Pharmacological Notch pathway inhibition leads to cell cycle arrest and stimulates ascl1 and neurogenin2 genes expression in dental pulp stem cells-derived neurospheres
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Human dental pulp-derived stem cells (hDPSCs) are becoming an attractive source for cell-based neurorestorative therapies. As such, it is important to understand the molecular mechanisms that regulate the differentiation of hDPSCs toward the neuronal fate. Notch signaling plays key roles in neural stem/progenitor cells (NS/PCs) maintenance and prevention of their differentiation. The aim of this study was to address the effects of Notch signaling inhibition on neurosphere formation of hDPSCs and neuronal differentiation of hDPSCs-neurospheres.
hDPSCs were isolated from third molar teeth. The cultivated hDPSCs highly expressed CD90 and CD44 and minimally presented CD34 and CD45 surface markers. The osteo/adipogenic differentiation of hDPSCs was documented. hDPSCs were cultured in neural induction medium and N-[N-(3,5-difluorophenacetyl-l-alanyl)]-Sphenylglycine t-butyl ester (DAPT) was applied to impede Notch signaling during transformation into spheres or on the formed neurospheres. Our results showed that the size and number of neurospheres decreased and the expression profile of nestin, sox1 and pax6 genes reduced provided DAPT. Treatment of the formed neurospheres with DAPT resulted in the cleaved Notch1 reduction, G0/G1 arrest and a decline in L-lactate production. DAPT significantly reduced hes1 and hey1 genes, while ascl1 and neurogenin2 expressions augmented. The number of MAP2 positive cells improved in the DAPT-treated group.
Our findings demonstrated the Notch activity in hDPSCs-neurospheres. DAPT treatment positively regulated proneural genes expression and increased neuronal-like differentiation.
KeywordshDPSCs Notch signaling Gamma-secretase inhibitor Neurosphere Differentiation ascl1 Neurogenin2
This study was financially supported by a Research Project (Grant No. 9223) of Research Vice-Chancellery of Ardabil University of Medical Sciences, Iran.
hDPSCs isolation and culture, neural induction, MTT assay and cell cycle were done by AN. hDPSCs characterization was done by VM. Real time-PCR was performed and interpreted by AN and NN. Western blotting and immunofluorescent staining were performed by PM and HGH, respectively. The l-lactate assay was performed by MSP. The paper was written principally by AN with input from all the other authors especially PM. Study design and statistically analyzing of data were done by AN and HGH.
Compliance with ethical standards
Conflict of interest
The authors declared no potential conflicts of interest.
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