Isolation and characterization of multipotent mesenchymal stromal cells from the gingiva and the periodontal ligament of the horse

BACKGROUND The equine periodontium provides tooth support and lifelong tooth eruption on a remarkable scale. These functions require continuous tissue remodeling. It is assumed that multipotent mesenchymal stromal cells (MSC) reside in the periodontal ligament (PDL) and play a crucial role in regulating physiological periodontal tissue regeneration. The aim of this study was to isolate and characterize equine periodontal MSC. Tissue samples were obtained from four healthy horses. Primary cell populations were harvested and cultured from the gingiva, from three horizontal levels of the PDL (apical, midtooth and subgingival) and for comparison purposes from the subcutis (masseteric region). Colony-forming cells were grown on uncoated culture dishes and typical in vitro characteristics of non-human MSC, i.e. self-renewal capacity, population doubling time, expression of stemness markers and trilineage differentiation were analyzed. RESULTS Colony-forming cell populations from all locations showed expression of the stemness markers CD90 and CD105. In vitro self-renewal capacity was demonstrated by colony-forming unit fibroblast (CFU-F) assays. CFU-efficiency was highest in cell populations from the apical and from the mid-tooth PDL. Population doubling time was highest in subcutaneous cells. All investigated cell populations possessed trilineage differentiation potential into osteogenic, adipogenic and chondrogenic lineages. CONCLUSIONS Due to the demonstrated in vitro characteristics cells were referred to as equine subcutaneous MSC (eSc-MSC), equine gingival MSC (eG-MSC) and equine periodontal MSC (eP-MSC). According to different PDL levels, eP-MSC were further specified as eP-MSC from the apical PDL (eP-MSCap), eP-MSC from the mid-tooth PDL (eP-MSCm) and eP-MSC from the subgingival PDL (eP-MSCsg). Considering current concepts of cell-based regenerative therapies in horses, eP-MSC might be promising candidates for future clinical applications in equine orthopedic and periodontal diseases.