Hair cell formation in cultures of dissociated cells from the vestibular sensory epithelium of the bullfrog.
OBJECTIVE
Bullfrog vestibular hair cells are capable of regenerating in vitro.


BACKGROUND
Recent studies have established that sensory organs in the inner ear of vertebrates continue to produce hair cells after birth. However, the mechanisms responsible for the regulation of this process are not well understood. The current study reports the development of a novel method for the culture of dispersed cells from the bullfrog inner ear.


METHODS
New hair cell formation in this in vitro preparation was shown by sequential photomicroscopy. Studies with the selective marker for mitotic activity 5-bromo-2-deoxyuridine (BrdU) were done to estimate the level of cell proliferation and to quantify postmitotic hair cell formation. Finally, confirmation of cell type was obtained by scanning electron microscopy and by the use of specific markers for hair cells.


RESULTS
Once the optimal culture conditions were established in the initial experiments, the formation of new hair cells was directly visualized in all unstained live cultures and fixed preparations without exception. Asymmetric division of progenitor cells, with subsequent differentiation of one of the daughter cells into new hair cells, also was documented by photomicroscopy. Approximately 12% of the cells were labeled with BrdU, of which 6% were hair cells, showing that new hair cell formation was subsequent to mitotic division in vitro. The identity of newly formed hair cells was verified as follows: 1) morphologically by scanning electron microscopy; 2) by positive labeling with phalloidin-rhodamine, a marker for actin; and 3) by positive calmodulin immunocytochemistry.


CONCLUSIONS
This study reports the development of an in vitro culture preparation in which undifferentiated epithelial cells proliferate to become new hair cells. Evidence is provided of division of hair cell progenitors and subsequent differentiation of the daughter cells as one of the mechanisms involved in new hair cell formation in the culture preparation. This newly developed cell culture technique provides a powerful tool for further study of the process of hair cell formation in the vestibular end organ.
Cristobal
R
R
Victor Goodhill Ear Center, Division of Head and Neck Surgery, University of California, Los Angeles School of Medicine, 90095-1624, USA.
Lopez
I
I
Chiang
S
S
Honrubia
D
D
Zamora
C
C
Espinosa de los Monteros
A
A
Micevych
P
P
Honrubia
V
V
D000818:Animals; D018929:Cell Culture Techniques; D002465:Cell Movement / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology; D006198:Hair Cells, Auditory / Q000166:cytology*; D006801:Humans; D007150:Immunohistochemistry; D011892:Rana catesbeiana / Q000502:physiology; D012038:Regeneration; D014722:Vestibule, Labyrinth / Q000166:cytology*