Multi-lineage Differentiation Potential of Pericytes Isolated from Human Dental Pulp

Objectives: Current literature has shown the existence of stem cells within the dental pulp cavity, although the cells' exact location has not been identified. We hypothesized that the stem cells within this area are pericytes that surround the vascular tissue of the dental pulp. Using different cell markers, we characterized, isolated and differentiated these cells to explore their osteo, chondro and adipogenic potential.

Methods: The dental pulp was taken from third molars of human subjects, ages 14-30 and digested with collagenase. Cells were then marked with surface markers and sorted using Fluorescent Activated Cell Sorting (FACS). The isolated cells, as well as positive and negative controls, were characterized as well as tested for their potential to differentiate into several lineages using differentiation media and supplements. Osteogenic and adipogenic cells were grown in a monolayer culture for 2 and 7 weeks respectively, while chondrogenic cells were grown in a 3D pellet (250,000 cells/pellet) culture for 3 weeks.

Results: Pericytes harvested from the dental pulp show positive staining for CD146 (endothelial cell and pericyte), a-SMA (smooth muscle cell and pericyte). They are negative for CD34 (endothelial cell and hematopoietic progenitor), CD45 (hematopoietic cell) and CD56 (NK cell). Thus, by using a CD146+CD34-CD45-CD56- FACS sort, pericytes can be isolate for culture expansion in vitro. We observed osteogenic differentiation of dental pulp pericytes by Alizarin red, alkaline phosphatase, and von Kossa stainings. Chondrogenic differentiation was determined by Alcian blue, Toluidine blue and Safranin-O stainings on sections of the cell pellets. Positive Oil red-O staining showed successful adipogenesis of pericytes.

Conclusions: Our data shows that the isolated pericytes possess stem cell-like properties and show their ability to differentiate into cartilage, bone or adipose tissues. The dental pulp pericytes could be considered for therapeutic use in regeneration of oral tissues.