Stem cells are responsible for the development of cellular tissue from the embryo to adult tissue. Adult-stem cells are found throughout various niches of the body and localize to damaged tissue to initiate repair. Of specific interest are mesenchymal stem cells (MSCs) which have shown promising therapeutic potential due to their impressive ability to secrete immunomodulatory, angiogenic, and regenerative cytokines. Spontaneous assembly of MSCs into 3D aggregates enhances stem cell properties and enables formation of heterotypic organoids, which has significant implication in cell therapy and tissue engineering. While metabolic reprograming towards glycolysis is a salient feature of multicellular aggregates it has commonly been attributed to oxygen diffusion limitations; however, recent studies have instead observed a limited decline in oxygen tension, in MSC aggregates, challenging this view. Although aggregation of a dispersed cell population involves both changes in 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. Herein, it’s shown 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. These findings demonstrate that aggregation-induced biomechanical stress plays a central role in driving metabolic reprogramming. Additionally, protein homeostasis is critical for cellular function, as loss of homeostasis is attributed to aging and the accumulation of unwanted protein. In fact, proteome control and proteostasis especially, is required for stem cell function and maintenance of phenotype. When MSCs are expanded in vitro they are plated on stiff plastic and undergo culture adaptation, which results in aberrant proliferation, shifts in metabolism, and decreased autophagic activity. It has previously been shown that aggregation can reverse some of this damage by heightening autophagy and recovering the metabolic state back to a naïve phenotype. For this reason, the effects of aggregation on the proteome have been explored. Results showed a decrease in the EIF2 pathway, which is responsible for controlling the protein initiation complex, a component of the integrated stress response (ISR). This was further explored through protein quantification revealing that aggregated MSCs derived from bone-marrow and adipose established a new proteiostatic state through ISR, while differentiated cells such as fibroblasts did not. Aggregation based rejuvenation holds the potential for improving the therapeutic efficacy of expanded MSCs. Finally, the beneficial effects attributed to MSC treatment have been measured in in vitro and in small animal models; however, this potential has yet to be translated into human clinical trials. This is due to both the availability of MSCs at time of need and lack of viable expansion method resulting from culture adaptation. To combat the effect of culture adaptation cyclical aggregation was explored as a means of expanding MSCs while still maintain functionality. Cyclical aggregation consists of an aggregation phase followed by dissociation onto planar tissue culture plastic as a means to expand the cells. Indeed, cyclical aggregation does maintain proliferative capability, stem cell proteins, clonogenicity, and prevents the acquisition of senescence. To determine what part of aggregation was responsible for this phenomenon the integrated stress response pathway was probed with salubrial and GSK-2606414. Treatment with salubrial had no significant effect, while GSK-2606414 mitigated the effects of aggregation leading to in vitro aging effects. This system hold the potential to increase the clinical relevance of MSCs from small model systems such as rats and mice to humans, and may open the potential of patient derived MSCs being used for treatment there by removing the need for immunosuppressants.
