Enriching Stem/Progenitor Cells from Dental Pulp Cells by Low-density Culturing

Abstract

Background/aim: Clonogenicity is a key feature of stem/progenitor cells. The present study aimed to enrich stem/progenitor cells from dental pulp cells by means of culturing the cells at a low clonal density with spatial separation and the evaluate differentiation potential of the surviving cells.

Materials and methods: Pulp cells derived from wisdom teeth were seeded into wells of a 96-plate at a mean density of 1 cell/well and cultured for 2 weeks. Surviving cells were harvested, pooled together and subjected to differentiation into adipocytes, osteoblasts and neurons using respective inducing conditions for 3 weeks. The former two types of cells were examined by staining with Oil Red O and Alizarin Red, respectively. Neuron-like cells were inspected for their morphology and immunostained for microtubule-associated protein 2 and β-tubulin III.

Results: Vital cells were obtained in eight wells of a 96-well plate, corresponding to a survival rate of 8%. Since fewer than two wells would be expected to contain more than four cells at seeding, the majority of surviving cells likely grew from 1-3 cells, which is a very low density. These cells differentiated into functional adipocytes and osteoblasts, and morphologically neuron-like cells.

Conclusion: Low-density seeding with spatial separation enables statistical estimation of cell number in wells and provides an effective strategy for enriching stem/progenitor cells and for isolating clonal dental pulp cells.

Keywords: Iow-density seeding; Stem cells; cell enrichment; clonogenicity; dental pulp cells; differentiation; progenitor cells.