Abstract
Mesenchymal stem cell (MSC)-based therapy has shown great promises in various animal disease models. However, this therapeutic potency has not been well claimed when applied to human clinical trials. This is due to both the availability of MSCs at the time of administration and lack of viable expansion strategies. MSCs are very susceptible to in vitro culture environment and tend to adapt the microenvironment which could lead to cellular senescence and aging. Therefore, extended in vitro expansion induces loss of MSC functionality and its clinical relevance. To combat this effect, this work assessed a novel cyclical aggregation as a means of expanding MSCs to maintain stem cell functionality. The cyclical aggregation consists of an aggregation phase and an expansion phase by replating the dissociated MSC aggregates onto planar tissue culture surfaces. The results indicate that cyclical aggregation maintains proliferative capability, stem cell proteins, and clonogenicity, and prevents the acquisition of senescence. To determine why 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. This method holds the potential to increase the clinical relevance of MSC therapeutic effects from small model systems (such as rats and mice) to humans, and may open the potential of patient-derived MSCs for treatment thereby removing the need for immunosuppression.
Highlights
Mesenchymal stem cell (MSC)-based therapy has shown great promises in various animal disease models
MSCs are very sensitive to their microenvironment and could adapt cellular events based on certain physiological cues such as oxygen or biophysical cues such as culture surface s tiffness[14]
Since integrated stress response (ISR) response is essential to maintaining stem cell function, and our study recently showed that aggregation of MSCs results in a heightened basal ISR response, the doubling time was assessed under ISR modulation with salubrinal, a growth arrest and DNA damage-inducible protein (GADD34) inhibitor which prevents ISR elevation, and GSK-2606414 (GSK), a phospho-protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor which prevents the cells from entering ISR
Summary
Mesenchymal stem cell (MSC)-based therapy has shown great promises in various animal disease models This therapeutic potency has not been well claimed when applied to human clinical trials. Of a breakdown in the integrated stress response (ISR), which is a complex pathway that recognizes misfolded proteins and initiates a lockdown on protein synthesis, increases folding chaperones, and if proteostasis cannot be recovered, provides signals for a utophagy[17,18] These effects can be mitigated through the MSC’s 3D aggregation process in which cells are integrated into a new cellular environment with a lower modulus similar to human tissue[19,20,21,22,23]. Cells within the aggregate have reduced proliferative potential, meaning that aggregation alone cannot solve the scale-up problem[30,32]
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