Osborn, Anya Helen (2020) Evaluation of cultured cochlear fibrocytes as a cell replacement therapy: comparison with native fibrocytes. Doctoral thesis, Keele University.

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Abstract

Fibrocyte degeneration in the cochlear lateral wall is one possible pathology of age-related metabolic hearing loss (presbyacusis). Fibrocytes play a role in potassium recycling and maintenance of the endocochlear potential (EP). It has been proposed that a cell replacement therapy could prevent fibrocyte degeneration in the CD/1 mouse model of hearing loss. One source of replacement cells is cultured spiral ligament fibrocytes (SLFs) as they do not require significant differentiation or development so may integrate into the cochlea better than stem cells. This research employs the techniques of whole-cell voltage clamping and immunolabelling to examine and compare the characteristics of native and cultured fibrocytes. The aim to see whether the cultured SLFs display similar features and characteristics to that of native SLFs, in aid of building the case of these cells being a suitable cell replacement to treat age-related hearing loss. Fibrocytes cultured as monolayers or on 3-D collagen I gels were compared with fibrocytes in cochlear slices or micro dissected spiral ligament (SL) from ~P7 cochlea of CD/1 mice.
Fibrocytes successfully grown for short periods on a 3-D collagen I matrix exhibited rounded cell bodies with extending processes, compared to a flatter morphology when grown in a monolayer. Immunofluorescence showed expression of aquaporin 1 (AQP1) which, in the cochlea, is confined to type III fibrocytes and to a lesser extent for S-100 found in several fibrocyte types. The cultured cells also expressed the inwardly-rectifying potassium channel Kir5.1, as well as labelling for the gap junction proteins connexin 26 and 31. Finally, wholecell voltage clamp recordings in both the cultured and putative native fibrocytes revealed outwardly- rectifying potassium currents on depolarisation. There was also a large group of linear response recordings, and a subset showing both inward and outward-rectification. There was no significant difference in the resting membrane potentials between the native and cultured cells in any of the linear or outwardly-rectifying group recordings. Thus 3-D cultured fibrocytes show morphology that more closely resembles that in vivo compared to monolayer cultures and appear to possess functional potassium channels. Thus far, this research indicates that these cells are suitable for transplantation into the lateral wall of the cochlea in a cell therapy to treat age-related metabolic presbyacusis.

Item Type: Thesis (Doctoral)
Subjects: R Medicine > RM Therapeutics. Pharmacology
Divisions: Faculty of Natural Sciences > School of Life Sciences
Contributors: Furness, DN (Thesis advisor)
Evans, MG (Thesis advisor)
Caruana, Douglas (Thesis advisor)
Depositing User: Lisa Bailey
Date Deposited: 20 Mar 2020 16:04
Last Modified: 20 Mar 2020 16:04
URI: https://eprints.keele.ac.uk/id/eprint/7825

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