Role of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is a process that takes place during embryonic development, wound healing, and under some pathological processes, including fibrosis and tumor progression. The molecular changes occurring within epithelial cells during transformation to a mesenchymal phenotype have been well studied. However, to date, the mechanism of EMT induction remains to be fully elucidated. Recent findings in the field of intercellular communication have shed new light on this process and indicate the need for further studies into this important mechanism. New evidence supports the hypothesis that intercellular communication between mesenchymal stroma/stem cells (MSCs) and resident epithelial cells plays an important role in EMT induction. Besides direct interactions between cells, indirect paracrine interactions by soluble factors and extracellular vesicles also occur. Extracellular vesicles (EVs) are important mediators of intercellular communication, through the transfer of biologically active molecules, genetic material (mRNA, microRNA, miRNA, siRNA, DNA), and EMT inducers to the target cells, which are capable of reprogramming recipient cells. In this review, we discuss the role of intercellular communication by EVs to induce EMT and the acquisition of stemness properties by normal and tumor epithelial cells

Construction of Fusion Protein for Enhanced Small RNA Loading to Extracellular Vesicles

Extracellular vesicles (EVs) naturally carry cargo from producer cells, such as RNA and protein, and can transfer these messengers to other cells and tissue. This ability provides an interesting opportunity for using EVs as delivery vehicles for therapeutic agents, such as for gene therapy. However, endogenous loading of cargo, such as microRNAs (miRNAs), is not very efficient as the copy number of miRNAs per EV is quite low. Therefore, new methods and tools to enhance the loading of small RNAs is required. In the current study, we developed fusion protein of EV membrane protein CD9 and RNA-binding protein AGO2 (hCD9.hAGO2). We show that the EVs engineered with hCD9.hAGO2 contain significantly higher levels of miRNA or shRNA (miR-466c or shRNA-451, respectively) compared to EVs that are isolated from cells that only overexpress the desired miRNA or shRNA. These hCD9.hAGO2 engineered EVs also transfer their RNA cargo to recipient cells more efficiently. We were not able to detect changes in gene expression levels in recipient cells after the EV treatments, but we show that the cell viability of HUVECs was increased after hCD9.hAGO2 EV treatments. This technical study characterizes the hCD9.hAGO2 fusion protein for the future development of enhanced RNA loading to EVs

Storage stability and delivery potential of cytochalasin B induced membrane vesicles

Cell-free therapies based on extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are considered as a promising tool for stimulating regeneration and immunomodulation. The need to develop a practical approach for large-scale production of vesicles with homogenous content led to the implementation of cytochalasin B-induced to induce microvesicles (CIMVs) biogenesis. CIMVs mimic natural EVs in size and composition of the surrounding cytoplasmic membrane. Previously we observed that MSC derived CIMVs demonstrate the same therapeutic angiogenic and immunomodulatory activity as the parental MSCs, making them a potentially scalabale cell-free therapeutic option. However, little is known about their storage stability and delivery potential. Therefore, in this study, we determined the effects of different storage conditions (+37°C in serum, +4°C,-20°, +25°C in saline, as well as freeze-drying prior to storage at-20°C) on the integrity and effective delivery of CIMVs derived from human MSCs. We determined that different storage conditions alter the protein concentration within the solution used to store CIMVs over time, this concided with a decrease in the amount of CIMVs due to gradual degradation. We established that freezing and storage CIMVs in saline at-20°C reduces degredation and prolongs their shelf life. Additionally, we found that freeze-thawing preserved the CIMVs morphology better than freeze drying and subsequent rehydration which resulted in aggregation of CIMVs. Collectively our data demonstrates for the first time, that the most optimal method of CIMVs storage is freezing at-20°C, to preserve the CIMVs in the maximum quantity and quality with retention of effective delivery. These findings will benefit the formation of standardized protocols for the use of CIMVs for both basic research and clinical application

Microvesicles of mesenchymal stem cells demonstrate immunosuppressive activity in vivo

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Construction of Fusion Protein for Enhanced Small RNA Loading to Extracellular Vesicles

Extracellular vesicles (EVs) naturally carry cargo from producer cells, such as RNA and protein, and can transfer these messengers to other cells and tissue. This ability provides an interesting opportunity for using EVs as delivery vehicles for therapeutic agents, such as for gene therapy. However, endogenous loading of cargo, such as microRNAs (miRNAs), is not very efficient as the copy number of miRNAs per EV is quite low. Therefore, new methods and tools to enhance the loading of small RNAs is required. In the current study, we developed fusion protein of EV membrane protein CD9 and RNA-binding protein AGO2 (hCD9.hAGO2). We show that the EVs engineered with hCD9.hAGO2 contain significantly higher levels of miRNA or shRNA (miR-466c or shRNA-451, respectively) compared to EVs that are isolated from cells that only overexpress the desired miRNA or shRNA. These hCD9.hAGO2 engineered EVs also transfer their RNA cargo to recipient cells more efficiently. We were not able to detect changes in gene expression levels in recipient cells after the EV treatments, but we show that the cell viability of HUVECs was increased after hCD9.hAGO2 EV treatments. This technical study characterizes the hCD9.hAGO2 fusion protein for the future development of enhanced RNA loading to EVs