Characterization of Human Gingiva- and PDL-derived Progenitor Cells in Xeno-free Conditions

Objectives: Periodontal tissues represent a clinically relevant and less invasive source of progenitor cells compared to bone marrow, for periodontal- and/or alveolar bone-tissue engineering (P-/BTE). The safety and efficacy of clinical-grade stem cells can be enhanced via substitution of xenogeneic supplements and three-dimensional (3-D) cell culture, respectively, to simulate the in vivo microenvironment more closely. The objective of this study was to comprehensively characterize progenitor cells derived from human gingiva (GPCs) and periodontal-ligament (PDL; PPCs) in xeno-free conditions for use in P-/BTE.
Methods: In preliminary experiments, pooled human platelet lysate (PL) was identified as the optimal xeno-substitute for fetal bovine serum (FBS) in stem cell cultures. Primary GPCs and PPCs were isolated and expanded as monolayers in PL- and FBS-supplemented media; passage 3-5 cells were used in experiments. Bone marrow mesenchymal stem cells (BMSCs) were used as a reference. Growth kinetics were compared via cell proliferation and colony forming-unit (CFU) assays. GPCs and PPCs were characterized via cytometric expression of stromal markers, multi-lineage (osteogenic, adipogenic and chondrogenic) differentiation potential, and secretory cytokine profiles. 3-D sphere cultures of GPCs and PPCs were established, and the expression of stemness- and osteogenesis-related markers in 3-D and monolayer cultures was evaluated via gene expression and immunocytochemistry.
Results: GPCs and PPCs in both FBS and PL showed characteristic fibroblastic morphology and stromal phenotype (highly positive for CD105/CD90/CD73 and negative for CD34/CD45/HLA-DR). Cell proliferation and CFU efficiency were superior in PL compared to FBS. GPCs and PPCs expanded in both PL and FBS showed multi-lineage differentiation comparable to BMSCs; notably, osteogenic differentiation was enhanced in GPCs expanded in PL. 3-D spheres of GPCs and PPCs were formed and maintained for up to 7 days in xeno-free suspension culture. Notably, expression of stemness- (Sox2, Oct4, Nanog) and osteogenesis-related markers (Runx2, Osx, BMP2) was significantly upregulated in GPC- and PPC-derived spheres vs. monolayers.
Conclusions: PPCs and particularly GPCs, due to easy access, cultured in 3-D xeno-free conditions represents a promising strategy for P-/BTE.