Size-dependent Cortical Compaction Induces Metabolic Adaptation in Mesenchymal Stem Cell Aggregates.

Spontaneous assembly of human mesenchymal stem cells (hMSCs) into three-dimensional aggregates enhances stem cell properties and enables formation of heterotypic organoids, which has significant implication in cell therapy and tissue engineering. Although metabolic reprograming toward glycolysis is a salient feature of multicellular aggregates and it has commonly been attributed to oxygen diffusion limitations, recent studies have instead observed a limited decline in oxygen tension, in hMSC aggregates, challenging this view. Although aggregation of a dispersed cell population involves changes in both the physical and molecular environment, most studies to date have focused on molecular gradients with limited investigation in biomechanical stress on the fate of aggregated cells. The objective of this study is to investigate how the mechanical gradient arising from aggregation-induced cortical compaction alters the metabolic profile of human adipose-derived mesenchymal stem cells by testing the hypothesis that size-dependent compaction in aggregates leads to differential cortical stress, which induces metabolic reprogramming. Herein, we show that aggregation of multiple sizes covering a wide range of interest does not lead to hypoxic core formation but instead varying levels of cortical compaction, indicated by the balance between stress fiber formation and the deposition of extracellular matrix proteins, resulting in corresponding levels of metabolic reconfiguration. Increased glycolytic metabolism, increased mitochondrial fission, and increased release of aldolase A were all observed as a result of cortical compaction. Chemical inhibition with Gleevec, Wortmannin, and Y27632 almost completely abolishes the cortical stress-induced enhancement in glycolytic properties. Our findings demonstrate that aggregation-induced biomechanical stress plays a central role in driving metabolic reprogramming. Impact Statement This study reveals that multicellular aggregation induces metabolic reprogramming via mechanical compaction in lieu of formation of a hypoxic core. Utilizing biomechanical knowledge gained from planar culture, we set forth a novel three-dimensional (3D) model of size-dependent cortical compaction and demonstrated its role in metabolic reconfiguration. Ultimately, this study establishes mechanical compaction and its spatial gradients as key regulatory factors and design parameters in the development of 3D human adipose-derived mesenchymal stem cell aggregates.