Harvesting, expanding, and re‐implanting osteogenic mesenchymal stem cells (MSCs) avoids the donor‐site morbidity associated with autogenous grafting from bone marrow. Mesenchymal stem cells sourced from the palatal periosteum could be an alternative to isolation of such cells using bone marrow aspiration procedures. For safe use in human therapy, MSCs should be expanded in culture medium that is free from animal or human‐derived serum. In this study we localized, quantified, and characterized MSCs from palatal periosteum cultured in serum‐free, xeno‐free Essential 8 medium. A portion of the palatal periosteum tissues from three patients were dual‐immunostained with MSC‐specific markers (CD105, CD90, and CD73). The remaining portions were expanded in culture, and the isolated MSCs were analyzed using flow cytometry and tri‐lineage differentiation. Palatal periosteum sections were found to contain CD105‐, CD90‐, and CD73‐positive cells. The cultured cells were 73.0 ± 6.7% (mean ± SD) positive for all three MSC‐specific markers and were without hematopoietic stem cell (HSC) markers 0.5 ± 0.3% (mean ± SD). Tri‐lineage differentiation analysis confirmed that palatal periosteum cells could become adipoblasts, chondroblasts, and osteoblasts. The results demonstrate that palatal‐derived MSCs could be detected in situ within small niches, and when expanded in serum‐free, xeno‐free medium represent a viable source of MSCs for clinical use.
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