Research Laboratory for Embryology and Stem Cells, Department of Anatomical Sciences, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran.
Department of Anatomy and Cell Biology, Immunogenetic Research Center, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
Biotechnol Lett. 2019 Jul;41(6-7):873-887. doi: 10.1007/s10529-019-02687-1. Epub 2019 May 9.
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.
人牙髓干细胞（hDPSCs）正成为细胞神经修复治疗的有吸引力的来源。因此，了解调控 hDPSCs 向神经元命运分化的分子机制非常重要。Notch 信号在神经干细胞/祖细胞（NS/PCs）的维持和防止其分化中起着关键作用。本研究旨在探讨 Notch 信号抑制对 hDPSCs 神经球形成和 hDPSCs-神经球神经元分化的影响。
从第三磨牙中分离出 hDPSCs。培养的 hDPSCs 高度表达 CD90 和 CD44，很少表达 CD34 和 CD45 表面标志物。记录了 hDPSCs 的成骨/成脂分化。hDPSCs 在神经诱导培养基中培养，并用 N-[N-(3,5-二氟苯乙酰基)-L-苯丙氨酸]-SP 酯（DAPT）抑制 Notch 信号，以阻止其在转化为球体或形成神经球时的信号传递。我们的结果表明，DAPT 降低了神经球的大小和数量，降低了巢蛋白、sox1 和 pax6 基因的表达谱。用 DAPT 处理形成的神经球导致 Notch1 裂解减少，G0/G1 停滞和 L-乳酸产生减少。DAPT 显著降低了 hes1 和 hey1 基因的表达，同时增加了 ascl1 和 neurogenin2 的表达。DAPT 处理组 MAP2 阳性细胞数量增加。
我们的研究结果表明，Notch 活性存在于 hDPSCs-神经球中。DAPT 处理正向调控神经前体细胞基因表达，并增加神经元样分化。
