Conclusions
Limiting dilution method is suggested when isolating NPMSCs as a means of improving cell activity and plasticity.
Methods
Nucleus pulposus tissues were isolated from 12-week-old male Sprague-Dawley rats and NPMSCs were isolated using limiting dilution method. Cells were then classified into 3 groups according to plating density. Cell morphologies were observed, and colony-forming units, migration abilities, proliferative capacities, cell cycle percentages, multilineage differentiation capacities, stem cell biomarker expression levels, and immunophenotyping were also examined in each group.
Results
Low density group (LD) had higher morphological homogeneity, stronger colony-forming ability, higher cell proliferation capacity, and enhanced cell migration ability relative to the other two groups (p < 0.05). Moreover, LD had more cells entering S phase, with fewer cells arrested in G0/G1 phase (p < 0.05). While all three density groups showed a multilineage differentiation potential, LD showed a higher degree of observed and semiquantified lineage specific staining (p < 0.05). Furthermore, LD displayed higher expression levels of stem cell biomarkers (Nanog, Oct4, and Sox2) and showed higher percentages of CD29+, CD44+, and CD90+ cells (p < 0.05) following flow cytometry analysis. Conclusions: Limiting dilution method is suggested when isolating NPMSCs as a means of improving cell activity and plasticity.