Human periosteum-derived cells maintain phenotypic stability and chondrogenic potential throughout expansion regardless of donor age

OBJECTIVE: To assess the in vitro chondrogenic potential of adult human periosteum-derived cells (PDCs) with regard to the number of cell passages and the age of the donor. METHODS: Cells were enzymatically released from the periosteum of the proximal tibia obtained from adult human donors and expanded in monolayer. PDCs were harvested at multiple passages for total RNA extraction and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) gene expression analysis. For the chondrogenesis assay, cells were plated in micromass and treated with transforming growth factor beta1 (TGFbeta1) in a chemically defined medium. At different time points, micromasses were either harvested for RT-PCR analysis for cartilage and bone markers or fixed, paraffin-embedded, and stained for cartilage matrix, and immunostained for type II collagen. RESULTS: At the first 2 passages, human PDCs from young donors formed chondrogenic nodules. This spontaneous chondrogenic activity was lost upon passaging, and it was not observed in donors older than 30 years. Using a panel of marker genes, PDCs were shown to be phenotypically stable during cell expansion. Regardless of donor age or cell passage, chondrogenesis could be induced consistently by combining micromass culture and TGFbeta1 treatment. Histochemical and immunohistochemical analyses demonstrated the hyaline-like cartilage phenotype of the tissue generated in vitro. Other TGFbeta superfamily members, such as growth differentiation factor 5/cartilage-derived morphogenetic protein 1, and bone morphogenetic proteins 2, 4, and 7, were poorly chondrogenic under the same culture conditions. CONCLUSION: Adult human PDCs have the potential to differentiate toward the chondrocytic lineage in vitro, retaining this property even after extensive subculture. Human PDCs are easily accessible, expandable, and maintain their chondrogenic potential, and are therefore promising progenitor cells for use in the repair of joint surface defects.