High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy.

Bacterial extracellular vesicles (BEVs), including outer membrane vesicles, have emerged as a promising new class of vaccines and therapeutics to treat cancer and inflammatory diseases, among other applications. However, clinical translation of BEVs is hindered by a current lack of scalable and efficient purification methods. Here, we address downstream BEV biomanufacturing limitations by developing a method for orthogonal size- and charge-based BEV enrichment using tangential flow filtration (TFF) in tandem with high performance anion exchange chromatography (HPAEC). The data show that size-based separation coisolated protein contaminants, whereas size-based TFF with charged-based HPAEC dramatically improved purity of BEVs produced by probiotic Gram-negative Escherichia coli and Gram-positive lactic acid bacteria (LAB). Escherichia coli BEV purity was quantified using established biochemical markers while improved LAB BEV purity was assessed via observed potentiation of anti-inflammatory bioactivity. Overall, this work establishes orthogonal TFF + HPAEC as a scalable and efficient method for BEV purification that holds promise for future large-scale biomanufacturing of therapeutic BEV products.

Differentiation state and culture conditions impact neural stem/progenitor cell-derived extracellular vesicle bioactivity.

Extracellular vesicles (EVs) derived from neural progenitor/stem cells (NPSCs) have shown promising efficacy in a variety of preclinical models. However, NPSCs lack critical neuroregenerative functionality such as myelinating capacity. Further, culture conditions used in NPSC EV production lack standardization, limiting reproducibility challenging and potentially potency of the overall approach via lack of optimization. Here, we assessed whether oligodendrocyte precursor cells (OPCs) and immature oligodendrocytes (iOLs), which are further differentiated than NPSCs and which both give rise to mature myelinating oligodendrocytes, could yield EVs with neurotherapeutic properties comparable or superior to those from NPSCs. We additionally examined the effects of extracellular matrix (ECM) coating materials and the presence or absence of growth factors in cell culture on the ultimate properties of EVs. The data show that OPC EVs and iOL EVs performed similarly to NPSC EVs in cell proliferation and anti-inflammatory assays, but NPSC EVs performed better in a neurite outgrowth assay. Additionally, the presence of nerve growth factor (NGF) in culture was found to maximize NPSC EV bioactivity among the conditions tested. NPSC EVs produced under rationally-selected culture conditions (fibronectin + NGF) enhanced axonal regeneration and muscle reinnervation in a rat nerve crush injury model. These results highlight the need for standardization of culture conditions for neurotherapeutic NPSC EV production.

Induced Pluripotent Stem Cell-Derived Extracellular Vesicles Promote Wound Repair in a Diabetic Mouse Model via an Anti-Inflammatory Immunomodulatory Mechanism.

Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have recently been explored in clinical trials for treatment of diseases with complex pathophysiologies. However, production of MSC EVs is currently hampered by donor-specific characteristics and limited ex vivo expansion capabilities before decreased potency, thus restricting their potential as a scalable and reproducible therapeutic. Induced pluripotent stem cells (iPSCs) represent a self-renewing source for obtaining differentiated iPSC-derived MSCs (iMSCs), circumventing both scalability and donor variability concerns for therapeutic EV production. Thus, it is initially sought to evaluate the therapeutic potential of iMSC EVs. Interestingly, while utilizing undifferentiated iPSC EVs as a control, it is found that their vascularization bioactivity is similar and their anti-inflammatory bioactivity is superior to donor-matched iMSC EVs in cell-based assays. To supplement this initial in vitro bioactivity screen, a diabetic wound healing mouse model where both the pro-vascularization and anti-inflammatory activity of these EVs would be beneficial is employed. In this in vivo model, iPSC EVs more effectively mediate inflammation resolution within the wound bed. Combined with the lack of additional differentiation steps required for iMSC generation, these results support the use of undifferentiated iPSCs as a source for therapeutic EV production with respect to both scalability and efficacy.

Induced pluripotent stem cell-derived extracellular vesicles promote wound repair in a diabetic mouse model via an anti-inflammatory immunomodulatory mechanism.

Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have recently been widely explored in clinical trials for treatment of diseases with complex pathophysiology. However, production of MSC EVs is currently hampered by donor-specific characteristics and limited ex vivo expansion capabilities before decreased potency, thus restricting their potential as a scalable and reproducible therapeutic. Induced pluripotent stem cells (iPSCs) represent a self-renewing source for obtaining differentiated iPSC-derived MSCs (iMSCs), circumventing both scalability and donor variability concerns for therapeutic EV production. Thus, we initially sought to evaluate the therapeutic potential of iMSC EVs. Interestingly, while utilizing undifferentiated iPSC EVs as a control, we found that their vascularization bioactivity was similar and their anti-inflammatory bioactivity was superior to donor-matched iMSC EVs in cell-based assays. To supplement this initial in vitro bioactivity screen, we employed a diabetic wound healing mouse model where both the pro-vascularization and anti-inflammatory activity of these EVs would be beneficial. In this in vivo model, iPSC EVs more effectively mediated inflammation resolution within the wound bed. Combined with the lack of additional differentiation steps required for iMSC generation, these results support the use of undifferentiated iPSCs as a source for therapeutic EV production with respect to both scalability and efficacy.

Bacterial Extracellular Vesicles and the Gut-Microbiota Brain Axis: Emerging Roles in Communication and Potential as Therapeutics.

Bacterial extracellular vesicles (BEVs) have emerged as candidate signaling vectors for long-distance interkingdom communication within the gut-microbiota brain axis. Most bacteria release these nanosized vesicles, capable of signaling to the brain via their abundant protein and small RNA cargo, possibly directly via crossing the blood-brain barrier. BEVs have been shown to regulate brain gene expression and induce pathology at most stages of neuroinflammation and neurodegeneration, and thus they may play a causal role in diseases such as Alzheimer's, Parkinson's, and depression/anxiety. On the other hand, BEVs have intrinsic therapeutic properties that may be relevant to probiotic therapy and can also be engineered to function as drug delivery vehicles and vaccines. Thus, BEVs may be both a cause of and solution to neuropathological conditions. In this review, current knowledge of the physiological roles of BEVs as well as state of the art pertaining to the development of therapeutic BEVs in the context of the microbiome-gut-brain axis are summarized.