Isolation of sensory hair cell specific exosomes in human perilymph

Abstract

Evaluation of hearing loss patients using clinical audiometry has been unable to give a definitive cellular or molecular diagnosis, hampering the development of treatments of sensorineural hearing loss. However, biopsy of inner ear tissue without losing residual hearing function for pathologic diagnosis is extremely challenging. In a clinical setting, perilymph can be accessed, so alternative methods for molecular characterization of the inner ear may be developed. Recent approaches to improving inner ear diagnostics have been focusing on the evaluation of the proteomic or miRNA profiles of perilymph. Inspired by recent characterization and classification of many neurodegenerative diseases using exosomes which not only are produced in locally in diseased tissue but are transported beyond the blood brain barrier, we demonstrate the isolation of human inner ear specific exosomes using a novel ultrasensitive immunomagnetic nano pom-poms capture-release approach. Using perilymph samples harvested from surgical procedures, we were able to isolate exosomes from sensorineural hearing loss patients in only 2-5 micro liter of perilymph. By isolating sensory hair cell derived exosomes through their expression level of myosin VII, we for the first time sample material from hair cells in the living human inner ear. This work sets up the first demonstration of immunomagnetic capture-release nano pom-pom isolated exosomes for liquid biopsy diagnosis of sensorineural hearing loss. With the ability to isolate exosomes derived from different cell types for molecular characterization, this method also can be developed for analyzing exosomal biomarkers from more accessible patient tissue fluids such as plasma.

Automated western blot analysis of ototoxin-induced prestin burst in the blood after cyclodextrin exposure

“…A closely related SLC26 gene family protein, Slc26a4 -pendrin, has been shown to be present in exosomes from urine [28], indicating that structurally similar proteins can be EV-associated. Hair cells are known to produce exosomes, which encase hair cell-speci c proteins and can be detected in perilymph [29,30]. Cochlear insult, such as with noise, is known to lead to increased prestin expression in the remaining OHCs [31].…”

Characterization of ototoxic-induced prestin burst in the blood and implications for developing prestin as an ototoxicity biomarker

“…The nding that the ~ 134 kDa prestin peak represents a glycosylated form of prestin is also key as glycosylated proteins are generally enriched in secreted EVs [25,26]. Hair cells are known to produce exosomes, which encase hair cell-speci c proteins and can be detected in perilymph [27,28]. Cochlear insult, such as with noise, is known to lead to increased prestin expression in the remaining OHCs [18].…”

Overcoming barriers: a review on innovations in drug delivery to the middle and inner ear

“…The precise physiological role of exosomes is unclear, however they have been demonstrated to have pleiotropic roles in cell-cell communication, signaling, proliferation, angiogenesis, immune response and metabolism and have been shown to carry proteins, nucleic acids and metabolites (Kalluri and LeBleu, 2020;Warnecke et al, 2022). Exosomes have been recently identified, isolated and characterized from the perilymph of adult patients with various forms of SNHL or mixed hearing loss, with the study suggesting a hair cell origin of the exosomes by their expression of MYO7A (Zhuang et al, 2021). On the therapeutic side, a study from Athanasia Warnecke and colleagues demonstrated protective effects of bone marrow mesenchymal stromal cell-derived extracellular vesicles on a mouse model of NIHL, which additionally promoted neurite outgrowth in vitro (Warnecke et al, 2020).…”