Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs) into osteoblastic lineage are passage dependent.

Objective: The effect of in vitro expansion of human adipose-derived stem cells (ASCs) on stem cell properties is controversial. We examined serial subcultivation with expansion on the ability of ASCs to grow and differentiate into osteoblastic lineages.

Design: Flow cytometric analysis, growth kinetics, cell population doubling time, light microscopy and confocal analysis, and osteogenesis induction were performed to assess growth and osteogenic potential of subcultivated ASCs at passages 2 (P2), P4 and P6.

Results: Flow cytometric analysis revealed that ASCs at P2 express classical mesenchymal stem cell markers including CD44, CD73, and CD105, but not CD14, CD19, CD34, CD45, or HLA-DR. Calcium deposition and alkaline phosphatase activity were the highest at P2 but completely abrogated at P4. Increased passage number impaired cell growth; P2 cultures exhibited exponential growth, while cells at P4 and P6 showed near linear growth with cell population doubling times increased from 3.2 at P2 to 4.8 d at P6. Morphologically, cells in various subcultivation stages showed flattened shape at low density but spindle-like structures at confluency as judged by phalloidin staining.

Conclusions: Osteogenic potential of ASCs is impaired by successive passaging and may not serve as a useful clinical source of osteogenic ASCs past P2.