Nogo-A Regulates the Fate of Human Dental Pulp Stem Cells toward Osteogenic, Adipogenic, and Neurogenic Differentiation

Abstract

Human teeth are highly innervated organs that contain a variety of mesenchymal stem cell populations that could be used for cell-based regenerative therapies. Specific molecules are often used in these treatments to favorably modulate the function and fate of stem cells. Nogo-A, a key regulator of neuronal growth and differentiation, is already used in clinical tissue regeneration trials. While the functions of Nogo-A in neuronal tissues are extensively explored, its role in teeth still remains unknown. In this work, we first immunohistochemically analyzed the distribution of Nogo-A protein in the dental pulp of human teeth. Nogo-A is localized in a variety of cellular and structural components of the dental pulp, including odontoblasts, fibroblasts, neurons and vessels. We also cross-examined Nogo expression in the various pulp cell clusters in a single cell RNA sequencing dataset of human dental pulp, which showed high levels of expression in all cell clusters, including that of stem cells. We then assessed the role of Nogo-A on the fate of human dental pulp stem cells and their differentiation capacity in vitro. Using immunostaining, Alizarin Red S, Nile Red and Oil Red O staining we showed that Nogo-A delayed the differentiation of cultured dental pulp stem cells toward the osteogenic, adipogenic and neurogenic lineages, while addition of the blocking anti-Nogo-A antibody had opposite effects. These results were further confirmed by qRT-PCR, which demonstrated overexpression of genes involved in osteogenic (RUNX2, ALP, SP7/OSX), adipogenic (PPAR-γ2, LPL) and neurogenic (DCX, TUBB3, NEFL) differentiation in the presence of the anti-Nogo-A antibody. Conversely, the osteogenic and adipogenic genes were downregulated by Nogo-A. Taken together, our results show that the functions of Nogo-A are not restricted to neuronal cells but are extended to other cell populations, including dental pulp stem cells. We show that Nogo-A regulates their fates toward osteogenic, adipogenic and neurogenic differentiation, thus indicating its potential use in clinics.

Trigeminal ganglion and tooth innervation modifications following genetic and pharmacological Nogo-A inhibition

“…Within the dental pulp of young adult mice, Nogo-A-expressing neurites are part of the sub- odontoblastic neuronal plexus, in close association with the dentine-producing odontoblasts. These results are in agreement with our previous findings showing Nogo-A expression in the neurons of human permanent teeth (88).…”

“…During postnatal development, the trigeminal axons innervate the connective tissues of the tooth while they continue to express Nogo-A. Within the dental pulp of young adult mice, Nogo-A-expressing neurites are part of the subresults are in agreement with our previous findings showing Nogo-A expression in the neurons of human permanent teeth(88).…”

“…It is also involved in the regulation of non-neuronal biological processes such as angiogenesis and vascular remodelling, macrophage activation, and stem cell behaviour (87). Surprisingly, the functions of Nogo-A in the highly innervated dental tissues were not assessed until recently (88). In the present study, we analysed the effects of Nogo-A deletion in the embryonic and postnatal trigeminal ganglia (tgg), and for the first Tooth innervation is regulated by neuroregulatory molecules that either attract or repulse the growing trigeminal nerves (43).…”

“…Identifying molecules involved in the establishment of connections between the peripheral nerves and dental tissues is essential for the development of novel therapeutic approaches for optimal recovery of tooth physiology. Indeed, in teeth like in many organs, the innervation is emerging as a key player in organ development, homeostasis, repair and/or regeneration (88–91). Our in vitro results suggest the potential use of Nogo-A blockers for the treatment of dental tissues in a clinical setting.…”

Mesenchymal stem cells under epigenetic control – the role of epigenetic machinery in fate decision and functional properties

“…3 ). Runt-related transcription factor 2 (RUNX2, also known as a core-binding factor-α-CBFA1) and Osterix (OSX; also known as Sp7) represent the main transcription factors for osteogenic differentiation [ 74 – 76 ]. Their expression is accompanied by increased miR-27b, miR-130a, activity that directly targets PPARγ expression [ 77 ].…”