Mesenchymal multipotency of adult human periosteal cells demonstrated by single-cell lineage analysis

Objective. To investigate whether periosteal cells from adult humans have features of multipotent mesenchymal stem cells (MSCs) at the single-cell level. Methods. Cell populations were enzymatically released from the periosteum of the proximal tibia obtained from adult human donors and then expanded in monolayer. Single-cell-derived clonal populations were obtained by limiting dilution. Culture-expanded periosteal cell populations were tested for their growth potential and for expression of conventional markers of MSCs and were subjected to in vitro assays to investigate their multilineage potential. To assess their multipotency in vivo, periosteal cells were injected into a regenerating mouse tibialis anterior muscle for skeletal myogenesis or were either seeded into an osteoinductive matrix and implanted subcutaneously into nude mice for osteogenesis or implanted in a joint surface defect under a periosteal nap into goats for chondrogenesis. Cell phengtypes were analyzed by histochemistry and immunohistochemistry and by reverse transcription-polymerase chain reaction for the expression of lineagerelated marker genes. Results. Regardless of donor age, periosteal cells were clonogenic and could be expanded extensively in monolayer, maintaining linear growth curves over at least 30 population doublings. They displayed long telomeres and expressed markers of MSCs. Under specific conditions, both parental and single-cell-derived clonal cell populations differentiated to the chondrocyte, osteoblast, adipocyte, and skeletal myocyte lineages in vitro and in vivo. Conclusion. Our study demonstrates that, regardless of donor age, the adult human periosteum contains cells that, upon enzymatic release and culture expansion, are multipotent MSCs at the single-cell level.