Exosomes from iPSCs Delivering siRNA Attenuate Intracellular Adhesion Molecule-1 Expression and Neutrophils Adhesion in Pulmonary Microvascular Endothelial Cells

Abstract

The pro-inflammatory activation of pulmonary microvascular endothelial cells resulting in continuous expression of cellular adhesion molecules, and subsequently recruiting primed neutrophils to form a firm neutrophils-endothelium (PMN-EC) adhesion, has been examined and found to play a vital role in acute lung injury (ALI). RNA interference (RNAi) is a cellular process through harnessing a natural pathway silencing target gene based on recognition and subsequent degradation of specific mRNA sequences. It opens a promising approach for precision medicine. However, this application was hampered by many obstacles, such as immunogenicity, instability, toxicity problems, and difficulty in across the biological membrane. In this study, we reprogrammed urine exfoliated renal epithelial cells into human induced pluripotent stem cells (huiPSCs) and purified the exosomes (Exo) from huiPSCs as RNAi delivery system. Through choosing the episomal system to deliver transcription factors, we obtained a non-integrating huiPSCs. Experiments in both vitro and vivo demonstrated that these huiPSCs possess the pluripotent properties. The exosomes of huiPSCs isolated by differential centrifugation were visualized by transmission electron microscopy (TEM) showing a typical exosomal appearance with an average diameter of 122 nm. Immunoblotting confirmed the presence of the typical exosomal markers, including CD63, TSG 101, and Alix. Co-cultured PKH26-labeled exosomes with human primary pulmonary microvascular endothelial cells (HMVECs) confirmed that they could be internalized by recipient cells at a time-dependent manner. Then, electroporation was used to introduce siRNA against intercellular adhesion molecule-1 (ICAM-1) into exosomes to form an Exo/siRNA compound. The Exo/siRNA compound efficiently delivered the target siRNA into HMVECs causing selective gene silencing, inhibiting the ICAM-1 protein expression, and PMN-EC adhesion induced by lipopolysaccharide (LPS). These data suggest that huiPSCs exosomes could be used as a natural gene delivery vector to transport therapeutic siRNAs for alleviating inflammatory responses in recipient cells.

Effects of tumor necrosis factor‐α‐induced exosomes on the endothelial cellular behavior, metabolism and bioenergetics

“…This method is widely accepted in the studies on the exosomes. 5,11,26,27 Previous study using nanoparticle tracking analysis showed that TNFα did not alter the size distribution of the exosomes, 11 suggesting that the protein content is well related to the enumeration (concentration) of the exosomes. However, the exosomes should be ideally counted by a particle analyzer, although a high cost for the instruments including nanoparticle tracking analysis or high-resolution flow cytometer restricts their use in exosomes-related studies.…”

“…In the current study, we used the protein content of the exosomes to represent the concentration of exosomes. This method is widely accepted in the studies on the exosomes . Previous study using nanoparticle tracking analysis showed that TNF‐α did not alter the size distribution of the exosomes, suggesting that the protein content is well related to the enumeration (concentration) of the exosomes.…”

Extracellular Vesicles in ARDS: New Insights into Pathogenesis with Novel Clinical Applications

“…In mouse models of lung injury, mesenchymal stromal cell extracellular vesicles have similar efficacy to mesenchymal stromal cells themselves. Finally, exogenous extracellular vesicles could be modified to package custom drugs or protective RNA cargo, which could be delivered to specific cell types as determined by the extracellular vesicle surface markers [47].…”

Extracellular Vesicles: The New Frontier of Stem Cell Regenerative Medicine?

“…siRNA loading is similar to that of miRNA loading, with electroporation being the most common method of active loading [209]. In a model of acute lung injury, EVs isolated from iPSCs derived by reprogramming renal epithelial cells were subjected to electroporation to introduce siRNA against ICAM-1, which is involved in the inflammatory response [210].…”