Stem Cell Reviews and Reports

, Volume 6, Issue 3, pp 438–449 | Cite as

Generation of Human Embryonic Stem Cell Reporter Lines Expressing GFP Specifically in Neural Progenitors

  • Parinya Noisa
  • Alai Urrutikoetxea-Uriguen
  • Meng Li
  • Wei CuiEmail author


Generation of lineage-specific human embryonic stem cell (hESC) reporter lines will facilitate the real time monitoring of differentiation in live cells and the identification of factors governing these processes. It will also enable researchers to purify specific cell populations from heterogeneous differentiated hESC progeny. Here we report the generation of clonally derived nestin-EGFP reporter hESC lines that express GFP under the control of the neuroepithelial specific nestin 2nd intron enhancer. We show that the nestin-EGFP hESC reporter lines retain the features of undifferentiated hESCs, are able to self-renew in hESC culture conditions and to differentiate into cells of all three germ layers. The nestin-EGFP reporter exhibited high expression in neural progenitor cells upon differentiation, although it is detectable at a low level in the undifferentiated state. Furthermore, the expression of the transgene is exclusively confined to the neural progenitors after differentiation. The specific expression of the transgene is determined by collaborative binding motifs of POU and SOX transcription factors in the nestin enhancer. Deletion of either of the binding elements resulted in a significant reduction of enhancer/promoter activity. Taken together, the nestin-EGFP reporter hESC lines are invaluable not only for the study of the neural differentiation process from hESCs but also for the enrichment of neural progenitor cells from other cell lineages.


Human embryonic stem cells GFP reporter Neural differentiation Neural progenitor Nestin 



We thank Ms Leigh Rogers for technical assistance. PN is a Royal Thai Government funded PhD student. This work has been supported by fundings for the ESTOOLS consortium under the Sixth Research Framework Programme of the European Union contract LSHG-CT-2006-018739 and the IOG Trust grant.

Conflicts of Interest

The authors declare no potential conflicts of interest.


  1. 1.
    Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282(5391), 1145–1147.Google Scholar
  2. 2.
    Daley, G. Q., & Scadden, D. T. (2008). Prospects for stem cell-based therapy. Cell, 132(4), 544–548.Google Scholar
  3. 3.
    Dvash, T., & Benvenisty, N. (2004). Human embryonic stem cells as a model for early human development. Best Pratice and Research Clinical Obstetrics and Gynaecology, 18(6), 929–940.Google Scholar
  4. 4.
    Okada, Y., Matsumoto, A., Shimazaki, T., et al. (2008). Spatiotemporal recapitulation of central nervous system development by murine embryonic stem cell-derived neural stem/progenitor cells. Stem Cells, 26(12), 3086–3098.Google Scholar
  5. 5.
    Aubert, J., Stavridis, M. P., Tweedie, S., et al. (2003). Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1-gfp knock-in mice. Proceedings of the National Academy of Sciences of the United States of America, 100(Suppl 1), 11836–11841.Google Scholar
  6. 6.
    Ganat, Y. M., Silbereis, J., Cave, C., et al. (2006). Early postnatal astroglial cells produce multilineage precursors and neural stem cells in vivo. Journal of Neuroscience, 26(33), 8609–8621.Google Scholar
  7. 7.
    Kawaguchi, A., Miyata, T., Sawamoto, K., et al. (2001). Nestin-EGFP transgenic mice: visualization of the self-renewal and multipotency of CNS stem cells. Molecular and Cellular Neurosciences, 17(2), 259–273.Google Scholar
  8. 8.
    Pratt, T., Sharp, L., Nichols, J., Price, D. J., & Mason, J. O. (2000). Embryonic stem cells and transgenic mice ubiquitously expressing a tau-tagged green fluorescent protein. Developments in Biologicals, 228(1), 19–28.Google Scholar
  9. 9.
    Zhao, S., Maxwell, S., Jimenez-Beristain, A., et al. (2004). Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons. The European Journal of Neuroscience, 19(5), 1133–1140.Google Scholar
  10. 10.
    Placantonakis, D. G., Tomishima, M. J., Lafaille, F., et al. (2009). BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage. Stem Cells, 27(3), 521–532.Google Scholar
  11. 11.
    Xue, H., Wu, S., Papadeas, S. T., et al. (2009). A targeted neuroglial reporter line generated by homologous recombination in human embryonic stem cells. Stem Cells, 27(8), 1836–1846.Google Scholar
  12. 12.
    Lendahl, U., Zimmerman, L. B., & McKay, R. D. (1990). CNS stem cells express a new class of intermediate filament protein. 60(4), Cell, 585–595.Google Scholar
  13. 13.
    Mujtaba, T., Mayer-Proschel, M., & Rao, M. S. (1998). A common neural progenitor for the CNS and PNS. Developments in Biologicals, 200(1), 1–15.Google Scholar
  14. 14.
    Lothian, C., & Lendahl, U. (1997). An evolutionarily conserved region in the second intron of the human nestin gene directs gene expression to CNS progenitor cells and to early neural crest cells. The European Journal of Neuroscience, 9(3), 452–462.Google Scholar
  15. 15.
    Lothian, C., Prakash, N., Lendahl, U., & Wahlstrom, G. M. (1999). Identification of both general and region-specific embryonic CNS enhancer elements in the nestin promoter. Experimental Cell Research, 248(2), 509–519.Google Scholar
  16. 16.
    Zimmerman, L., Parr, B., Lendahl, U., et al. (1994). Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors. Neuron, 12(1), 11–24.Google Scholar
  17. 17.
    Gerrard, L., Zhao, D., Clark, A. J., & Cui, W. (2005). Stably transfected human embryonic stem cell clones express OCT4-specific green fluorescent protein and maintain self-renewal and pluripotency. Stem Cells, 23(1), 124–133.Google Scholar
  18. 18.
    Xu, C., Inokuma, M. S., Denham, J., et al. (2001). Feeder-free growth of undifferentiated human embryonic stem cells. Nature Biotechnology, 19(10), 971–974.Google Scholar
  19. 19.
    Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., et al. (2000). Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Molecular Medicine, 6(2), 88–95.Google Scholar
  20. 20.
    Gerrard, L., Rodgers, L., & Cui, W. (2005). Differentiation of human embryonic stem cells to neural lineages in adherent culture by blocking bone morphogenetic protein signaling. Stem Cells, 23(9), 1234–1241.Google Scholar
  21. 21.
    Kioussis, D., & Festenstein, R. (1997). Locus control regions: overcoming heterochromatin-induced gene inactivation in mammals. Current Opinion in Genetics and Development, 7(5), 614–619.Google Scholar
  22. 22.
    Pera, M. F., Andrade, J., Houssami, S., et al. (2004). Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin. Journal of Cell Science, 117(Pt 7), 1269–1280.Google Scholar
  23. 23.
    Xu, R. H., Chen, X., Li, D. S., et al. (2002). BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nature Biotechnology, 20(12), 1261–1264.Google Scholar
  24. 24.
    Jin, Z., Liu, L., Bian, W., et al. (2009). Different transcription factors regulate nestin gene expression during P19 cell neural differentiation and central nervous system development. Journal of Biological Chemistry 284(12), 8160–8173.Google Scholar
  25. 25.
    Tanaka, S., Kamachi, Y., Tanouchi, A., Hamada, H., Jing, N., & Kondoh, H. (2004). Interplay of SOX and POU factors in regulation of the Nestin gene in neural primordial cells. Molecular and Cellular Biology, 24(20), 8834–8846.Google Scholar
  26. 26.
    Yasuhara, N., Shibazaki, N., Tanaka, S., et al. (2007). Triggering neural differentiation of ES cells by subtype switching of importin-alpha. Nature Cell Biology, 9(1), 72–79.Google Scholar
  27. 27.
    Wu, J. Q., Habegger, L., Noisa, P., et al. (2010). Dynamic transcriptomes during neural differentiation of human embryonic stem cells revealed by short, long, and paired-end sequencing. Proceedings of the National Academy of Sciences of the United States of America, 107(11), 5254–5259.Google Scholar
  28. 28.
    Johansson, C. B., Lothian, C., Molin, M., Okano, H., & Lendahl, U. (2002). Nestin enhancer requirements for expression in normal and injured adult CNS. Journal of Neuroscience Research, 69(6), 784–794.Google Scholar
  29. 29.
    Lenka, N., Lu, Z. J., Sasse, P., Hescheler, J., & Fleischmann, B. K. (2002). Quantitation and functional characterization of neural cells derived from ES cells using nestin enhancer-mediated targeting in vitro. Journal of Cell Science, 115(Pt 7), 1471–1485.Google Scholar
  30. 30.
    Boyer, L. A., Lee, T. I., Cole, M. F., et al. (2005). Core transcriptional regulatory circuitry in human embryonic stem cells. Cell, 122(6), 947–956.Google Scholar
  31. 31.
    Chambers, I., & Tomlinson, S. R. (2009). The transcriptional foundation of pluripotency. Development, 136(14), 2311–2322.Google Scholar
  32. 32.
    Kondoh, H., & Kamachi, Y. (2010). SOX-partner code for cell specification: Regulatory target selection and underlying molecular mechanisms. The International Journal of Biochemistry and Cell Biology, 42(3), 391–399.Google Scholar
  33. 33.
    Giudice, A., & Trounson, A. (2008). Genetic modification of human embryonic stem cells for derivation of target cells. Cell Stem Cell, 2(5), 422–433.Google Scholar
  34. 34.
    Alami, R., Greally, J. M., Tanimoto, K., et al. (2000). Beta-globin YAC transgenes exhibit uniform expression levels but position effect variegation in mice. Human Molecular Genetics, 9(4), 631–636.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Parinya Noisa
    • 1
  • Alai Urrutikoetxea-Uriguen
    • 1
  • Meng Li
    • 2
  • Wei Cui
    • 1
    Email author
  1. 1.Institute of Reproductive and Developmental Biology, Department of Surgery and CancerImperial College LondonLondonUK
  2. 2.MRC Clinical Sciences Centre; Faculty of MedicineImperial College LondonLondonUK

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