EV products obtained from iPSC-derived MSCs show batch-to-batch variations in their ability to modulate allogeneic immune responses in vitro

Abstract

Mesenchymal stromal cells (MSCs) have demonstrated therapeutic potential in diverse clinical settings, largely due to their ability to produce extracellular vesicles (EVs). These EVs play a pivotal role in modulating immune responses, transforming pro-inflammatory cues into regulatory signals that foster a pro-regenerative milieu. Our previous studies identified the variability in the immunomodulatory effects of EVs sourced from primary human bone marrow MSCs as a consistent challenge. Given the limited proliferation of primary MSCs, protocols were advanced to derive MSCs from GMP-compliant induced pluripotent stem cells (iPSCs), producing iPSC-derived MSCs (iMSCs) that satisfied rigorous MSC criteria and exhibited enhanced expansion potential. Intriguingly, even though obtained iMSCs contained the potential to release immunomodulatory active EVs, the iMSC-EV products displayed batch-to-batch functional inconsistencies, mirroring those from bone marrow counterparts. We also discerned variances in EV-specific protein profiles among independent iMSC-EV preparations. Our results underscore that while iMSCs present an expansive growth advantage, they do not overcome the persistent challenge of functional variability of resulting MSC-EV products. Once more, our findings accentuate the crucial need for batch-to-batch functional testing, ensuring discrimination of effective and ineffective MSC-EV products for considered downstream applications.

Evaluation of Batch-To-Batch Variability and Efficacy Of IPSC-Derived Mesenchymal Stromal Derived Extracellular Vesicles in Mitigating Osteoarthritis Associated Inflammation In Vitro

“…Additionally, there was an enrichment of larger vesicles in late passages of both MSCs and iMSCs, potentially re ecting cellular aging processes. This nding underscores the importance of understanding the dynamic changes in EV properties over culture passages for optimizing their therapeutic e cacy (35). Importantly, our characterization of EV morphology via transmission electron microscopy con rms the e cient isolation of EVs from both MSCs and iMSCs, validating their structural integrity.…”

High throughput screening of mesenchymal stromal cell morphological response to inflammatory signals for bioreactor-based manufacturing of extracellular vesicles that modulate microglia

“…Selecting priming conditions corresponding to unique morphologies in the exploratory and validation HTS resulted in a range of potency in the microglia modulation assay as expected. Validation of +CTL, Hit 2, and Hit 4 as high potency priming conditions demonstrated the ability of morphological screening combined with priming as an approach to reduce not only MSC functional heterogeneity, but also EV functional heterogeneity [ 127 , 128 ].…”

Large-scale separation and purification of exosomes using ionexchange chromatography

“…The potential application value of exosomes has increasingly garnered attention 17, 18 . Exosomes are heterogeneous in terms of their size, content, source, and function 19 . Thus, effective isolation and enrichment of exosomes will assist in evaluating their corresponding biological functions.…”