Isolation of dental pulp progenitor cells utilising stem cell markers

Adult stem cells are found in the dental pulp, remaining quiescent until recruited during reparative dentinogenesis. Isolation and clonal expansion of dental pulp cells demonstrates variable odontogenic potential, suggesting more than one progenitor population exists. Objectives: To establish clonal colonies of progenitor / stem cells from rat dental pulp using two different methods of isolation and examine characteristics relative to stem cell behaviour. Methods: Single cell suspensions were enzymically obtained from dental pulps of male Wistar rats. Neural crest cells are proposed to persist within adult dental pulp. Cells with high expression of low affinity nerve growth factor receptor (p75NGFR) surface protein (p75 cells) were isolated from disintegrated pulps using magnetic column sorting. High levels of ß1 integrin are reported for primitive cells. These progenitor cells were isolated from disintegrated pulps by preferential adherence to a fibronectin for 20 minutes (FNA cells). For both populations, colony forming efficiency (CFE) was recorded and population doublings (PD) were calculated on passaging. Vimentin levels and p75NGRF expression for each progenitor population were examined using immunocytochemistry and RT-PCR. Migration and differentiation of progenitor cells towards collagen gels impregnated with dentine matrix extract was examined using “boyden chambers” and immuo-fluoresence. Results: From 40,000 cells seeded, p75 cells demonstrated a higher CFE, yielding 101 colonies (average), whilst FNA cells produced 23 colonies (average). Following expansion of clonal colonies, both progenitor populations demonstrated high PD. FNA cells had higher expression of vimentin, whilst higher expression of p75NGFR was seen in p75 cells. FNA progenitor cell migrated into collagen gels with chemotactic response demonstrating a positive correlation to DME concentration. Conclusions: Early data suggests the potential presence of more than one population of progenitor cell which vary in terms of their cellular characteristics and behaviour. The differential responses progenitor populations may indicate different roles in tertiary dentinogenesis.