Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, P.O. Box 9196, One Medical Center Drive, Morgantown, WV, 26506-9196, USA.
Drug Deliv Transl Res. 2012 Oct;2(5):363-74. doi: 10.1007/s13346-012-0094-y.
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/cm(2)). 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.
我们在这项研究中的假设是，低接种密度扩增可以保留人滑膜来源干细胞（hSDSC）的“干性”，其定义为更高的增殖和多向分化能力；“干性”的保留可能通过丝裂原活化蛋白激酶（MAPK）信号通路发生。hSDSCs 在常规塑料培养瓶中以低或高密度（30 或 3000 个细胞/cm²）连续传代两次进行扩增。通过流式细胞术、生化分析、组织学、免疫染色和实时聚合酶链反应，评估接种密度对细胞形态、增殖、凋亡、干细胞表面标志物和多向分化能力（软骨形成、脂肪形成和成骨分化）的影响。还使用 Western blot 评估 MAPK 信号通路（Erk1/2、p38 和 JNK）和衰老相关标志物（p21 和窖蛋白）在基于细胞密度的单层扩增中的作用。我们的数据表明，与在高接种密度下生长的细胞相比，低接种密度扩增产生的 hSDSCs 具有增强的增殖和多向分化能力，尽管在高和低密度下扩增的细胞的成骨能力较低。低接种密度还下调了 Erk1/2 和 JNK 的表达，上调了 p38 的表达，这可能是低接种密度扩增细胞中保留“干性”的原因。低接种密度扩增可以保留 hSDSC 的增殖和多向分化能力，并防止细胞发生复制性衰老。
