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Cellular phenotypes of human model neurons (NT2) after differentiation in aggregate culture

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Abstract

The well-characterized human teratocarcinoma line Ntera2 (NT2) can be differentiated into mature neurons. We have significantly shortened the time-consuming process for generating postmitotic neurons to approximately 4 weeks by introducing a differentiation protocol for free-floating cell aggregates and a subsequent purification step. Here, we characterize the neurochemical phenotypes of the neurons derived from this cell aggregate method. During differentiation, the NT2 cells lose immunoreactivity for vimentin and nestin filaments, which are characteristic for the immature state of neuronal precursors. Instead, they acquire typical neuronal markers such as β-tubulin type III, microtubule-associated protein 2, and phosphorylated tau, but no astrocyte markers such as glial fibrillary acidic protein. They grow neural processes that express punctate immunoreactivity for synapsin and synaptotagmin suggesting the formation of presynaptic structures. Despite their common clonal origin, neurons cultured for 2–4 weeks in vitro comprise a heterogeneous population expressing several neurotransmitter phenotypes. Approximately 40% of the neurons display glutamatergic markers. A minority of neurons is immunoreactive for serotonin, gamma-amino-butyric acid, and its synthesizing enzyme glutamic acid decarboxylase. We have found no evidence for a dopaminergic phenotype. Subgroups of NT2 neurons respond to the application of nitric oxide donors with the synthesis of cGMP. A major subset shows immunoreactivity to the cholinergic markers choline acetyl-transferase, vesicular acetylcholine transporter, and the non-phosphorylated form of neurofilament H, all indicative of motor neurons. The NT2 system may thus be well suited for research related to motor neuron diseases.

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Abbreviations

BrdU:

5-bromo-2’-deoxyuridine

ChAT:

choline acetyl-transferase

cGMP:

cyclic guanosine-monophosphate

DAPI:

4',6-diamidino-2'-phenylindol-dihydrochloride

GABA:

gamma-amino butyric acid

GAD:

glutamate decarboxylase

GFAP:

glial fibrillary acidic protein

MAP-2:

microtubule-associated protein 2

NF-H:

neurofilament H

NO:

nitric oxide

RA:

retinoic acid

sGC:

soluble guanylyl-cyclase

TH:

tyrosine hydroxylase

VAChT:

vesicular acetylcholine transporter

VG1uT1:

vesicular glutamate transporter 1

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Acknowledgements

We are grateful to Jan de Vente, Maastricht University for the gift of the cGMP antiserum, to O. P. Ottersen, Department of Anatomy, University of Oslo for the gift of the glutamate antiserum, to Alexandra Kotsiari, Daniela Ragancokova, and Martin Stangel, Hannover Medical School for the gift of primary rat astrocytes, to the Department of Physiology, University of Veterinary Medicine, Hannover for the gift of mouse CNS tissue, and to Sabine Knipp for helpful discussions.

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Authors and Affiliations

  1. Div. of Cell Biology, Institute of Physiology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173, Hannover, Germany

    Grzegorz Podrygajlo, Million A. Tegenge, Andrea Gierse, Saime Tan, Gerd Bicker & Michael Stern

  2. Center for Systems Neuroscience (ZSN), Hannover, Germany

    Grzegorz Podrygajlo, Million A. Tegenge & Gerd Bicker

  3. Centre for Ophthalmology, Institute for Ophthalmic Research, Division of Experimental Ophthalmology, University of Tübingen, Röntgenweg 11, 72076, Tübingen, Germany

    Francois Paquet-Durand

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  1. Grzegorz Podrygajlo
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  2. Million A. Tegenge
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  3. Andrea Gierse
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  4. Francois Paquet-Durand
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  5. Saime Tan
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  6. Gerd Bicker
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  7. Michael Stern
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Correspondence to Michael Stern.

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G. Podrygajlo is a Marie Curie Actions Fellow and M.A. Tegenge received a Georg Christoph Lichtenberg Fellowship from the Federal State of Lower Saxony. M. Stern and G. Bicker were supported by the Federal Ministry for Education and Research (BMBF). F. Paquet-Durand was supported by grants from the Kerstan Foundation, Tübingen, Germany and the German Research Council (DFG; PA 1751/1-1).

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Podrygajlo, G., Tegenge, M.A., Gierse, A. et al. Cellular phenotypes of human model neurons (NT2) after differentiation in aggregate culture. Cell Tissue Res 336, 439–452 (2009). https://doi.org/10.1007/s00441-009-0783-0

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