Low-density expansion protects human synovium-derived stem cells from replicative senescence: a preliminary study

Our hypothesis in this study is that low seeding density expansion could retain human synovium-derived stem cell (hSDSC) “stemness”, defined as higher proliferation and multi-differentiation capacity; retention of “stemness” probably occurs through the mitogen-activated protein kinase (MAPK) signaling pathway. hSDSCs were expanded in conventional plastic flasks for two consecutive passages at either low or high density (30 or 3,000 cells/cm2). Expanded cells were assessed for the effect of seeding density on their morphology, proliferation, apoptosis, stem cell surface markers, and multi-lineage differentiation capacity (chondrogenic, adipogenic, and osteogenic differentiation) using flow cytometry, biochemical analysis, histology, immunostaining, and real-time polymerase chain reaction. The MAPK signaling pathway (Erk1/2, p38, and JNK) and senescence-associated markers (p21 and caveolin) were also evaluated for their role in cell density-based monolayer expansion using western blot. Our data suggested that low seeding density expansion yielded hSDSCs with enhanced proliferation and multi-differentiation capacity compared to those grown at high seeding density, despite the fact that the cells expanded at both high and low density had lower osteogenic capacity. Low seeding density also down-regulated Erk1/2 and JNK expression and up-regulated p38 expression, which might be responsible for the retained “stemness” in the cells expanded at low density. Low seeding density expansion could retain hSDSC proliferation and multi-differentiation capacity and protect cells from replicative senescence.