Skip to main content

Advertisement

SpringerLink
Log in

Efficient Generation of Schwann Cells from Human Embryonic Stem Cell-Derived Neurospheres

  • Published:
Stem Cell Reviews and Reports Aims and scope Submit manuscript

Abstract

Schwann cells (SC), the glial cells of peripheral nerves, are involved in many diseases including Charcot Marie Tooth and neurofibromatosis, and play a pivotal role in peripheral nerve regeneration. Although it is possible to obtain human SC from nerve biopsies, they are difficult to maintain and expand in culture. Here we describe an efficient system for directing the differentiation of human embryonic stem cells (hESC) into cells with the morphological and molecular characteristics of SC. Neurospheres were generated from hESC using stromal cell induction and grown under conditions supportive of SC differentiation. After 8 weeks, hESC-derived SC expressed characteristic markers GFAP, S100, HNK1, P75, MBP and PMP-22, and were observed in close association with hESC-derived neurites. ~60% of the cells were double-immunostained for the SC markers GFAP/S100. RT-PCR analysis confirmed the expression of GFAP, S100, P75, PMP-22 and MBP and demonstrated expression of the SC markers P0, KROX20 and PLP in the cultures. Expression of CAD19 was observed in 2 and 4 week cultures and then was down-regulated, consistent with its expression in SC precursor, but not mature stages. Co-culture of hESC-derived SC with rat, chick or hESC-derived axons in compartmentalized microfluidic chambers resulted in tight association of the SC with axons. Apparent wrapping of the axons by SC was occasionally observed, suggestive of myelination. Our method for generating SC from hESC makes available a virtually unlimited source of human SC for studies of their role in nerve regeneration and modeling of disease.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. LaBonne, C., & Bronner-Fraser, M. (1999). Molecular mechanisms of neural crest formation. Annual Review of Cell and Developmental Biology, 15, 81–112.

    Article  PubMed  CAS  Google Scholar 

  2. Jessen, K., & Mirsky, R. (2005). The origin and development of glial cells in peripheral nerves. Nature Reviews. Neuroscience, 6(9), 671–682.

    Article  PubMed  CAS  Google Scholar 

  3. Lee, Y., Chang, M., & Lin, K. (2008). Charcot-Marie-tooth disease. Acta Neurologica Taiwanica, 17(3), 203–213.

    PubMed  CAS  Google Scholar 

  4. Carroll, S. L., & Ratner, N. (2008). How does the Schwann cell lineage form tumors in NF1? Glia, 56(14), 1590–1605.

    Article  PubMed  Google Scholar 

  5. Chen, Z., Yu, W., & Strickland, S. (2007). Peripheral regeneration 2. Annual Review of Neuroscience, 30, 209–233.

    Article  PubMed  Google Scholar 

  6. Raivich, G., & Makwana, M. (2007). The making of successful axonal regeneration: genes, molecules and signal transduction pathways 1. Brain Research Reviews, 53(2), 287–311.

    Article  PubMed  CAS  Google Scholar 

  7. Blakemore, W. F., & Crang, A. J. (1985). The use of cultured autologous Schwann cells to remyelinate areas of persistent demyelination in the central nervous system. Journal of the Neurological Sciences, 70(2), 207–223.

    Article  PubMed  CAS  Google Scholar 

  8. Kohama, I., Lankford, K. L., Preiningerova, J., White, F. A., Vollmer, T. L., & Kocsis, J. D. (2001). Transplantation of cryopreserved adult human Schwann cells enhances axonal conduction in demyelinated spinal cord. The Journal of Neuroscience, 21(3), 944–950.

    PubMed  CAS  Google Scholar 

  9. Takami, T., Oudega, M., Bates, M. L., Wood, P. M., Kleitman, N., & Bunge, M. B. (2002). Schwann cell but not olfactory ensheathing glia transplants improve hindlimb locomotor performance in the moderately contused adult rat thoracic spinal cord. The Journal of Neuroscience, 22(15), 6670–6681.

    PubMed  CAS  Google Scholar 

  10. Pearse, D. D., Pereira, F. C., Marcillo, A. E., Bates, M. L., Berrocal, Y. A., Filbin, M. T., et al. (2004). cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury. Natural Medicines, 10(6), 610–616.

    Article  CAS  Google Scholar 

  11. Mosahebi, A., Fuller, P., Wiberg, M., & Terenghi, G. (2002). Effect of allogeneic Schwann cell transplantation on peripheral nerve regeneration. Experimental Neurology, 173(2), 213–223.

    Article  PubMed  CAS  Google Scholar 

  12. Hill, C. E., Moon, L. D. F., Wood, P. M., & Bunge, M. B. (2006). Labeled Schwann cell transplantation: cell loss, host Schwann cell replacement, and strategies to enhance survival. Glia, 53(3), 338–343.

    Article  PubMed  Google Scholar 

  13. Morrissey, T. K., Kleitman, N., & Bunge, R. P. (1991). Isolation and functional characterization of Schwann cells derived from adult peripheral nerve. The Journal of Neuroscience, 11(8), 2433–2442.

    PubMed  CAS  Google Scholar 

  14. McKenzie, I. A., Biernaskie, J., Toma, J. G., Midha, R., & Miller, F. D. (2006). Skin-derived precursors generate myelinating Schwann cells for the injured and dysmyelinated nervous system. The Journal of Neuroscience, 26(24), 6651–6660.

    Article  PubMed  CAS  Google Scholar 

  15. Dezawa, M., Takahashi, I., Esaki, M., Takano, M., & Sawada, H. (2001). Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells. The European Journal of Neuroscience, 14(11), 1771–1776.

    Article  PubMed  CAS  Google Scholar 

  16. Thomson, J., Itskovitz-Eldor, J., Shapiro, S., Waknitz, M., Swiergiel, J., et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–1147.

    Article  PubMed  CAS  Google Scholar 

  17. Reubinoff, B., Pera, M., Fong, C., Trounson, A., & Bongso, A. (2000). Embryonic stem cell lines from human blastocysts:somatic differentiation in vitro. Nature Biotechnology, 18, 399–404.

    Article  PubMed  CAS  Google Scholar 

  18. Friedrich Ben-Nun, I., & Benvenisty, N. (2006). Human embryonic stem cells as a cellular model for human disorders. Molecular and Cellular Endocrinology, 252(1–2), 154–159.

    PubMed  Google Scholar 

  19. Pomp, O., Brokhman, I., Ben-Dor, I., Reubinoff, B., & Goldstein, R. (2005). Generation of peripheral sensory and sympathetic neurons and neural crest cells from human embryonic stem cells. Stem Cells, 23(7), 923–930.

    Article  PubMed  CAS  Google Scholar 

  20. Brokhman, I., Gamarnik-Ziegler, L., Pomp, O., Aharonowiz, M., Reubinoff, B. E., & Goldstein, R. S. (2008). Peripheral sensory neurons differentiate from neural precursors derived from human embryonic stem cells. Differentiation, 76(2), 145–155.

    Article  PubMed  CAS  Google Scholar 

  21. Pomp, O., Brokhman, I., Ziegler, L., Almog, M., Korngreen, A., Tavian, M., et al. (2008). PA6-induced human embryonic stem cell-derived neurospheres: a new source of human peripheral sensory neurons and neural crest cells. Brain Research, 1230, 50–60.

    Article  PubMed  CAS  Google Scholar 

  22. Hotta, R., Pepdjonovic, L., Anderson, R. B., Zhang, D., Bergner, A. J., Leung, J., et al. (2009). Small-molecule induction of neural crest-like cells derived from human neural progenitors. Stem Cells, 27(12), 2896–2905.

    PubMed  CAS  Google Scholar 

  23. Jiang, X., Gwye, Y., McKeown, S. J., Bronner-Fraser, M., Lutzko, C., & Lawlor, E. R. (2009). Isolation and characterization of neural crest stem cells derived from in vitro-differentiated human embryonic stem cells. Stem Cells and Development, 18(7), 1059–1070.

    Article  PubMed  CAS  Google Scholar 

  24. Lee, G., Kim, H., Elkabetz, Y., Al Shamy, G., Panagiotakos, G., Barberi, T., et al. (2007). Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nature Biotechnology, 25(12), 1468–1475.

    Article  PubMed  CAS  Google Scholar 

  25. Eldridge, C. F., Bunge, M. B., Bunge, R. P., & Wood, P. M. (1987). Differentiation of axon-related Schwann cells in vitro. I. Ascorbic acid regulates basal lamina assembly and myelin formation. The Journal of Cell Biology, 105(2), 1023–1034.

    Article  PubMed  CAS  Google Scholar 

  26. Yang, I. H., Siddique, R., Hosmane, S., Thakor, N., & Höke, A. (2009). Compartmentalized microfluidic culture platform to study mechanism of paclitaxel-induced axonal degeneration. Experimental Neurology, 218(1), 124–128.

    Article  PubMed  CAS  Google Scholar 

  27. Campenot, R. (1977). Local control of neurite development by nerve growth factor 71. Proceedings of the National Academy of Sciences of the United States of America, 74(10), 4516–4519.

    Article  PubMed  CAS  Google Scholar 

  28. Adcock, K. H., Brown, D. J., Shearer, M. C., Shewan, D., Schachner, M., Smith, G. M., et al. (2004). Axon behaviour at Schwann cell-astrocyte boundaries: manipulation of axon signalling pathways and the neural adhesion molecule L1 can enable axons to cross. The European Journal of Neuroscience, 20(6), 1425–1435.

    Article  PubMed  Google Scholar 

  29. Spiegel, I., Adamsky, K., Eshed, Y., Milo, R., Sabanay, H., Sarig-Nadir, O., et al. (2007). A central role for Necl4 (SynCAM4) in Schwann cell-axon interaction and myelination. Nature Neuroscience, 10(7), 861–869.

    Article  PubMed  CAS  Google Scholar 

  30. Shea, T. B., & Beermann, M. L. (1994). Respective roles of neurofilaments, microtubules, MAP1B, and tau in neurite outgrowth and stabilization. Molecular Biology of the Cell, 5(8), 863–875.

    PubMed  CAS  Google Scholar 

  31. Beqqali, A., Kloots, J., Ward-van, O. D., Mummery, C., & Passier, R. (2006). Genome-wide transcriptional profiling of human embryonic stem cells differentiating to cardiomyocytes. Stem Cells, 24(8), 1956–1967.

    Article  PubMed  CAS  Google Scholar 

  32. Wood, P. M. (1976). Separation of functional Schwann cells and neurons from normal peripheral nerve tissue. Brain Research, 115(3), 361–375.

    Article  PubMed  CAS  Google Scholar 

  33. Lobsiger, C. S., Taylor, V., & Suter, U. (2002). The early life of a Schwann cell. Biological Chemistry, 383(2), 245–253.

    Article  PubMed  CAS  Google Scholar 

  34. Park, J., Koito, H., Li, J., & Han, A. (2009). Microfluidic compartmentalized co-culture platform for CNS axon myelination research. Biomedical Microdevices, 11(6), 1145–1153.

    Article  PubMed  CAS  Google Scholar 

  35. Höke, A., Redett, R., Hameed, H., Jari, R., Zhou, C., Li, Z. B., et al. (2006). Schwann cells express motor and sensory phenotypes that regulate axon regeneration. The Journal of Neuroscience, 26(38), 9646–9655.

    Article  PubMed  Google Scholar 

  36. Taylor, A. M., Blurton-Jones, M., Rhee, S. W., Cribbs, D. H., Cotman, C. W., & Jeon, N. L. (2005). A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nature Methods, 2(8), 599–605.

    Article  PubMed  CAS  Google Scholar 

  37. Agudo, M., Woodhoo, A., Webber, D., Mirsky, R., Jessen, K. R., & McMahon, S. B. (2008). Schwann cell precursors transplanted into the injured spinal cord multiply, integrate and are permissive for axon growth. Glia, 56(12), 1263–1270.

    Article  PubMed  CAS  Google Scholar 

  38. Roth, T. M., Ramamurthy, P., Ebisu, F., Lisak, R. P., Bealmear, B. M., & Barald, K. F. (2007). A mouse embryonic stem cell model of Schwann cell differentiation for studies of the role of neurofibromatosis type 1 in Schwann cell development and tumor formation. Glia, 55(11), 1123–1133.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

Supported by the Dr. Miriam and Sheldon Adelson Medical Research Foundation and Israel Science Foundation grant #158/07 (RSG). and a Maryland Technology Development Corporation Grant 104307 (NVT). Thanks to Chaya Morgenstern for technical and logistic support.

Disclosures

The authors declare that the work described in this report was not supported by commercial entities and that there are no conflicts of interests. RSG, IHY and NVT have applied for intellectual property protection on some of the techniques described here.

Author information

Authors and Affiliations

  1. Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Gonda Building, Old Campus, 52900, Ramat-Gan, Israel

    Lina Ziegler, Sergei Grigoryan & Ronald S. Goldstein

  2. Department of Biomedical Engineering, School of Medicine, Johns Hopkins University, 720 Rutland Ave. Traylor #715, Baltimore, MD, 21205, USA

    In Hong Yang & Nitish V. Thakor

Authors
  1. Lina Ziegler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sergei Grigoryan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. In Hong Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nitish V. Thakor
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ronald S. Goldstein
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ronald S. Goldstein.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Figure 1

Scheme depicting hESC differentiation into SC and their interaction with neurons. Neurospheres were generated using the PA6 induction method, grown in suspension for 4 wk and differentiated under adherent conditions into putative SC for a further 8 wk. In some experiments, differentiated SC were co-cultured with chick / rat or hESC-derived neurons (GIF 484 kb)

High resolution image (TIFF 1324 kb)

Supplementary Figure 2

Oil red staining of cultures of hESC-derived SC. After fixation with 4% paraformaldehyde and rinsing with 60% isopropanol, 8-wk cultures were stained with freshly prepared Oil Red and lightly counterstained with hematoxylin. The red staining of many cells indicates high concentrations of lipids, indicative of myelination (GIF 144 kb)

High resolution image (TIFF 1827 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ziegler, L., Grigoryan, S., Yang, I.H. et al. Efficient Generation of Schwann Cells from Human Embryonic Stem Cell-Derived Neurospheres. Stem Cell Rev and Rep 7, 394–403 (2011). https://doi.org/10.1007/s12015-010-9198-2

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12015-010-9198-2

Keywords

Search

Navigation