Abstract
Approaches which differentiate embryonic stem (ES) cells into neurons have recently garnered greater attention mainly due to their importance in physiological research and possible applications in regenerative medicine. However, much effort is being marshaled to generate uniform neuronal populations, but a completely reliable method has yet to be developed. Herein, methods which have been reported are summarized and discussed. Based on the principle of isolating glutamatergic neurons, they can be classified into approaches of immunoisolation, gene manipulation, and ectoderm dissociation. Each approach claims to be able to obtain uniform glutamatergic neurons. This chapter summarizes the three methods, describes detailed isolation procedures, and discusses functionally related studies. Some concerns with and disadvantages of each method still remain as protocols are further developed for clinical application. Nevertheless, establishing these methods has confirmed that neurons derived from ES cell differentiation are excellent cellular models for investigating properties related to the central nervous system. They also have great potential for applications to regenerative medicine.
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References
Aoyama K, Watabe M, Nakaki T (2008) Regulation of neuronal glutathione synthesis. J Pharmacol Sci 108:227–238
Bibel M, Richter J, Lacroix E, Barde YA (2007) Generation of a defined and uniform population of CNS progenitors and neurons from mouse embryonic stem cells. Nat Protoc 2:1034–1043
Cao Q, Benton RL, Whittemore SR (2002) Stem cell repair of central nervous system injury. J Neurosci Res 68:501–510
Chuang JH, Tung LC, Lee-Chen GJ, Yin Y, Lin Y (2011) An approach for differentiating uniform glutamatergic neurons from mouse embryonic stem cells. Anal Biochem 410:149–151
Cook A, Hippensteel R, Shimizu S, Nicolai J, Fatatis A, Meucci O (2010) Interactions between chemokines: regulation of fractalkine/CX3CL1 homeostasis by SDF/CXCL12 in cortical neurons. J Biol Chem 285:10563–10571
Copi A, Jungling K, Gottmann K (2005) Activity- and BDNF-induced plasticity of miniature synaptic currents in ES cell-derived neurons integrated in a neocortical network. J Neurophysiol 94:4538–4543
Dubinsky JM (1993) Intracellular calcium levels during the period of delayed excitotoxicity. J Neurosci 13:623–631
Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156
Fischer G, Kunemund V, Schachner M (1986) Neurite outgrowth patterns in cerebellar microexplant cultures are affected by antibodies to the cell surface glycoprotein L1. J Neurosci 6:605–612
Hirsch JA, Gibson GE (1984) Selective alteration of neurotransmitter release by low oxygen in vitro. Neurochem Res 9:1039–1049
Huang EJ, Liu W, Fritzsch B, Bianchi LM, Reichardt LF, Xiang M (2001) Brn3a is a transcriptional regulator of soma size, target field innervation and axon pathfinding of inner ear sensory neurons. Development 128:2421–2432
Jungling K, Nagler K, Pfrieger FW, Gottmann K (2003) Purification of embryonic stem cell-derived neurons by immunoisolation. FASEB J 17:2100–2102
Jungling K, Eulenburg V, Moore R, Kemler R, Lessmann V, Gottmann K (2006) N-cadherin transsynaptically regulates short-term plasticity at glutamatergic synapses in embryonic stem cell-derived neurons. J Neurosci 26:6968–6978
Karis A, Pata I, van Doorninck JH, Grosveld F, de Zeeuw CI, de Caprona D, Fritzsch B (2001) Transcription factor GATA-3 alters pathway selection of olivocochlear neurons and affects morphogenesis of the ear. J Comp Neurol 429:615–630
Kondo T, Sheets PL, Zopf DA, Aloor HL, Cummins TR, Chan RJ, Hashino E (2008) Tlx3 exerts context-dependent transcriptional regulation and promotes neuronal differentiation from embryonic stem cells. Proc Natl Acad Sci U S A 105:5780–5785
Lang RJ, Haynes JM, Kelly J, Johnson J, Greenhalgh J, O’Brien C, Mulholland EM, Baker L, Munsie M, Pouton CW (2004) Electrical and neurotransmitter activity of mature neurons derived from mouse embryonic stem cells by Sox-1 lineage selection and directed differentiation. Eur J Neurosci 20:3209–3221
Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443:787–795
Manev H, Favaron M, Guidotti A, Costa E (1989) Delayed increase of Ca2+ influx elicited by glutamate: role in neuronal death. Mol Pharmacol 36:106–112
Markowitz AJ, White MG, Kolson DL, Jordan-Sciutto KL (2007) Cellular interplay between neurons and glia: toward a comprehensive mechanism for excitotoxic neuronal loss in neurodegeneration. Cellscience 4:111–146
McNutt P, Celver J, Hamilton T, Mesngon M (2011) Embryonic stem cell-derived neurons are a novel, highly sensitive tissue culture platform for botulinum research. Biochem Biophys Res Commun 405:85–90
Nicholls DG (2009) Spare respiratory capacity, oxidative stress and excitotoxicity. Biochem Soc Trans 37:1385–1388
Palmada M, Centelles JJ (1998) Excitatory amino acid neurotransmission. Pathways for metabolism, storage and reuptake of glutamate in brain. Front Biosci 3:d701–d718
Rathjen FG, Schachner M (1984) Immunocytological and biochemical characterization of a new neuronal cell surface component (L1 antigen) which is involved in cell adhesion. EMBO J 3:1–10
Reyes JH, O’Shea KS, Wys NL, Velkey JM, Prieskorn DM, Wesolowski K, Miller JM, Altschuler RA (2008) Glutamatergic neuronal differentiation of mouse embryonic stem cells after transient expression of neurogenin 1 and treatment with BDNF and GDNF: in vitro and in vivo studies. J Neurosci 28:12622–12631
Rohwedel J, Guan K, Wobus AM (1999) Induction of cellular differentiation by retinoic acid in vitro. Cells Tissues Organs 165:190–202
Stavridis MP, Smith AG (2003) Neural differentiation of mouse embryonic stem cells. Biochem Soc Trans 31:45–49
Tong M, Hernandez JL, Purcell EK, Altschuler RA, Duncan RK (2010) The intrinsic electrophysiological properties of neurons derived from mouse embryonic stem cells overexpressing neurogenin-1. Am J Physiol Cell Physiol 299:C1335–C1344
Varga BV, Hadinger N, Gocza E, Dulberg V, Demeter K, Madarasz E, Herberth B (2008) Generation of diverse neuronal subtypes in cloned populations of stem-like cells. BMC Dev Biol 8:89–106
Wernig M, Benninger F, Schmandt T, Rade M, Tucker KL, Bussow H, Beck H, Brustle O (2004) Functional integration of embryonic stem cell-derived neurons in vivo. J Neurosci 24:5258–5268
Acknowledgements
This work was partially supported by grants (NSC97-2311-B-003-001 and NSC98-2311-B-003-MY3) to Y. Lin from the National Science Council, Taipei, Taiwan. We are grateful to the support from the Office of Research and Development (96B01) and Image Core, National Taiwan Normal University, Taipei, Taiwan. Special thanks also go to Dr. Joseph Avruch and Dr. Yi Yin of Mass General Hospital, Boston, MA, USA for providing some reagents.
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Lin, Y. (2012). Differentiation of Embryonic Stem Cells into Glutamatergic Neurons (Methods). In: Hayat, M. (eds) Stem Cells and Cancer Stem Cells, Volume 6. Stem Cells and Cancer Stem Cells, vol 6. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-2993-3_5
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DOI: https://doi.org/10.1007/978-94-007-2993-3_5
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