(599cj) Actin-Mediated Contractility in Three-Dimensional Aggregates of Human Mesenchymal Stem Cells

Ang-Chen Tsai, Yijun Liu, Xuegang Yuan, and Teng Ma

Chemical and Biomedical Engineering, College of Engineering, Florida State University, Tallahassee, FL, USA

Introduction

Human mesenchymal stem cells (hMSCs) are an important cell source for therapeutic applications because of their ability of self-renewal and secretion of trophic factors. In translation to clinical application, a major challenge is to maintain the functions of the culture-expanded hMSCs. Prior studies have shown that formation of 3D hMSC aggregates improved a range of biological properties, including multilineage potential, secretion of therapeutic factors, and resistance against ischemic condition.  To date, however, the underlying mechanism for the aggregate-mediated functional enhancement is unclear.  The objective of the current study is to investigate the role of  actin-mediated contractility in regulating aggregate assembly, contraction, fusion, apoptosis and functional activation.

Materials and Methods

HMSCs from monolayer culture were trypsinized and added in each well of an ultra-low attachment 96-well plate with round-bottom for overnight.  Suspended hMSCs spontaneously assembled into one aggregate per well. The aggregates were cultured and tracked individually for up to 7 days with media change every two days.  Actin modulators, such as Cytochalasin D (cytoD), lysophosphatidic acid (LPA), Y-27632, were added into culture media to control the cell contractility, which were measured as packing density, cells per unit volume.  Cell numbers per aggregates were determined by DNA assay. Aggregate volumes were calculated by the analysis of aggregate project area with ImageJ software.  Caspase 3/7 activity was measured by Caspase-Glo 3/7 assay following manufacturer’s instruction and expression of cytokine such as PGE-2 and IL-6 were determined using ELISA.

Results and conclusion:

Formation of multi-cellular hMSC aggregates involves sequential steps of cell detachment, initial cell-cell contacts, cadherin accumulation, and aggregate compaction, in which actin-mediated contractility plays a prominent role.  The addition of actin polymerization inhibitor, CytoD, disrupted hMSC aggregation, indicating that actin is required in hMSC aggregation. Treatments with CytoD or Y-27632 (ROCK inhibitor) after hMSC aggregation prevented cell contraction, but failed to increase cell viability, suggesting that actin-mediated contractility is the major force in aggregate compaction but not the primary factor leading to cell death. LPA, an actin polymerization enhancer, treated aggregates maintained similar properties as the control.  The addition of microtubule polymerization inhibitor, nocodazole, significantly alters hMSC morphology on 2D culture but has no effect on 3D aggregate compaction. We also found hMSC aggregates readily fuse and spread on solid surface, but CytoD treatment effectively abolished fusion and spreading, suggesting actin polymerization changes the hMSC aggregate viscoelastic behavior from liquid state to solid state.  To investigate cell apoptosis in hMSC functional activation, we found that hMSCs cultured as aggregates have much higher caspase 3/7 expression, suggesting elevated apoptosis in the aggregates. The treatments with Q-VD-OP, a pan-caspase inhibitor, however, increased cell viability and reduced PGE-2 and IL-6 secretion, suggesting a caspase-dependent mechanism for the enhancement cytokine secretion. 

In summary, actin-mediated contractility plays a major role in regulating hMSC aggregate formation, compaction, fusion, and spreading on solid surface but its inhibition failed to inhibit apoptosis. On the other hand, formation of 3D aggregates significantly increased PGE-2 and IL-6 secretion via an apoptosis-dependent mechanism.  Taken together, formation of multi-cellular aggregates is an effective strategy for hMSC functional enhancement.

Reference:

1. Kim  J  and  Ma  T,  Endogenous  Extracellular  Matrices Enhance  Human  Mesenchymal  Stem  Cell  Aggregate Formation  and  Survival,  Biotechnology  Progress,  29,  2, 441-451, 2013
2. Sart, S., Tsai, A.-C., Li, Y. and Ma, T. Three-dimensional aggregates of mesenchymal stem cells: cellular mechanisms, biological properties, and applications. Tissue Engineering Part B: Reviews, 2013 (In press) DOI: 10.1089/ten.teb.2013.0537