Serum Deprivation of Mesenchymal Stem Cells Improves Exosome Activity and Alters Lipid and Protein Composition

Abstract

Summary Exosomes can serve as delivery vehicles for advanced therapeutics. The components necessary and sufficient to support exosomal delivery have not been established. Here we connect biochemical composition and activity of exosomes to optimize exosome-mediated delivery of small interfering RNAs (siRNAs). This information is used to create effective artificial exosomes. We show that serum-deprived mesenchymal stem cells produce exosomes up to 22-fold more effective at delivering siRNAs to neurons than exosomes derived from control cells. Proteinase treatment of exosomes stops siRNA transfer, indicating that surface proteins on exosomes are involved in trafficking. Proteomic and lipidomic analyses show that exosomes derived in serum-deprived conditions are enriched in six protein pathways and one lipid class, dilysocardiolipin. Inspired by these findings, we engineer an “artificial exosome,” in which the incorporation of one lipid (dilysocardiolipin) and three proteins (Rab7, Desmoplakin, and AHSG) into conventional neutral liposomes produces vesicles that mimic cargo delivering activity of natural exosomes.

Angiogenic Effects of Human Dental Pulp and Bone Marrow-Derived Mesenchymal Stromal Cells and their Extracellular Vesicles

“…Similar results have been obtained by the addition of growth factors, such as platelet-derived growth factor (PDGF) to the culture medium or by genetically overexpressing important angiogenic pathways including HIF-1α or Akt signalling in the stem cells [26,57,71]. A recent study of Haraszti et al could also show a significant impact of serum-free culturing on the number, proteome and activity of BM-MSC-derived EVs, with opposite effects on exosomal and microvesicle fractions [68]. Therefore, more standardisation is needed in EV isolation protocols and culture conditions, in order to be able to compare different findings between studies.…”

“…EVs reflect the actual status of the parental cell, which is very susceptible to minor fluctuations in internal and external factors, including culture medium [65], passage number [66,67], seeding density [66], serum concentrations [68] and oxygen levels [69,70], which could explain inconsistencies between EV studies. Culturing MSCs under hypoxic conditions has indeed been observed to increase their EV production and functional effects on endothelial cell proliferation and tube formation [69,70].…”

Exosome-Mediated siRNA pool Inhibits Axonal Growth Inhibitor in Cortical Neurons of Spinal Cord Injury in Rats

“…[24] A recent study has reported that exosomes from serum-deprived mesenchymal stem cells were utilized to deliver siRNAs to neurons. [25] In line with Wang's discovery, exosomes/TRPP2 siRNA complex is capable of suppressing TRPP2 expression of Fadu cells, meanwhile, low expression of TRPP2 elicits an increase in E-cadherin, but downward expression levels of N-cadherin and vimentin. [26] Reports have revealed that rehabilitation of SCI rats achieved satisfactory results via injecting intravenously with exosomes (control exosomes, miRNA-29b exosomes) and BMSCs (miR NC, miRNA-29b) to the tail vein.…”

Extracellular Proteasomes

“…Проте накопичено чимало даних, які свідчать, що виділення протеасом у міжклітинне середовище відбувається внаслідок секреції позаклітинних везикул, зокрема екзосом. Протеасомні комплекси та їх протеазна активність неодноразово були виявлені у екзосомах [28,40,[49][50][51].…”

“…Крім того, було виявлено три специфічні субодиниці імунопротеасоми. Функціональні протеасоми виділялися спільно із екзосомами мезенхімальних стовбурових клітин мишей, що підтверджує безпосередній зв'язок протеасомних комплексів та позаклітинних везикул [49][50][51].…”