Simultaneous PKC and cAMP activation induces differentiation of human dental pulp stem cells into functionally active neurons.
The plasticity of dental pulp stem cells (DPSCs) has been demonstrated by several studies showing that they appear to self-maintain through several passages, giving rise to a variety of cells. The aim of the present study was to differentiate DPSCs to mature neuronal cells showing functional evidence of voltage gated ion channel activities in vitro. First, DPSC cultures were seeded on poly-l-lysine coated surfaces and pretreated for 48h with a medium containing basic fibroblast growth factor and the demethylating agent 5-azacytidine. Then neural induction was performed by the simultaneous activation of protein kinase C and the cyclic adenosine monophosphate pathway. Finally, maturation of the induced cells was achieved by continuous treatment with neurotrophin-3, dibutyryl cyclic AMP, and other supplementary components. Non-induced DPSCs already expressed vimentin, nestin, N-tubulin, neurogenin-2 and neurofilament-M. The inductive treatment resulted in decreased vimentin, nestin, N-tubulin and increased neurogenin-2, neuron-specific enolase, neurofilament-M and glial fibrillary acidic protein expression. By the end of the maturation period, all investigated genes were expressed at higher levels than in undifferentiated controls except vimentin and nestin. Patch clamp analysis revealed the functional activity of both voltage-dependent sodium and potassium channels in the differentiated cells. Our results demonstrate that although most surviving cells show neuronal morphology and express neuronal markers, there is a functional heterogeneity among the differentiated cells obtained by the in vitro differentiation protocol described herein. Nevertheless, this study clearly indicates that the dental pulp contains a cell population that is capable of neural commitment by our three step neuroinductive protocol.
Király
Marianna
M
Department of Oral Biology, Semmelweis University, Budapest, Hungary.
Porcsalmy
Balázs
B
Pataki
Agnes
A
Kádár
Kristóf
K
Jelitai
Márta
M
Molnár
Bálint
B
Hermann
Péter
P
Gera
István
I
Grimm
Wolf-Dieter
WD
Ganss
Bernhard
B
Zsembery
Akos
A
Varga
Gábor
G
D001374:Azacitidine / Q000008:administration & dosage; D001483:Base Sequence; D002454:Cell Differentiation; D002478:Cells, Cultured; D003470:Culture Media; D000242:Cyclic AMP / Q000378:metabolism*; D017931:DNA Primers; D003782:Dental Pulp / Q000166:cytology* / Q000201:enzymology; D004789:Enzyme Activation; D016222:Fibroblast Growth Factor 2 / Q000008:administration & dosage; D006801:Humans; D007150:Immunohistochemistry; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology*
D003470:Culture Media; D017931:DNA Primers; D016222:Fibroblast Growth Factor 2; D000242:Cyclic AMP; D011493:Protein Kinase C; D001374:Azacitidine