Human mesenchymal stem cells (hMSCs) are primary candidates in cell therapy and tissue engineering and are being tested in clinical trials for a wide range of diseases. Originally isolated and expanded as plastic adherent cells, hMSCs have intriguing properties of in vitro self-assembly into three-dimensional (3D) aggregates that improve a range of biological properties, including multilineage potential, secretion of therapeutic factors, and resistance against ischemic condition. While cellācell contacts and cellāextracellular matrix interactions mediate 3D cell aggregation, the adaptive changes of hMSC cytoskeleton during self-assembly and associated metabolic reconfiguration may also influence aggregate properties and functional activation. In this study, we investigated the role of actin in regulating 3D hMSC aggregate compaction, fusion, spreading and functional activation. Individual hMSC aggregates with controlled initial cell number were formed by seeding a known number of hMSCs (500, 2000, and 5000 cells/well) in multi-well plates of an ultra-low adherent surface to form multicellular aggregates in individual wells. To assess the influence of actin-mediated contractility on hMSC aggregation and properties, actin modulators, including cytochalasin D (cytoD), nocodazole, lysophosphatidic acid (LPA), and Y-27632, were added at different stages of aggregation and their impacts on hMSC aggregate compaction and apoptosis were monitored. The results suggest that actin-mediated contractility influences hMSC aggregation, compaction, fusion, and spreading on adherent surface. Formation of multi-cellular aggregates significantly upregulated caspase 3/7 expression, expression of C-X-C chemokine receptor type 4 (CXCR-4), cell migration, secretion of prostaglandin E2 (PGE-2) and interleukin 6 (IL-6), and resistance to in vitro ischemic stress. The functional enhancement, however, is dependent on caspase activation, because treatment with Q-VD-OPh, a pan-caspase inhibitor, attenuated CXCR-4 and cytokine secretion. Importantly, comparable ATP/cell levels and significantly reduced mitochondrial membrane potential in aggregates of different sizes suggest that altered mitochondria bioenergetics on 3D aggregation is the primary inducer for apoptosis. Together, the results suggest multicellular aggregation as an effective and nongenetic strategy for hMSC functional activation.
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