Notch/Wnt cross-signalling regulates stemness of dental pulp stem cells through expression of neural crest and core pluripotency factors

Abstract

Dental pulp stem cells (DPSCs) from adult teeth express neural crest (NC) markers together with core transcriptional factors associated with stem cell pluripotency, such as Oct4a, Sox2, Rex1, Stella/ Dppa3, Ssea1/Fut4, Lin28 and Nanog. The possibility to boost the natural stemness features of DPSCs by mild methods, that do not involve gene and/or chromatin modification or gene transfection, is highly desirable for cell therapy. Canonical Wnt and Notch are two highly conserved developmental signalling pathways that are involved in NC emergence and stem cell self-renewal. We determined that both pathways coordinate to regulate the expression of core pluripotency and NC factors in DPSCs. Pharmacological inhibition of the Notch pathway for 48 h, by the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), abolished the expression of NC and core factors. In addition, it induced a silencing of the canonical Wnt signalling and a clear reduction in the stemness potential of DPSCs, as shown by a reduced ability to generate mature, fully differentiated osteoblasts and adipocytes. Conversely, pharmacological activation of the Wnt pathway for 48 h, by either the glycogen synthase kinase 3 beta (GSK3-β) inhibitor 6-bromoindirubin-3´-oxime (BIO) or the human recombinant protein Wnt-3a, not only largely increased the expression of NC and core factors, but also increased the efficiency of DPSCs to differentiate into mature osteoblasts and adipocytes. These results showed that a short preconditioning activation of Wnt/Notch signalling by small molecules and/or recombinant proteins enhanced the stemness and potency of DPSCs in culture, which could be useful for optimising the therapeutic use of these and other tissue-specific stem cells.

Wnt-3a Induces Epigenetic Remodeling in Human Dental Pulp Stem Cells

“…Notch and Wnt signaling were known to affect the proliferative ability of DPSCs [13]. To precisely evaluate the impact of these treatments over the cell cycle, the relative rates of G0/G1, S and G2/M cell cycle phases by flow cytometry were studied after exposure of DPSCs to DAPT, BIO and WNT-3A.…”

“…Dental pulp stem cells or DPSCs arise from the neural crest (NC) as many other cells of craniomaxillofacial tissues [1][2][3][4][5][6][7]. Interestingly, DPSCs present some advantages with respect to other multipotent stem cell populations found in the adult human body [8][9][10], and they show a relatively high expression of core pluripotency factors like OCT4, SOX2, KLF4, LIN28, SSEA1, and NANOG [2,[9][10][11][12][13]. The expression of those core factors regulates stem cell pluripotency [14][15][16][17][18].…”

“…The canonical Notch and Wnt signaling pathways are widely regarded as important regulators of stemness [39][40][41][42][43] and cell differentiation [44][45][46] in DPSCs and many other stem cell types. It is known that dental stem cells present particularly high levels of Wnt and Notch pathway activity, which relates to their high expression of pluripotency core factors and high ability for cell reprogramming [2,9,13,47,48]. However, the epigenetic regulations that these pathways may exert on DPSCs are still unclear.…”

Human Dental Pulp Stem Cells Grown in Neurogenic Media Differentiate into Endothelial Cells and Promote Neovasculogenesis in the Mouse Brain

“…RNA extraction from cell pellets, reverse transcription and QPCR were performed as previously described 64 . The molecular weights of the amplification products were checked by electrophoresis in a 2 % agarose gel.…”

Wnt-5a Promotes Neural Development and Differentiation by Regulating CDK5 via Ca2+/Calpain Pathway

“…Most Wnt ligands have critical functions that contribute to neuronal differentiation of embryonal carcinoma and embryonic stem cells [12]. Wnt also affects several other signaling pathways, including Notch and PI3K/ AKT, whose activities affect neuronal development and differentiation [13]. Canonical Wnt signaling regulates axon genesis and the differentiation of cerebellar granule neuron(CGN) progenitors.…”