Quantitative comparison of the mRNA content of human iPSC‐derived motor neurons and their extracellular vesicles

Abstract

Extracellular vesicles (EVs) contain various cargo molecules, including RNAs and proteins. EVs, which include exosomes, are predicted to be suitable surrogates of their source cells for liquid biopsy to measure biomarkers. Several studies have performed qualitative comparisons of cargo molecule repertoires between source cells and their EVs. However, quantitative comparisons have not been reported so far. Furthermore, many studies analyzed microRNAs or proteins in EVs, but not mRNAs. In this study, we analyzed mRNAs in motor neurons and their EVs. Normal human-induced pluripotent stem cells were differentiated into motor neurons, and comprehensive analysis of mRNAs in the cells and their EVs was performed by RNA sequencing. Differential analysis between cellular and EV mRNAs was performed by edgeR after normalization of read count. The results suggest that signatures in the abundance of EV mRNAs were different from those of cellular mRNAs. Comparison of intracellular vesicle and EV mRNA abundance showed negatively and positively biased genes in the EVs. Gene Ontology analysis revealed that the genes showing negatively biased abundance in the EVs were enriched in many functions regarding neuronal development. In contrast, the positively biased genes were enriched in functions regarding cellular metabolism and protein synthesis. These results suggest that mRNAs in motor neurons are loaded into EVs to regulate certain mechanisms, which are yet to be elucidated. Exosomes are known as a representative subset of extracellular vesicles (EVs) that are secreted from any type of cell [1]. Exosomes are secreted from the cells via the formation of multivesicular bodies in endosomes and their fusion to the plasma membrane [2]. Microvesicles are also a subset of EVs, and they are secreted by a budding of plasma membrane [3]. The size of exosomes ranges from 30 to 200 nm, while that of microvesicles ranges from 100 to 1 lm [4]. However, it is difficult to completely separate and distinguish exosomes from other EV subsets because their diameters, marker molecules and cargos are rather overwrapped. Therefore, EVs are used as a general term [5]. EVs carry functional cargo molecules, such as RNAs and proteins, inside and outside of their membranes [6]. Generally, cargo molecules in EVs are considered to reflect at least a portion

Extracellular Vesicles fromPRPF31+/−iPSC-Derived RPE Have a Distinct RNA Profile Compared to Those From Healthy RPE

“…Other studies have reported similar differences in RNA profiles and abundance when comparing EVs to their source cells (35,36). Although the mechanism of RNA sorting and loading into EVs is currently unknown, some studies suggest the contents of EVs are loaded by source cells as a mechanism of controlling intracellular availability of certain RNAs (23,33,34).…”

Extracellular Vesicles in Amyotrophic Lateral Sclerosis

“…There are fewer in vitro studies of messenger RNA (mRNA) expression in EVs in ALS. Similar to miRNA, mRNA expression in EVs from iPSC derived motor neurons is markedly different from that of the iPSC cells themselves, being enriched for genes regulating cellular metabolism and protein homeostasis [ 106 ].…”