Geneeskundige Stichting Koningin Elisabeth ... - GSKE - FMRE

Cell biology for prevention and treatment of sensori-neural deafness
Malgrange, B. 1 , Lefebvre, P.P. 1 , Belachew, S. 1 , Rigo, J.M. 1 , Nguyen, L. 1 , Van de Water, T.R. 2 ,
Thiry, M. 3 and Moonen, G. 1
1 Centre de Neurobiologie Cellulaire et Moléculaire, Université de Liège, Belgique
2 Albert Einstein College of medicine, New York, USA
3 Service de Biologie Cellulaire, Université de Liège, Belgique
The organ of Corti is a complex structure containing an invariant mosaic of hair cells
(HCs) and supporting cells (SCs). The signals regulating the production if HCs and SCs are
actually not known. During development in mice, the production of these cells is completed by
E16 and the capacity to produce new cells following an injury is limited to this neonatal period.
In order to understand the mechanism underlying this production of new hair cells in the
cochlea, we have studied the production of supernumerary hair cells in late embryonic and
early postnatal rat organ of Corti. We have shown that supernumerary HCs are produced in
the neonatal organ of Corti in culture in response to an incubation in the presence of transforming
growth factor ∃ (TGF∃) or epidermal growth factor (EGF). This neoformation of sensory
cells does not occur through a cell proliferation because of the lack of inhibition by cytosine arabinoside
(arac 10 -5M) of supernumerary cell production. Conversely, when E19 rat organs of
Corti are cultured for 5 days in the presence of arac supernumerary HCs and SCs are observed.
One possible mechanism underlying this supernumerary cell production is the inhibition of
cyclin-dependent kinases (cdks) particularly cdk 1, 2 and 5, which will induced HCs and SCs
differentiation. In order to test this hypothesis, we used specific cdk inhibitors (cdki). Roscovitine
(10 :M) and olomoucine (30 :M), two specific cdk1, 2and 5 inhibitors, induced the production
of supernumerary HCs and SCs. This phenomenon is dose-dependent and reach a maximum for
E19 rat organ of Corti in the developmental study that we have carried out. In the presence of
brdU (10-5M) for 18 hours, no proliferative cell was observed in the sensory cell area,demonstrating
that the HCs and SCs production is independant from an inhibition of cell proliferation.
Another theoretical mechanism which can account for the production of supernumerary hair
cell is the induction or the inhibition of apoptotic cell death. When organs of Corti are cultured
either in control condition or in the presence of cdk inhibitors, no apoptotic nuclei is seen. The
supernumerary HCs and SCs should arise from a direct differentiation of a cell progenitor. This
hypothesis is reinforced by previous findings on other cell types showing that roscovitine has the
ability to induce cell differentiation through cdk2 inhibition (Lee et al., 1999).
In a parallel investigation, we tried to identify HC progenitors. Analysis of semithin sections
has revealed that the supernumerary HCs are produced at the outer edge of the organ of
Corti. Ultrastructural studies show that the tectal cells present a morphology which is intermediate
between that of Hensen’s cell and the HCs, and that Hensen’s cells are very similar to
Deiters’ cells. Quantitative analysis demonstrates that, when the number of HCs increases in the
organ of Corti, 1) the total number of HCs + Deiters’ cells + tectal cells + undertectal cells +
Hensen’s cells remains constant, 2) the number of Deiters’ cells increases, 3) the number of tectal
cells decreases, and 4) the number of Hensen’s cells decreases. Immunolabeling of organ of
Corti’s explants using antibodies specifically directed against a protein expressed early in the
133