Korchak, Jennifer A.

Extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) exhibit great therapeutic potential in ischemia-associated conditions and diseases such as myocardial infarction, ischemic stroke, and wound healing. Enhancing the therapeutic efficacy of MSC-EVs could advance their clinical application. Diverse cargos (proteins, mRNA, microRNA, etc.) in MSC-EVs contribute to the therapeutic effects in various diseases. Vascular endothelial growth factor (VEGF) is one of the primary driving molecules in promoting angiogenesis and protecting endothelial cells lining blood vessels from apoptosis. In this study, we explored the feasibility of engineering parent MSCs with VEGF mRNA to potentiate therapeutic effects of their derived EVs. We first detected elevated levels of VEGF mRNA and protein in transfected MSCs and demonstrated the bioactivity of secreted VEGF by an angiogenesis assay. Furthermore, EVs derived from VEGF mRNA-engineered MSCs (VEGF-MSC-EVs) contained high levels of VEGF mRNA and protein and showed superior ability to protect human umbilical vein endothelial cells (HUVECs) from apoptosis compared to EVs derived from control MSCs (control MSC-EVs). To our knowledge, this is the first report demonstrating that VEGF-MSC-EVs boost therapeutic efficacy by promoting endothelial cell survival. Our findings offer a novel approach for cell-free therapy in ischemia-associated conditions and diseases.

Abstract Background Extracellular vesicles (EVs) have gained interest in the field of regenerative and transfusion medicine. Specifically, current Good Manufacturing Practice (cGMP)‐grade EVs produced by mesenchymal stromal cells (MSCs) are an intriguing option for cell‐free therapeutics. With the development of cGMP‐grade EV products, a simple and reliable method for batch‐to‐batch consistency is needed. Study Design and Methods The objective of this study was to validate a method to semiquantitatively assess the batch‐to‐batch consistency of isolated EVs. A multiplex bead‐based flow cytometric assay containing 37 surface markers and 2 assay control antibody‐coated capture beads was validated. Detection limits ( n = 10 buffer samples), repeatability ( n = 9 EV samples), and intra‐observer reproducibility over 2 days ( n = 10 EV batches) were assessed. A Spearman correlation matrix was used to evaluate the batch‐to‐batch consistency of independently isolated EV products ( n = 37 surface markers). Batches with a Spearman correlation coefficient ≥0.9 and p < 0.05 were considered statistically indistinguishable from previous batches. Results This assay demonstrated robust repeatability as well as intra‐ and inter‐assay reproducibility. In‐house batches of EVs were significantly correlated (r ≥ 0.90; p ≤ 1×10 −14 ). Compared with buffer, EV batches had correlation coefficients near zero (r ≤ −0.10; p ≥ 0.12). Commercially sourced EVs significantly correlated with in‐house EV batches, but fell below the 90% correlation cutoff (r ≤ 0.71; p ≤ 0.0004). Discussion This time‐efficient and technically simple assay offers a robust method of quality control for assessing the batch‐to‐batch reproducibility of cGMP‐grade EV products.

Mesenchymal stem cells (MSCs) are used in various studies to induce immunomodulatory effects in clinical conditions associated with immune dysregulation such as graft versus host disease (GvHD). However, most of these clinical trials failed to go beyond early phase 2 studies because of limited efficacy. Various methods have been assessed to increase the potency of MSCs. IL-10 is an anti-inflammatory cytokine that is known to modulate immune responses in GvHD. In this study, we evaluated the feasibility of transfecting IL-10 mRNA to enhance MSC therapeutic potential. IL-10 mRNA engineered MSCs (eMSCs-IL10) maintained high levels of IL-10 expression even after freezing and thawing. IL-10 mRNA transfection did not appear to alter MSC intrinsic characteristics. eMSCs-IL10 significantly suppressed T cell proliferation relative to naïve MSCs in vitro. In a mouse model for GvHD, eMSCs-IL10 induced a decrease in plasma level of potent pro-inflammatory cytokines and inhibited CD4+ and CD8+ T cell proliferation in the spleen. In summary, our studies demonstrate the feasibility of potentiating MSCs to enhance their immunomodulatory effects by IL-10 mRNA transfection. The use of non-viral transfection may generate a safe and potent MSC product for treatment of clinical conditions associated with immune dysregulation such as GvHD.