Adult Neural Crest-Derived Stem Cells (NCSCS) for comparative research

Abstract

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Adult craniofacial tissues in vertebrates contain post-migratory neural crest-derived stem cells (NCSCs). These cells, originated from ectoderma, undergo an epithelial-mesenchymal transition in embryogenesis, while keeping their multipotency. The optimal source of NCSC is the tissues of the oral cavity - alveolar and palatine mucous membranes, dental pulp, periodontal ligaments, etc. In each tissue, stem cells (SC) are located in different microenvironment that affects their properties. NCSC from the oral cavity of standard laboratory rodents differ from human SC. We hypothesized that the ovine palate contains NCSCs with a developmental potential equivalent to their human counterparts. Objective: to investigate the morphofunctional properties of NCSC from the oral cavity of human and sheep. The study was carried out using primary cultures of dental pulp SC (DPSC) and SC from human exfoliated deciduous teeth (SHED), as well as SC of human periodontal ligaments (PDLSC) and SC of the sheep hard palate mucosa. Cells were isolated both by adhesion to plastic and by immunomagnetic separation. The proliferative activity and immunophenotype of the populations were determined. To study the features of osteogenic differentiation, we compared the intensity of staining with alizarin, the timing of calcifications and transcription of the late and early response osteogenic genes (POSTN, OSX, ALP, Cbfa1, bGlAP), as well as odontoblast markers (dentinsialoprotein, DSPP; Dentin matrix acidic phosphoprotein 1 precursor - DMP-1). The ability of the isolated cells to form neurospheres was evaluated. Human PDLSC had the maximum proliferative activity, DPSC proliferated at a slower rate. The cells of all samples possessed had surface markers of mesenchymal stromal cells. However up to 20% of cell at early (2-3) passages were positive for CD117+. Pulp SCs (DpSc) were the fastest responding to osteogenic stimuli; in this case, odontoblast marker genes were transcribed at a higher level as compared to PDLSC. Sheep NCSC proliferated at a rate comparable to human PDLSC. Both human SC and sheep SC were capable of forming neurospheres and expressing NCSC markers (e. g. HNK-1, Slug, CD271). Thus, despite the fact that the properties of NCSC depend on the their niche, sheep NCSC have properties similar to human cells and can be used as a model object in pre-clinical studies of the efficacy and safety of cellular products based on NCSC.