(160c) Aggregation of Mesenchymal Stem Cells Rejuvenates Functionality Following in Vitro Aging through Induction of the Integrated Stress Response

Mesenchymal stem cells (MSCs) have shown promising therapeutic potential due to their impressive ability to secrete inflammatory modulatory, angiogenic, and regenerative cytokines along with their ability to differentiate and aid in regeneration. Most work with MSCs though has been done in model systems requiring only a small amount of MSCs. Scaled-up expansion of isolated MSCs is carried out on stiff culture plastic which increases the surface modulus one thousand-fold [1]and limits cell-cell interactions. This type of culture results in in vitro aging characterized by increased senescence, decreased glycolytic metabolism, reduced autophagic activity, and loss of multipotent differentiation potential [2]. For these reasons understanding the mechanism of culture induced changes is critical for maintaining MSC phenotype during expansion and proper implementation of MSC therapy.

Protein homeostasis is critical for cells functional maintenance and retention of stemness. As cells age, they loss control over proteasome activity leading to the accumulation of misfolded proteins that would normally be handled by the integrated stress response (ISR). Normally, when misfolded proteins occur they are coated in chaperone proteins such as binding immunoglobin protein (BiP). BiP normally prevents activation of eukaryotic initiation factor 2 alpha kinase 3 (PERK), but as soluble BiP decreases PERK dimerizes/auto-phosphorylates and signals a reduction in protein initiation complex activity causing a cessation of protein synthesis. The misfolded protein is then ubiquinated and lysed. Proteasome autophagy is especially critical in maintaining cell stemness and fighting senescence. It has recently been shown that when autophagy was inhibited in naïve muscle-stem cells they took on the phenotype of in vitro aged cells, and when aged cells had autophagy restored they re-established their naïve phenotype [3]. Previously, it has also been shown that embryonic stem cells hyper-control their proteome to maintain stemness by limiting initiation, increasing folding through chaperone protein content, and increasing proteasome activity to degrade build-up of misfolded proteins and prevent defects from going into daughter cells.

It has previously been shown that aggregation of MSCs leads to heightened autophagy [3], and a reversal of metabolic phenotype back to aerobic glycolysis [4]. For this reason, we have explored what effects aggregation has on the proteome to determine what pathways are elevated by aggregation in bone-marrow derived MSCs (bMSCs), adipose derived MSCs (ASCs), and dermal fibroblasts (FB). Pathway analysis showed a decrease in the eIF2 pathway; however, western blot and ingenuity pathway analysis revealed that bMSC and ASCs had augmented eIF2α and eIF2γ. Specifically, eIF2α was significantly phosphorylated and eIF2γ was decreased showing heightened initiation control following aggregation. Since phosphorylation of eIF2α leads to the subsequent translation of activating transcription factor 4 (ATF) which in turn starts a cascade increasing autophagy. Measured levels of ATF in bMSCs and ASCs decreased following aggregation, while FB didn’t shift. Downstream proteins DNA-damage inducible transcript 3 (CHOP) showed no shift though autophagy substrate microtubule associated light chain protein 3B (LC3B) showed a significant upregulation following aggregation in bMSCs and ASC. CHOP is also known to regulate growth arrest and DNA-damage inducible protein (GADD34), which is known to dephosphorylate eIF2α, showed lower levels in bMSC and ASC aggregates. This would lock the aggregated stem cells into a higher basal ISR state and recovering the in vivo homeostatic state. Further proteomic analysis revealed that MSCs and ASCs showed heightened proteasome control, while FB had higher chaperon content showing a distinct difference in stem and differentiated cells ISR mechanism.

This work has illustrated the importance of 3D culture in the form of aggregation for the maintenance of MSC stem cell properties. This was carried out in MSCs by induction of the ISR pathway. The ISR pathway in vivo allows cells to mitigate cellular stress, in particular stress associated with protein misfolding, which is a problem commonly encountered during in vitro aging. Aggregation of FB did not result in the same trend of ISR upregulation suggesting a loss of sensitivity following differentiation. Aggregation induce ISR in MSCs lead to heightened basal levels as the in vivo tempering protein GADD34 was not upregulated, this heighten basal ISR also lead to increased autophagy and increased stem protein levels. Taken together these results show a promising alternative to traditional expansion culture for MSCs which leads to loss of function. This will allow for more effective scale-up thereby increasing the therapeutic efficacy of MSC treatments.

References

[1]	Z. Cesarz, J. L. Funnell, J. Guan and K. Tamama, "Soft Elasticity-Associated Signaling and Bone Morphogenic Protein 2 Are Key Regulators of Mesenchymal Stem Cell Spheroidal Aggregates," Stem Cells and Development, vol. 25, no. 8, pp. 622-35, 2016.
[2]	A. Banfi, A. Muraglia, B. Dozin, M. Mastrogiacomo, R. Cancedda and R. Quarto, "Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy," Experimental Hematology, vol. 28, no. 6, pp. 707-15, 2000.
[3]	L. Garcia-Prat, M. Martinez-Vicente, E. Perdiguero, L. Ortet, J. Rodriguez-Ubreva, E. Rebollo, V. Ruiz-Bonilla, S. Gutarra, E. Ballestar, A. L. Serrano, M. Sandri and P. Munoz-Canoves, "Autophagy maintains stemness by preventing senescence," Nature, vol. 529, no. 7584, 2016.
[4]	R. Pennock, E. Bray, P. Pryor, S. James, P. McKeegan, R. Sturmey and P. Genever, "Human cell dedifferentiation in mesenchymal condensates through controlled autophagy," Scientific Reports, 2015.
[5]	Y. Liu, N. Munoz, A.-C. Tsai, T. M. Logan and T. Ma, "Metabolic Reconfiguration Supports Reacquisition of Primitive Phenotype in Human Mesenchymal Stem Cell Aggregates," Stem Cells, vol. 35, no. 2, pp. 398-410, 2017.