Objectives: Embryonic stem cells (ESCs) have wide-ranging therapeutic applications in tissue repair/regeneration. However, ethical concerns surround their use and hence alternatives are sought. Adult stem cells (ASCs) have been isolated from adult tissues including the oral mucosa lamina propria (OMLP). This study aims to isolate ASCs from the OMLP, reprogram these cells to induced pluripotent stem cells (iPSCs) and determine the potential for both to differentiate into functional neurons.
Methods: OMLP-progenitor cells (OMLP-PCs) were isolated by differential adhesion to fibronectin, nucleofected with plasmids containing pluripotency factors (oct-4, sox-2, nanog, klf-4, lin-28, c-myc) to reprogram to iPSCs and stained for alkaline phosphatase or pluripotency markers. Cells were expanded in ESC medium as neurospheres and differentiated in DMEM/F12 with brain derived neurotrophic factor, nerve growth factor and neurotrophin-3 (all 10ng/mL). Immunocytochemistry was performed on cells to determine the proportion expressing neural markers and Ca2+ imaging was employed to determine the presence of functional Ca2+ and ligand-gated ion channels.
Results: Differentiated OMLP-PCs demonstrated expression of neural markers; microtubule-associated protein-2 (≈93%+ve), glial fibrillary acidic protein (≈85%+ve), neurofilament medium (≈67%+ve) and βIII tubulin (≈88%+ve). Treatment with 50mM KCl or 300mM GABA resulted in Ca2+ influx, whilst application of 100mM ATP evoked rapid Ca2+ influx through P2X purinoceptors. Nucleofection of OMLP-PCs resulted in colonies similar to ESCs which demonstrated alkaline phosphatase staining and SSEA-4, SSEA-5, TRA-1-60 and TRA-1-81 expression suggesting that these were iPSCs. Further work is ongoing to determine the potential of these iPSCs to form functional neurons.
Conclusions: Differentiated OMLP-PCs express neuronal markers, respond appropriately to certain ligands and depolarize suggesting strongly that these cells have a functional neuronal phenotype. OMLP-PCs can be reprogrammed to iPSCs and could potentially be a better source of neurons. Together, these data suggest that OMLP-PCs have a great potential for regenerative medicine applications.