Characterisation of human gingival neural crest-derived stem cells in monolayer and neurosphere cultures

Abstract

Neural crest (NC)-derived stem cells (NCSC) have an exceptionally wide differentiation potential, but their use in regenerative therapy has been hampered by their scarcity in adult tissues and complex isolation protocols. Human oral mucosal gingiva may provide an attractive source of these cells as it contains NC-derived cells, the tissue is easily accessible and wound healing is fast and scarless with very little morbidity. To this end, we first investigated whether NC-derived cells are retained in adult gingiva by examining 8-months-old NC-reporter Wnt1-Cre/R26RYFP mice. We then hypothesised that gingival cell NC-like phenotype can be further enhanced by floating neurosphere cultures generated from standard human gingival fibroblast (GF) and pooled CFU-F (GSC) cultures. Findings showed that NC-derived cells are retained in the gingival connective tissue of aged mice. Human GFs and GSCs expressed NC-related genes nestin, Snai1, Twist1, Pax3, Sox9 and FoxD3, and generated neurospheres. This was mediated via calcium-and connexin 43-dependent cell communication, which is similar to neurospheres formed by neural progenitors. Cells in the spheres showed significantly increased expression of NC-related genes, and down regulation of fibroblast-related type I collagen. Structurally, the neurospheres were polarised with nestin positive cells located on the outer layers underlined with an extracellular matrix rich in molecules typical to embryonic NC. Sphere-derived cells expressed significantly elevated levels of neural markers, and differentiated into Tau, neurofilament-M and GFAP-positive cells suggesting neural differentiation potential. Thus, human GF and GSC cultures may provide an efficient source of NC-derived cells via enrichment by floating sphere cultures.

Establishment and neural differentiation of neural crest-derived stem cells from human dental pulp in serum-free conditions

“…Our findings led us to hypothesize that heterogeneity in our hDPC cultures could lead to inefficacy in differentiation and that an intermediate population may be required for neural maturation, as shown by observations from the literature on hDPC-neural differentiation. 14,[29][30][31] Together with increasing reports obtaining NCSCs from other dental-derived stem cells, 7,8,32,33 it is suggested that a neural crest or neurogenic population needs to be induced or enriched. In this regard, sphere aggregation has been used to enrich for a neural crest-like phenotype in other tissues.…”

Extracellular Vesicles Derived from Human Gingival Mesenchymal Stem Cells: A Transcriptomic Analysis

“…The oral cavity has been identified as an easily-accessible reservoir of human mesenchymal stem cells (hMSCs) [1,2]. The oral hMSCs were successfully isolated and characterized from a variety of oral tissues, including dental pulp [3], apical papilla [4], exfoliated deciduous teeth [5], dental follicle [6], periodontal ligament [7], and gingiva [8]. Oral stem cells originate from neural crests and represent a transient population of embryonic pluripotent stem cells [9,10].…”

Cannabidiol Activates Neuronal Precursor Genes in Human Gingival Mesenchymal Stromal Cells

“…In recent years, the tissues of oral cavity have been identified as a rich sources of MSCs. Until now, MSCs from six different type of oral tissues have been reported: dental pulp [Gronthos et al, ], exfoliated deciduous teeth [Miura et al, ], periodontal ligament [Seo et al, ; Trubiani et al, ; Diomede et al, ], apical papilla [Sonoyama et al, ], dental follicle [Morsczeck et al, ], and gingiva [Fournier et al, , ]. A key advantage of these oral tissues‐derived MSCs is that unlike bone marrow tissues collection for the extraction of MSCs, they can be isolated from the oral tissues with restricted morbidity and without additional risks to the donor using minimally invasive procedure.…”