CD73 activity of mesenchymal stromal cell-derived extracellular vesicle preparations is detergent-resistant and does not correlate with immunomodulatory capabilities

Bauer, Fabiola Nardi; Tertel, Tobias; Stambouli, Oumaima; Wang, Chen; Dittrich, Robin; Staubach, Simon; Börger, Verena; Hermann, Dirk M.; Brandau, Sven; Giebel, Bernd

doi:10.1016/j.jcyt.2022.09.006

Cited by 13 publications

(10 citation statements)

References 29 publications

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“…Here, upon pooling mononuclear cells from 12 different donors, strong allogenic immune responses are induced resulting in the activation of CD4 and CD8 T cells that can be recognized by their cell surface expression of CD25 and CD54. As reported in detail previously, the content of activated CD4 as well as of activated CD8 cells can be suppressed partially by immunomodulatory competent MSC-EV preparations ( Bremer et al, 2023 ; Nardi Bauer et al, 2023 ).…”

Section: Resultssupporting

confidence: 66%

“…The immunomodulatory activities of samples of the obtained iMSC-EV preparations were evaluated individually within the mdMLR assay exactly as described previously ( Bremer et al, 2023 ; Nardi Bauer et al, 2023 ). Briefly, peripheral blood mononuclear cells (PBMCs) from 12 different donors were isolated by conventional Ficoll density gradient centrifugation and pooled using identical cell numbers from each donor.…”

Section: Methodsmentioning

confidence: 99%

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

Tertel,

Dittrich,

Arsène

et al. 2023

Front. Cell Dev. Biol.

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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.

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Section: Resultssupporting

confidence: 66%

Section: Methodsmentioning

confidence: 99%

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

Tertel,

Dittrich,

Arsène

et al. 2023

Front. Cell Dev. Biol.

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“…For evaluation of the in vitro immunomodulatory function of MSC-EV products, 25-µg protein of EV preparations was tested in a multi-donor mixed lymphocyte reaction (mdMLR) assay as previously described [ 27 , 49 ]. Briefly, after thawing, 6 × 10 5 pooled peripheral blood mononuclear cells (PBMC) of 12 donors were seeded into each well of a 96-well u-bottom shape plate (Corning, Germany).…”

Section: Methodsmentioning

confidence: 99%

Extracellular vesicles from immortalized mesenchymal stromal cells protect against neonatal hypoxic-ischemic brain injury

et al. 2023

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Background Human mesenchymal stromal cell (MSC)-derived extracellular vesicles (EV) revealed neuroprotective potentials in various brain injury models, including neonatal encephalopathy caused by hypoxia–ischemia (HI). However, for clinical translation of an MSC-EV therapy, scaled manufacturing strategies are required, which is challenging with primary MSCs due to inter- and intra-donor heterogeneities. Therefore, we established a clonally expanded and immortalized human MSC line (ciMSC) and compared the neuroprotective potential of their EVs with EVs from primary MSCs in a murine model of HI-induced brain injury. In vivo activities of ciMSC-EVs were comprehensively characterized according to their proposed multimodal mechanisms of action. Methods Nine-day-old C57BL/6 mice were exposed to HI followed by repetitive intranasal delivery of primary MSC-EVs or ciMSC-EVs 1, 3, and 5 days after HI. Sham-operated animals served as healthy controls. To compare neuroprotective effects of both EV preparations, total and regional brain atrophy was assessed by cresyl-violet-staining 7 days after HI. Immunohistochemistry, western blot, and real-time PCR were performed to investigate neuroinflammatory and regenerative processes. The amount of peripheral inflammatory mediators was evaluated by multiplex analyses in serum samples. Results Intranasal delivery of ciMSC-EVs and primary MSC-EVs comparably protected neonatal mice from HI-induced brain tissue atrophy. Mechanistically, ciMSC-EV application reduced microglia activation and astrogliosis, endothelial activation, and leukocyte infiltration. These effects were associated with a downregulation of the pro-inflammatory cytokine IL-1 beta and an elevated expression of the anti-inflammatory cytokines IL-4 and TGF-beta in the brain, while concentrations of cytokines in the peripheral blood were not affected. ciMSC-EV-mediated anti-inflammatory effects in the brain were accompanied by an increased neural progenitor and endothelial cell proliferation, oligodendrocyte maturation, and neurotrophic growth factor expression. Conclusion Our data demonstrate that ciMSC-EVs conserve neuroprotective effects of primary MSC-EVs via inhibition of neuroinflammation and promotion of neuroregeneration. Since ciMSCs can overcome challenges associated with MSC heterogeneity, they appear as an ideal cell source for the scaled manufacturing of EV-based therapeutics to treat neonatal and possibly also adult brain injury.

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“…In contrast, when the desired effect is driven by synergistic functions of different EV subtypes, or co-isolates, there is no need for complex separation steps and basic isolation methods for yield optimization would be beneficial. Thus, increased knowledge about functional EV heterogeneity is of crucial importance to determine if and how EV treatments can be improved (Bauer et al, 2022). Batch-to-batch variability forms another important hurdle for the translation of EV therapeutics (Figure 4C).…”

Section: Challenges and Scientific Roadblocks For Translation To Clin...mentioning

confidence: 99%

“…Batch-to-batch variability forms another important hurdle for the translation of EV therapeutics (Figure 4C). It has been observed that independent MSC-EV batches produced in a standardized manner differ in EV activity (Bauer et al, 2022;Madel et al, 2020). Clonal drift, which is even seen in individual MSC batches (Ratajczak et al, 2006), and environmental factors may impact the production of different EV subtypes and their function on target tissues.…”

Section: Challenges and Scientific Roadblocks For Translation To Clin...mentioning

confidence: 99%

Extracellular Vesicle Heterogeneity and Its Impact for Regenerative Medicine Applications

et al. 2023

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Extracellular vesicles (EVs) are cell-derived membrane enclosed particles that are involved in physiological and pathological processes. EVs are increasingly being studied for therapeutic applications in the field of regenerative medicine. Therapeutic application of stem cell-derived EVs has shown great potential to stimulate tissue repair. However, the exact mechanisms through which they induce this effect has not been fully clarified. This may to a large extent be attributed to a lack of knowledge on EV heterogeneity. Recent studies suggest that EVs represent a heterogeneous population of vesicles with distinct functions. The heterogeneity of EVs can be attributed to differences in their biogenesis, and as such, they can be classified into distinct populations that can then be further subcategorized into various subpopulations. A better understanding of EV heterogeneity is crucial for elucidating their mechanisms of action in tissue regeneration. This review provides an overview of the latest insights on EV heterogeneity related to tissue repair, including the different characteristics that contribute to such heterogeneity, as well as the functional differences among EV subtypes. It also sheds light on the challenges that hinder clinical translation of EVs.Additionally, innovative EV isolation techniques for studying EV heterogeneity are discussed.Improved knowledge of active EV subtypes would promote the development of tailored EV therapies and aid researchers in the translation of EV-based therapeutics to the clinic.

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CD73 activity of mesenchymal stromal cell-derived extracellular vesicle preparations is detergent-resistant and does not correlate with immunomodulatory capabilities

Cited by 13 publications

References 29 publications

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

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

Extracellular vesicles from immortalized mesenchymal stromal cells protect against neonatal hypoxic-ischemic brain injury

Extracellular Vesicle Heterogeneity and Its Impact for Regenerative Medicine Applications

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