In Vitro Functional Characterization of Human Neurons and Astrocytes Using Calcium Imaging and Electrophysiology

  • Marita Grønning Hansen
  • Daniel Tornero
  • Isaac Canals
  • Henrik AhleniusEmail author
  • Zaal KokaiaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1919)


Recent progress in stem cell biology and epigenetic reprogramming has opened up previously unimaginable possibilities to study and develop regenerative approaches for neurological disorders. Human neurons and glial cells can be generated by differentiation of embryonic and neural stem cells and from somatic cells through reprogramming to pluripotency (followed by differentiation) as well as by direct conversion. All of these cells have the potential to be used for studying and treating neurological disorders. However, before considering using human neural cells derived from these sources for modelling or regenerative purposes, they need to be verified in terms of functionality and similarity to endogenous cells in the central nervous system (CNS).

In this chapter, we describe how to assess functionality of neurons and astrocytes derived from stem cells and through direct reprogramming, using calcium imaging and electrophysiology.

Key words

Calcium imaging Electrophysiology Neurons Astrocytes Stem cells Reprogramming Direct conversion 


  1. 1.
    Uchida N et al (2000) Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci U S A 97(26):14720–14725PubMedPubMedCentralCrossRefGoogle Scholar
  2. 2.
    Thomson JA et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282(5391):1145–1147CrossRefGoogle Scholar
  3. 3.
    Takahashi K et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872CrossRefGoogle Scholar
  4. 4.
    Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4):663–676CrossRefGoogle Scholar
  5. 5.
    Tao Y, Zhang SC (2016) Neural subtype specification from human pluripotent stem cells. Cell Stem Cell 19(5):573–586PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Pang ZP et al (2011) Induction of human neuronal cells by defined transcription factors. Nature 476(7359):220–223PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Vierbuchen T et al (2010) Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463(7284):1035–1041PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Lujan E et al (2012) Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells. Proc Natl Acad Sci U S A 109(7):2527–2532PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Yang N et al (2013) Generation of oligodendroglial cells by direct lineage conversion. Nat Biotechnol 31(5):434–439PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Caiazzo M et al (2015) Direct conversion of fibroblasts into functional astrocytes by defined transcription factors. Stem Cell Rep 4(1):25–36CrossRefGoogle Scholar
  11. 11.
    Zhang Y et al (2013) Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron 78(5):785–798PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Yang N et al (2017) Generation of pure GABAergic neurons by transcription factor programming. Nat Methods 14(6):621–628PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Rapid and efficient induction of functional astrocytes from human pluripotent stem cells. Canals I, Ginisty A, Quist E, Timmerman R, Fritze J, Miskinyte G, Monni E, Hansen MG, Hidalgo I, Bryder D, Bengzon J, Ahlenius H. Nat Methods. 2018 Sep;15(9):693–696. Epub 2018 Aug 20. PMID: 30127505PubMedCrossRefGoogle Scholar
  14. 14.
    Ehrlich M et al (2017) Rapid and efficient generation of oligodendrocytes from human induced pluripotent stem cells using transcription factors. Proc Natl Acad Sci U S A 114(11):E2243–E2252PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Kallur T et al (2006) Human fetal cortical and striatal neural stem cells generate region-specific neurons in vitro and differentiate extensively to neurons after intrastriatal transplantation in neonatal rats. J Neurosci Res 84(8):1630–1644PubMedCrossRefGoogle Scholar
  16. 16.
    Tornero D et al (2017) Synaptic inputs from stroke-injured brain to grafted human stem cell-derived neurons activated by sensory stimuli. Brain 140(3):692–706PubMedGoogle Scholar
  17. 17.
    Tornero D et al (2013) Human induced pluripotent stem cell-derived cortical neurons integrate in stroke-injured cortex and improve functional recovery. Brain 136(Pt 12):3561–3577PubMedCrossRefGoogle Scholar
  18. 18.
    Miskinyte G et al (2017) Direct conversion of human fibroblasts to functional excitatory cortical neurons integrating into human neural networks. Stem Cell Res Ther 8(1):207PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Berridge MJ, Lipp P, Bootman MD (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1(1):11–21PubMedCrossRefGoogle Scholar
  20. 20.
    Grienberger C, Konnerth A (2012) Imaging calcium in neurons. Neuron 73(5):862–885PubMedCrossRefGoogle Scholar
  21. 21.
    Newman EA, Zahs KR (1997) Calcium waves in retinal glial cells. Science 275(5301):844–847PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Parpura V et al (1994) Glutamate-mediated astrocyte-neuron signalling. Nature 369(6483):744–747PubMedCrossRefGoogle Scholar
  23. 23.
    Bazargani N, Attwell D (2016) Astrocyte calcium signaling: the third wave. Nat Neurosci 19(2):182–189PubMedCrossRefGoogle Scholar
  24. 24.
    Paredes RM et al (2008) Chemical calcium indicators. Methods 46(3):143–151PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Bedner P et al (2015) Astrocyte uncoupling as a cause of human temporal lobe epilepsy. Brain 138(Pt 5):1208–1222PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    Dallerac G, Chever O, Rouach N (2013) How do astrocytes shape synaptic transmission? Insights from electrophysiology. Front Cell Neurosci 7:159PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    Chung WS, Allen NJ, Eroglu C (2015) Astrocytes control synapse formation, function, and elimination. Cold Spring Harb Perspect Biol 7(9):a020370PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Faculty of Medicine, Department of Clinical Sciences Lund, Neurology, Lund Stem Cell CenterLaboratory of Stem Cells and Restorative Neurology, Lund University, Skåne University HospitalLundSweden
  2. 2.Faculty of Medicine, Department of Clinical Sciences Lund, Neurology, Lund Stem Cell CenterStem Cells, Aging and Neurodegeneration Group, Lund UniversityLundSweden

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