Oral Progenitor Cells as a Cell-based Treatment for Neuronal Damage

Objectives: Embryonic stem cells have the potential to make any cell type, giving them wide-ranging therapeutic applications in tissue repair/regeneration. However, ethical concerns surround their use and alternative solutions are being sought. Adult stem cells (ASCs) have been isolated from various adult tissues including the oral mucosa lamina propria (OMLP). The aim of this study was to isolate ASCs from the OMLP and determine their potential to differentiate into functional neurons.

Methods: OMLP-progenitor cells (PC) were isolated by differential adhesion to fibronectin, clonally isolated and expanded in ESC medium (+80ng/mL bFGF) on laminin-coated plates as neurospheres. Expanded OMLP-PC neurospheres were differentiated in DMEM/F12 supplemented with brain derived neurotrophic factor, nerve growth factor and neurotrophin-3 (all 10ng/mL), on matrigel-coated plates. Immunocytochemistry was performed on differentiated cells to determine the proportion expressing neural markers and ratiometric Ca²⁺ imaging using cell permeable Fura-2 was employed to determine whether the cells expressed functional Ca²⁺ and ligand-gated ion channels.

Results: OMLP-PCs were driven down a neuronal lineage, evidenced by the expression of the neural markers Mitogen activated protein-2 (≈93%+ve), glial fibrillary acidic protein (≈85%+ve), neurofilament medium (NF-M, ≈67%+ve) and βIII Tubulin (≈88%+ve). Depolarising cells with a solution containing 50mM KCl or application of 300mM GABA resulted in Ca²⁺ influx, indicative of activation of voltage-sensitive Ca²⁺ channels, whilst application of 100mM ATP evoked rapid Ca²⁺ influx through P2X purinoceptors.

Conclusions: OMLP-PCs can be successfully differentiated into a neuronal phenotype, assessed by expression of known neuronal markers, including NF-M, indicative of terminal neuronal differentiation. Moreover, the ability of cells to respond appropriately to certain ligands (GABA and ATP) and depolarize suggests strongly that these cells have, or are going towards, a differentiated functional neuronal phenotype. Together, these data suggest that OMLP-PC cells have great potential as a source of autologous graft tissue in regenerative medicine applications.