Three-dimensional Spheres Derived From Dental Pulp Cells Exhibit Enhanced Stemness

Objectives: Mesenchymal stem cells derived from dental tissues (dMSCs) are typically cultured as two-dimensional monolayer using conventional tissue-culture technique. However, this technique incompletely reproduces in vivo microenvironment of stem cells and results in the loss of stemness properties. Various three-dimensional culture techniques have been developed to recapitulate the in vivo microenvironment more accurately in vitro. For instance, formation of three-dimensional sphere, a spherical cluster of cells formed by self-renewal capacity in a defined condition medium, is one of the most widely used techniques for the three-dimensional culture. However, the sphere forming ability of human dMSCs and multi-lineage differentiation capacity of dMSC spheres have not been well investigated.
Methods: Dental pulp stem cells (DPSCs) were cultured and examined for sphere forming ability. Expression of genes was assayed by quantitative RT-PCR. Multi-lineage differentiation capacity was examined by alkaline phosphatase (ALP), Alizarin Red S (ARS) staining, and Oil Red-O (ORO) staining assays.
Results: We demonstrated that DPSCs form three-dimensional multicellular spheres. DPSC spheres exhibited robust expression of pluripotency transcription factors (i.e., Oct4, Nanog, KLF4, Lin28, and Sox2) compared to adherent monolayer DPSCs. When DPSC spheres were cultured as adherent monolayer cells, the pluripotency factors were significantly decreased, suggesting that DPSC spheres preserve higher stemness properties than adherent monolayer DPSCs. Functionally, DPSC spheres exhibited enhanced osteo/odontogenic differentiation and mineralization as demonstrated by ALP activity/staining as well as ARS staining when compared with their corresponding adherent monolayer cells. The expressions of Runx2, a master regulator of osteoblast differentiation, and odontogenic differentiation markers were also increased in DPSC spheres. Moreover, DPSC spheres showed elevated adipogenic differentiation potential as demonstrated by ORO staining of lipid droplets.
Conclusions: Collectively, our data indicate that spheres derived from DPSCs are enriched with stem cells and thus have higher multi-lineage differentiation capacity, suggesting that DPSC spheres may provide unique opportunity for pulp tissue regeneration.