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Characterization and differentiation potential of rat ventral mesencephalic neuronal progenitor cells immortalized with SV40 large T antigen

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An Erratum to this article was published on 11 June 2010

Abstract

Neuronal progenitor cells (NPCs) possess high potential for use in regenerative medicine. To overcome their limited mitotic competence, various immortalization strategies have been applied that allow their prolonged maintenance and expansion in vitro. Such immortalized cells can be used for the design and discovery of new cell-based therapies for neurodegenerative diseases, such as Parkinson’s disease. We immortalized rat ventral mesencephalic NPCs by using SV40 large T antigen (SV40Tag). All cell clones displayed a two- to three–fold higher proliferation rate compared with the primary cells. In order to induce dopaminergic differentiation of generated cell clones, both glial-derived neurotrophic factor and di-butyryl cyclic adenosine monophosphate were applied. Treated cells were then characterized regarding the expression of dopaminergic lineage markers, differentiation of various cell populations, calcium imaging in the presence of kainate, and immunohistochemistry after intrastriatal transplantation. Treated cells displayed morphological maturation, and calcium imaging revealed neuronal properties in the presence of kainate. These cells also expressed low mRNA levels of the dopamine transporter and tyrosine hydroxylase (TH), although no TH-immunopositive neurons were found. Intrastriatal transplantation into the neurotoxin-lesioned rats did not induce further differentiation. As an alternative approach, we silenced SV40Tag with short interfering RNA, but this was not sufficient to trigger differentiation into dopaminergic neurons. Nevertheless, neuronal and glial cells were detected as shown by β-tubulin type III and glial fibrillary acidic protein staining, respectively. SV40Tag cells are suitable for carrying out controlled genetic modifications as shown by overexpression of enhanced green fluorescence protein after efficient non-viral transfection.

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Abbreviations

AMPA:

α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid

BDNF:

brain-derived neurotropic factor

cAMP:

di-butyryl cyclic adenosine monophosphate

DA:

dopamine

DAPI:

4,6-diamidino-2-phenylindole

DAT:

dopamine transporter

EGFP:

enhanced green fluorescence protein

E12:

embryonic day 12

GAPDH:

glyceraldehyde-3-phosphate dehydrogenase

GDNF:

glial cell line-derived neurotrophic factor

GFAP:

glial fibrillary acidic protein

KA:

kainate

NPCs:

neuronal progenitor cells

PD:

Parkinson’s disease

PFA:

paraformaldehyde

SDS:

sodium dodecyl sulfate

SV40:

simian virus 40

Tag:

large T antigen

TH:

tyrosine hydroxylase

VM:

ventral mesencephalic

6-OHDA:

6-hydroxydopamine

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Acknowledgements

We thank the Department of Experimental Nephrology (Hannover Medical School) for permission to use the Amaxa Nucleofector device. We gratefully acknowledge the generous gift of the pCAGGS-EGFP vector from Dr. J.R. Whiteford. We also thank Kerstin Kuhlemann, Hella Brinkmann, Natascha Heidrich, and Silke Fischer for excellent technical support and Jürgen Wittek, Serguei Tkachuk, Kerstin Schwabe, and Johannes Haile for discussions.

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Authors and Affiliations

  1. Institute of Neuroanatomy, Hannover Medical School, 30625, Hannover, Germany

    André Nobre, Ieva Kalve, Konstantin Cesnulevicius, Andreas Ratzka, Nina Halfer, Maike Wesemann, Peter Claus & Claudia Grothe

  2. Department of Neurology, Hannover Medical School, 30625, Hannover, Germany

    Daniela Rangancokova & Klaus Krampfl

  3. Center for Systems Neuroscience Hannover, 30625, Hannover, Germany

    André Nobre, Ieva Kalve, Konstantin Cesnulevicius, Daniela Rangancokova, Klaus Krampfl, Peter Claus & Claudia Grothe

  4. Institute of Neuroanatomy, Center of Anatomy, OE 4140, Hannover Medical School, Carl-Neuberg-Strasse 1, 30623, Hannover, Germany

    Claudia Grothe

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  1. André Nobre
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  2. Ieva Kalve
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  3. Konstantin Cesnulevicius
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  10. Claudia Grothe
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Correspondence to Claudia Grothe.

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This work was supported by EU Marie Curie host fellowships (A.N.) for early stage researchers training—MEST-CT-2005-021 014 (“ZSN-PHD Program”) and by Georg-Christoph-Lichtenberg scholarships (I.K., K.C., and D.R.) provided by the Ministry of Science and Culture of Lower Saxony, as part of the international PhD program of the Center for Systems Neuroscience Hannover, Germany.

An erratum to this article can be found at http://dx.doi.org/10.1007/s00441-010-0999-z

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Nobre, A., Kalve, I., Cesnulevicius, K. et al. Characterization and differentiation potential of rat ventral mesencephalic neuronal progenitor cells immortalized with SV40 large T antigen. Cell Tissue Res 340, 29–43 (2010). https://doi.org/10.1007/s00441-010-0933-4

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