Actin-Mediated Contractility in Three-Dimensional Aggregates of Human Mesenchymal Stem Cells

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 reminiscent of skeletal condensation in vivo. Recent studies have shown that hMSC 3D aggregation improved a range of biological properties, including multilineage potential, secretion of therapeutic factors, and resistance against ischemic condition.  Although studies have suggested that hMSC aggregation is mediated through cell-cell contacts and cell-ECM interactions, the adaptive changes of hMSC cytoskeleton during self-assembly into 3D aggregates also play an important role that influences aggregate formation, compaction, functional activation, and multilineage differentiation.  In the current study, we investigated the actin-mediated contractility in regulating hMSC 3D aggregation, contraction, apoptosis, functional activation, spreading on adherent surface, and coalescing.  Individual hMSC aggregates with controlled initial cell number were formed by seeding a known number of hMSCs (500, 2,000, 5,000, and 20,000/well) in multi-well plates of ultra-low adherent (ULA) surface to form 3D aggregates.  Tracking of individual hMSC aggregates revealed size-dependent contraction and decreasing viability with larger aggregates having lower contraction and apoptosis compared to smaller aggregates.  Induction of osteogenic differentiation improved cell viability in hMSC aggregates while expressions of osteogenic markers were size-dependent.  To assess the influence of actin-mediated contractility on hMSC aggregation and properties, actin modulators (i.e., blebbistatin, cytochalasin D, jasplakinolide, lysophosphatidic acid (LPA), Y-27632) were added at different stages of aggregation and their impacts on hMSC aggregate size, contraction, apoptosis, and multilineage potential were monitored.  Interruption of actin polymerization by cytochalasin D prevented aggregate contraction but has minimal impact on cell viability in the aggregates.  In contrast, addition of ROCK inhibitor (Y-27632) and LPA prevented aggregate contraction with increased cell viability in the aggregates.  To further assess the role of actin and microtubule in mediating aggregate spreading and re-adhesion, aggregates treated by the actin modulators and nocodazole, a microtubule modulator, were placed on (a) adherent surface or (b) in close proximity on ULA surface.  The results show that interruption of actin polymerization by cytochalasin D significantly reduced cell motility with reduced spreading on adherent surface and aggregate coalescing.  On the other hand, hMSC aggregates treated with nocodazole, ROCK inhibitor (Y-27632), and LPA maintained their mobility on adherent surface and readily coalesced when in contact.  Together, these results suggest that reorganization of actin cortical network during cell aggregation plays a prominent role in mediating hMSC aggregate assembly, contraction, and motility.  The impact of actin-mediated contractility on hMSC phenotype selection and functional activation will also be presented.