Abstract
Therapeutic benefits of mesenchymal stem cells (MSCs) are now widely believed to come from their paracrine signalling, i.e. secreted factors such as cytokines, chemokines, and extracellular vesicles (EVs). Cell-free therapy using EVs is an active and emerging field in regenerative medicine. Typical 2D cultures on tissue culture plastic is far removed from the physiological environment of MSCs. The application of 3D cell culture allows MSCs to adapt to their cellular environment which, in turn, influences their paracrine signalling activity. In this study we evaluated the impact of 3D MSCs culture on EVs secretion, cargo proteome composition, and functional assessment in immunomodulatory, anti-inflammatory and anti-fibrotic properties.MSC-EVs from 2D and 3D cultures expressed classical EV markers CD81, CD63, and CD9 with particle diameter of <100 nm. There were distinct changes in immunomodulatory potencies where 3D cultures exhibited reduced indoleamine 2,3-dioxygenase (IDO) activity and significantly reduced macrophage phagocytosis. Administration of 2D and 3D EVs following double dose bleomycin challenge in aged mice showed a marked increase of bodyweight loss in 3D group throughout days 7–28. Histopathological observations of lung tissues in 3D group showed increased collagen deposition, myofibroblast differentiation and leukocytes infiltrations. Assessment of lung mechanics showed 3D group did not improve lung function and instead exhibited increased resistance and tissue damping. Proteome profiling of MSC-EV composition revealed molecular enrichment of EV markers (compared to parental cells) and differential proteome between EVs from 2D and 3D culture condition associated with immune-based and fibrosis/extracellular matrix/membrane organization associated function.This study provides insight into distinct variation in EV protein composition dependent on the cellular microenvironment of the parental cells, which could have implications in their therapeutic effect and potency. Overall, this work suggests that EVs produced from 3D MSC cultures did not enhance typical MSC-EV properties expected from 2D cultures (immunomodulation, anti-fibrotic, anti-inflammatory). The outcome highlights critical differences between MSC-EVs obtained from different culture microenvironments, which should be considered when scaling up MSC culture for clinical manufacturing.
Highlights
Mesenchymal stem cells (MSCs) are excellent candidates for a range of therapeutic applications (Levy et al, 2020)
Representative co-localisation data and particle size distribution confirmed that both 2D and 3D MSC-extracellular vesicles (EVs) have an average particle size of
This study adds a new dimension to the current understanding of MSC secretome biology and highlights that the microenvironmental conditions will affect EV content and function
Summary
Mesenchymal stem cells (MSCs) are excellent candidates for a range of therapeutic applications (Levy et al, 2020). Much of the clinical interest in MSCs is due to their known anti-inflammatory and anti-fibrotic properties These are mediated by the secretion of various cytokines and soluble factors, which in turn modulate immune cell activity and promote tissue generation. The therapeutic capacity of MSC-EVs derived from different tissues have been tested in various disease models, demonstrating a similar or even superior functional potential to MSCs themselves (Burrello et al, 2016; Riazifar et al, 2017). EVs, in contrast, have no vascular obstructive effect or apparent adverse effects. These properties suggest that EVs could be safely and used in therapies such as for respiratory diseases (Abreu et al, 2016; Fujita et al, 2018; Claridge et al, 2021a)
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