong mediators in neural fate determination. Here, by use of the model of human embryonic stem cell (hESC)-derived neurogenesis, miRNAs involved in the differentiation from neural stem cells (hNSC) to neurons were profiled and identified. hNSC were differentiated into the neural lineage, out of which the neuronal subset was enriched through cell sorting based on select combinatorial biomarkers: CD15-/CD29Low/CD24High. This relatively pure and viable subpopulation expressed the neuronal marker β III-tubulin. The miRNA array demonstrated that a number of miRNAs were simultaneously induced or suppressed in neurons, as compared to hNSC. Real-time PCR further validated the decrease in levels of miR214, but increase in brain-specific miR7 and miR9 in the derived neurons. For functional studies, hNSC were stably transduced with lentiviral vectors carrying specific constructs to downregulate miR214 or to upregulate miR7. Manipulation of either miR214 or miR7 did not affect the expression of β III-tubulin or neurofilament, however miR7 overexpression gave rise to enhanced synapsin expression in the derived neurons. This indicated that miR7 might play an important role in neurite outgrowth and synapse formation. In conclusion, our data demonstrate that miRNAs function as important modulators in neural lineage determination. These studies shed light on strategies to optimize in vitro differentiation efficiencies to mature neurons for use in drug discovery studies and potential future clinical applications.


microRNAs Human neural stem cell Neurogenesis microRNA array 



Human embryonic stem cells


hESC derived neural stem cells


hESC (H9 line) derived neurons


Embryoid body



This work was supported by Shriners Hospital Fellowship to J. Liu and California Institute for Regenerative Medicine (CIRM TR1-01257) to J. Nolta. We thank Dr. Christoph Eicken at LC Sciences for his help with miRNA array data analysis and submission.

Conflicts of interest

The authors declare no potential conflicts of interest.


  1. 1.
    Shi, Y., Zhao, X., Hsieh, J., et al. (2010). MicroRNA regulation of neural stem cells and neurogenesis. Journal of Neuroscience, 30, 14931–14936.PubMedCrossRefGoogle Scholar
  2. 2.
    Krichevsky, A. M., Sonntag, K. C., Isacson, O., & Kosik, K. S. (2006). Specific microRNAs modulate embryonic stem cell-derived neurogenesis. Stem Cells, 24, 857–864.PubMedCrossRefGoogle Scholar
  3. 3.
    Wayman, G. A., Davare, M., Ando, H., et al. (2008). An activity-regulated microRNA controls dendritic plasticity by down-regulating p250GAP. Proceedings of the National Academy of Sciences of the United States of America, 105, 9093–9098.PubMedCrossRefGoogle Scholar
  4. 4.
    Edbauer, D., Neilson, J. R., Foster, K. A., et al. (2010). Regulation of synaptic structure and function by FMRP-associated microRNAs miR-125b and miR-132. Neuron, 65, 373–384.PubMedCrossRefGoogle Scholar
  5. 5.
    Smith, B., Treadwell, J., Zhang, D., Ly, D., McKinnell, I., Walker, P. R., & Sikorska, M. (2010). Large-scale expression analysis reveals distinct microRNA profiles at different stages of human neurodevelopment. PLoS One, 5, e11109.PubMedCrossRefGoogle Scholar
  6. 6.
    Sempere, L. F., Freemantle, S., Pitha-Rowe, I., Moss, E., Dmitrovsky, E., & Ambros, V. (2004). Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation. Genome Biology, 5, R13.PubMedCrossRefGoogle Scholar
  7. 7.
    Pruszak, J., Ludwig, W., Blak, A., Alavian, K., & Isacson, O. (2009). CD15, CD24, and CD29 define a surface biomarker code for neural lineage differentiation of stem cells. Stem Cells, 27, 2928–2940.PubMedGoogle Scholar
  8. 8.
    Yuan, S. H., Martin, J., Elia, J., et al. (2011). Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One, 6, e17540.PubMedCrossRefGoogle Scholar
  9. 9.
    Zhang, S. C., Wernig, M., Duncan, I. D., Brustle, O., & Thomson, J. A. (2001). In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nature Biotechnology, 19, 1129–1133.PubMedCrossRefGoogle Scholar
  10. 10.
    Koch, P., Opitz, T., Steinbeck, J. A., Ladewig, J., & Brustle, O. (2009). A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration. Proceedings of the National Academy of Sciences of the United States of America, 106, 3225–3230.PubMedCrossRefGoogle Scholar
  11. 11.
    Chen, H., Shalom-Feuerstein, R., Riley, J., et al. (2010). miR-7 and miR-214 are specifically expressed during neuroblastoma differentiation, cortical development and embryonic stem cells differentiation, and control neurite outgrowth in vitro. Biochemical and Biophysical Research Communications, 394, 921–927.PubMedCrossRefGoogle Scholar
  12. 12.
    Fornasiero, E. F., Bonanomi, D., Benfenati, F., & Valtorta, F. (2009). The role of synapsins in neuronal development. Cellular and Molecular Life Sciences, 67, 1383–1396.PubMedCrossRefGoogle Scholar
  13. 13.
    Tessmar-Raible, K., Raible, F., Christodoulou, F., Guy, K., Rembold, M., Hausen, H., & Arendt, D. (2007). Conserved sensory-neurosecretory cell types in annelid and fish forebrain: insights into hypothalamus evolution. Cell, 129, 1389–1400.PubMedCrossRefGoogle Scholar
  14. 14.
    Saydam, O., Senol, O., Wurdinger, T., et al. (2010). miRNA-7 attenuation in Schwannoma tumors stimulates growth by upregulating three oncogenic signaling pathways. Cancer Research, 71, 852–861.PubMedCrossRefGoogle Scholar
  15. 15.
    Kefas, B., Godlewski, J., Comeau, L., et al. (2008). microRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway and is down-regulated in glioblastoma. Cancer Research, 68, 3566–3572.PubMedCrossRefGoogle Scholar
  16. 16.
    Junn, E., Lee, K. W., Jeong, B. S., Chan, T. W., Im, J. Y., & Mouradian, M. M. (2009). Repression of alpha-synuclein expression and toxicity by microRNA-7. Proceedings of the National Academy of Sciences of the United States of America, 106, 13052–13057.PubMedCrossRefGoogle Scholar
  17. 17.
    Doxakis, E. (2010). Post-transcriptional regulation of alpha-synuclein expression by mir-7 and mir-153. Journal of Biological Chemistry, 285, 12726–12734.PubMedCrossRefGoogle Scholar
  18. 18.
    Li, X., Cassidy, J. J., Reinke, C. A., Fischboeck, S., & Carthew, R. W. (2009). A microRNA imparts robustness against environmental fluctuation during development. Cell, 137, 273–282.PubMedCrossRefGoogle Scholar
  19. 19.
    Penna, E., Orso, F., Cimino, D., et al. (2011). microRNA-214 contributes to melanoma tumour progression through suppression of TFAP2C. EMBO Journal, 30, 1990–2007.PubMedCrossRefGoogle Scholar
  20. 20.
    Yang, H., Kong, W., & He, L. (2008). MicroRNA expression profiling in human ovarian cancer: miR-214 induces cell survival and cisplatin resistance by targeting PTEN. Cancer Research, 68, 425–433.PubMedCrossRefGoogle Scholar
  21. 21.
    Decembrini, S., Bressan, D., & Vignali, R. (2009). MicroRNAs couple cell fate and developmental timing in retina. Proceedings of the National Academy of Sciences of the United States of America, 106, 21179–21184.PubMedCrossRefGoogle Scholar
  22. 22.
    Zhao, C., Sun, G., Li, S., Lang, M. F., Yang, S., Li, W., & Shi, Y. (2010). MicroRNA let-7b regulates neural stem cell proliferation and differentiation by targeting nuclear receptor TLX signaling. Proceedings of the National Academy of Sciences of the United States of America, 107, 1876–1881.PubMedCrossRefGoogle Scholar
  23. 23.
    Bock, C., Kiskinis, E., & Verstappen, G. (2011). Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. Cell, 144, 439–452.PubMedCrossRefGoogle Scholar
  24. 24.
    Boulting, G. L., Kiskinis, E., & Croft, G. F. (2011). A functionally characterized test set of human induced pluripotent stem cells. Nature Biotechnology, 29, 279–286.PubMedCrossRefGoogle Scholar
  25. 25.
    Kim, H., Lee, G., & Ganat, Y. (2011). miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells. Cell Stem Cell, 8, 695–706.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Jing Liu
    • 1
  • Jackline Githinji
    • 1
  • Bridget Mclaughlin
    • 1
  • Kasia Wilczek
    • 1
  • Jan Nolta
    • 1
  1. 1.University of California, Davis2921 Stockton Blvd., Room 1300SacramentoUSA

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