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Stem Cell Reviews and Reports

, Volume 9, Issue 4, pp 461–474 | Cite as

Human Pluripotent Stem Cell Differentiation into Authentic Striatal Projection Neurons

  • Alessia Delli Carri
  • Marco Onorati
  • Valentina Castiglioni
  • Andrea Faedo
  • Stefano Camnasio
  • Mauro Toselli
  • Gerardo Biella
  • Elena Cattaneo
Article

Abstract

Here we present the principles and steps of a protocol that we have recently developed for the differentiation of hES/iPS cells into the authentic human striatal projection medium spiny neurons (MSNs) that die in Huntington’s Disease (HD). Authenticity is judged by the convergence of multiple features within individual cells. Our procedure lasts 80 days and couples neural induction via BMP/TGF-β inhibition with exposure to the developmental factors sonic hedgehog (SHH) and dickkopf1 (DKK-1) to drive ventral telencephalic specification, followed by terminal differentiation [1]. Authenticity of the resulting neuronal population is monitored by the appearance of FOXG1+/GSX2+ progenitor cells of the lateral ganglionic eminence (LGE) at day 15–25 of differentiation, followed by appearance of CTIP2-, FOXP1- and FOXP2-positive cells at day 45. These precursor cells then mature into MAP2+/GABA+ neurons with 20 % of them ultimately co-expressing the DARPP-32 and CTIP2 diagnostic markers and carrying electrophysiological properties expected for fully functional MSNs.

The protocol is characterized by its replicability in at least three human pluripotent cell lines. Altogether this protocol defines a useful platform for in vitro developmental neurobiology studies, drug screening, and regenerative medicine approaches.

Keywords

Striatal neuronal differentiation Medium spiny neurons DARPP-32 Huntington’s disease Directed differentiation Human embryonic stem cells Human pluripotent stem cells 

Notes

Acknowledgments

Several other authors have participated in the original paper (Delli Carri et al., [9]). The authors list in this paper accounts for those that have designed and validated the protocol. The research described in our original paper (Delli Carri et al., [9]) and leading to the protocol herein detailed has received funding from NeuroStemcell, European Union Seventh Framework Programme grant agreement n° 222943, partially from the Ministero dell’Istruzione dell’Università e della Ricerca [MIUR, 2008JKSHKN] and from Cure Huntington's Disease Initiative (CHDI) ID: A-4529 to E.C. and by Fondo per gli Investimenti della Ricerca di Base [FIRB, RBFR10A01S] to M.O.; A.F. was supported by a Marie Curie fellowship. We acknowledge the important contribution of Tavola Valdese (2007–2010) and the support of Unicredit Banca S.p.A. (Italy). We also thank the families of HD patients for their continuous support.

Disclosures

The authors indicate no potential conflicts of interest.

References

  1. 1.
    Chambers, S. M., Fasano, C. A., Papapetrou, E. P., Tomishima, M., Sadelain, M., & Studer, L. (2009). Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature Biotechnology, 27, 275–280.PubMedCrossRefGoogle Scholar
  2. 2.
    Gaspard, N., & Vanderhaeghen, P. (2010). From stem cells to neural networks: recent advances and perspectives for neurodevelopmental disorders. Developmental Medicine and Child Neurology, 53, 13–17.PubMedCrossRefGoogle Scholar
  3. 3.
    Shi, Y., Kirwan, P., Smith, J., Robinson, H. P., & Livesey, F. J. (2012). Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. Nature Neuroscience, 15(S1), 477–486.PubMedCrossRefGoogle Scholar
  4. 4.
    Bissonnette, C. J., Lyass, L., Bhattacharyya, B. J., Belmadani, A., Miller, R. J., & Kessler, J. A. (2011). The controlled generation of functional basal forebrain cholinergic neurons from human embryonic stem cells. Stem Cells, 29, 802–811.PubMedCrossRefGoogle Scholar
  5. 5.
    Kriks, S., Shim, J. W., Piao, J., et al. (2011). Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature, 480, 547–551.PubMedGoogle Scholar
  6. 6.
    Kirkeby, A., Grealish, S., Wolf, D. A., et al. (2012). Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Report, 1, 703–714.CrossRefGoogle Scholar
  7. 7.
    Aubry, L., Bugi, A., Lefort, N., Rousseau, F., Peschanski, M., & Perrier, A. L. (2008). Striatal progenitors derived from human ES cells mature into DARPP32 neurons in vitro and in quinolinic acid-lesioned rats. Proceedings of the National Academy of Sciences of the United States of America, 105, 16707–16712.PubMedCrossRefGoogle Scholar
  8. 8.
    Ma, L., Hu, B., Liu, Y., et al. (2012). Human embryonic stem cell-derived GABA neurons correct locomotion deficits in quinolinic acid-lesioned mice. Cell Stem Cell, 10, 455–464.Google Scholar
  9. 9.
    Delli Carri, A., Onorati, M., Lelos, M. J., et al. (2013). Developmentally coordinated extrinsic signals drive human pluripotent stem cell differentiation toward authentic DARPP-32+ medium-sized spiny neurons. Development, 140, 301–312.CrossRefGoogle Scholar
  10. 10.
    Lupo, G., Harris, W. A., & Lewis, K. E. (2006). Mechanisms of ventral patterning in the vertebrate nervous system. Nature Reviews Neuroscience, 7, 103–114.PubMedCrossRefGoogle Scholar
  11. 11.
    Sousa, V. H., & Fishell, G. (2010). Sonic hedgehog functions through dynamic changes in temporal competence in the developing forebrain. Current Opinion in Genetics and Development, 20, 391–399.PubMedCrossRefGoogle Scholar
  12. 12.
    Schuurmans, C., & Guillemot, F. (2002). Molecular mechanisms underlying cell fate specification in the developing telencephalon. Current Opinion in Neurobiology, 12, 26–34.PubMedCrossRefGoogle Scholar
  13. 13.
    Camnasio, S., Carri, A. D., Lombardo, A., et al. (2012). The first reported generation of several induced pluripotent stem cell lines from homozygous and heterozygous Huntington’s disease patients demonstrates mutation related enhanced lysosomal activity. Neurobiology of Disease, 46, 41–51.PubMedCrossRefGoogle Scholar
  14. 14.
    Kirkeby, A., Nelander, J., & Parmar, M. (2012). Generating regionalized neuronal cells from pluripotency, a step-by-step protocol. Frontiers in Cellular Neuroscience, 6, 64.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Alessia Delli Carri
    • 1
  • Marco Onorati
    • 1
  • Valentina Castiglioni
    • 1
  • Andrea Faedo
    • 1
  • Stefano Camnasio
    • 1
  • Mauro Toselli
    • 2
  • Gerardo Biella
    • 2
  • Elena Cattaneo
    • 1
  1. 1.Department of Biosciences and Center for Stem Cell ResearchUniversità degli Studi di MilanoMilanItaly
  2. 2.Department of Biology and BiotechnologyUniversity of PaviaPaviaItaly

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