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Morphological and morphometric characterization of direct transdifferentiation of support cells into hair cells in ototoxin-exposed neonatal utricular explants
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
Umeå University, Faculty of Social Sciences, Umeå School of Business and Economics (USBE), Statistics.
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2015 (English)In: Hearing Research, ISSN 0378-5955, E-ISSN 1878-5891, Vol. 321, 1-11 p.Article in journal (Refereed) Published
Abstract [en]

We have studied aminoglycoside-induced vestibular hair-cell renewal using long-term culture of utricular macula explants from 4-day-old rats. Explanted utricles were exposed to 1 mM of gentamicin for 48 h, during 2nd and 3rd days in vitro (DIV), and then recovering in unsupplemented medium. Utricles were harvested at specified time points from the 2nd through the 28th DIV. The cellular events that occurred within hair cell epithelia during the culture period were documented from serial sectioned specimens. Vestibular hair cells (HCs) and supporting cells (SCs) were systematically counted using light microscopy (LM) with the assistance of morphometric software. Ultrastructural observations were made from selected specimens with transmission electron microscopy (TEM). After 7 DIV, i.e. four days after gentamicin exposure, the density of HCs was 11% of the number of HCs observed in non-gentamicin-exposed control explants. At 28 DIV the HC density was 61% of the number of HCs observed in the control group explant specimens. Simultaneously with this increase in HCs there was a corresponding decline in the number of SCs within the epithelium. The proportion of HCs in relation to SCs increased significantly in the gentamicin-exposed explant group during the 5th to the 28th DIV period of culture. There were no significant differences in the volume estimations of the gentamicin-exposed and the control group explants during the observed period of culture. Morphological observations showed that gentamicin exposure induced extensive loss of HCs within the epithelial layer, which retained their intact apical and basal linings. At 7 to 14 DIV (i.e. 3-11 days after gentamicin exposure) a pseudostratified epithelium with multiple layers of disorganized cells was observed. At 21 DIV new HCs were observed that also possessed features resembling SCs. After 28 DIV a new luminal layer of HCs with several layers of SCs located more basally characterized the gentamicin-exposed epithelium. No mitoses were observed within the epithelial layer of any explants. Our conclusion is that direct transdifferentiation of SCs into HCs was the only process contributing to the renewal of HCs after gentamicin exposure in these explants of vestibular inner ear epithelia obtained from the labyrinths of 4-day-old rats.

Place, publisher, year, edition, pages
2015. Vol. 321, 1-11 p.
National Category
URN: urn:nbn:se:umu:diva-102465DOI: 10.1016/j.heares.2014.12.011ISI: 000350933600001PubMedID: 25576788OAI: diva2:812654
Available from: 2015-05-19 Created: 2015-04-26 Last updated: 2016-08-18Bibliographically approved
In thesis
1. Hair cell regeneration in vestibular epithelia: a study in an in vitro model
Open this publication in new window or tab >>Hair cell regeneration in vestibular epithelia: a study in an in vitro model
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]


Hair cells (HCs) are the sensory receptors in both the auditory and the vestibular organs of the inner ear. Supporting cells (SCs) are non-sensory cells embracing the HCs. Injuries of the HCs by aging, acoustic trauma or ototoxic drugs (mainly aminoglycosides, e.g. gentamicin) and cisplatin, often cause permanent impairment of hearing and balance. Birds and amphibians can regenerate their auditory and vestibular HCs after injury through proliferation of SCs or direct transdifferentiation of a SC into a HC. For mammals this ability is limited and spontaneous HC regeneration occurs only in the vestibular sensory epithelia. The utricle is one of the five vestibular organs and contributes to our balance by registering linear acceleration and head tilts. The aim of this PhD thesis was to investigate morphological and morphometric events during spontaneous HC regeneration following gentamicin exposure in neonatal rat utricular explants.


Long-term organ culture of macula utriculi, which is stable and reproducible for up to 28 days in vitro (DIV), was used in all papers in the thesis. HC damage was induced by gentamicin. On 2 DIV the explanted utricular maculae were divided into two groups, a control group and a gentamicin-exposed group. In the latter group macular explants were exposed to gentamicin for 48 hours during 2-3 DIV and then allowed to recover. Morphologic and morphometric evaluations were done from utricles harvested at various time points during 28 DIV. Imaging techniques used were light microscopy, including immunohistochemistry, and transmission electron microscopy.


In the control group the epithelia were well preserved with a slight decline in HC density after 14 DIV. In the gentamicin-exposed group there was an initial substantial decline in HC density and thereafter the proportion of HCs in relation to SCs increased significantly. Using BrdU as a proliferation marker and myosin 7a as a HC marker, we found no cells that were double marked. At the ultrastructural level, the apical occlusion of the explanted epithelia was intact in both the control and the gentamicin exposed group during the entire in vitro period. Cells that seemed to be in a transitional state, transforming from SCs into HCs were observed in the gentamicin-exposed group. These cells had cytoplasmic extensions basally i.e. foot processes, an assembly of mitochondria basally in the cell or in these foot processes, and often apical SC extensions covering the HC. HCs classified as transitional cells had an increased number of SC connections to their basal parts compared to mature HCs.


In these neonatal rat utricular explants:

- The morphological structure of the sensory epithelia was well preserved during long-term culture.

- The renewal of hair cells after gentamicin exposure occurred through direct transdifferentiation of supporting cells into hair cells.

- There was also a proliferative response by the supporting cells, but this supporting cell proliferation did not contribute to the generation of new hair cells.

- Cells in a transitional state, showing a characteristic morphology, were observed during the process of transdifferentiation from supporting cells into hair cells.

- The tight junctional seal of the epithelia stayed morphologically intact also after gentamicin exposure.

- Gap junctions were observed in between supporting cells but not found in between hair cells and supporting cells or between transitional cells and supporting cells.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2016. 46 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1804
Vestibular hair cell, regeneration, transdifferentiation, proliferation, utricle, rat
National Category
Research subject
urn:nbn:se:umu:diva-124077 (URN)978-91-7601-524-7 (ISBN)
Public defence
2016-09-09, Hörsal D, Undervisningsnod T9, byggnad 1D, plan 9, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Available from: 2016-08-19 Created: 2016-07-13 Last updated: 2016-08-22Bibliographically approved

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