Fournier B P, Loison-Robert L S, Ferré F C, Owen G R, Larjava H, Häkkinen L
University of British Columbia, Faculty of Dentistry, Department of Oral Biological and Medical Sciences, 2199 Wesbrook Mall, Vancouver, BC, V6T 1Z3
Eur Cell Mater. 2016 Jan 5;31:40-58. doi: 10.22203/ecm.v031a04.
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.
神经嵴（NC）衍生的干细胞（NCSC）具有异常广泛的分化潜能，但由于其在成体组织中稀缺且分离方案复杂，限制了它们在再生治疗中的应用。人类口腔黏膜牙龈可能是这些细胞的一个有吸引力的来源，因为它含有NC衍生细胞，组织易于获取，伤口愈合快且无疤痕，发病率极低。为此，我们首先通过检查8个月大的NC报告基因Wnt1-Cre/R26RYFP小鼠，研究NC衍生细胞是否保留在成体牙龈中。然后我们假设，通过从标准人类牙龈成纤维细胞（GF）和集落形成单位-成纤维细胞（CFU-F）混合培养物（GSC）中生成的悬浮神经球培养，可以进一步增强牙龈细胞的NC样表型。研究结果表明，NC衍生细胞保留在老年小鼠的牙龈结缔组织中。人类GF和GSC表达NC相关基因巢蛋白、Snai1、Twist1、Pax3、Sox9和FoxD3，并生成神经球。这是通过钙和连接蛋白43依赖性细胞通讯介导的，类似于神经祖细胞形成的神经球。球内细胞显示NC相关基因的表达显著增加，而成纤维细胞相关的I型胶原蛋白表达下调。在结构上，神经球呈极化状态，巢蛋白阳性细胞位于外层，其下方是富含胚胎NC典型分子的细胞外基质。球衍生细胞表达的神经标志物水平显著升高，并分化为Tau、神经丝-M和GFAP阳性细胞，表明具有神经分化潜能。因此，人类GF和GSC培养物可能通过悬浮球培养富集，提供一种有效的NC衍生细胞来源。
