Isolation, growth and differentiation of hair cell progenitors from the newborn rat cochlear greater epithelial ridge.

Mammalian cochlear hair cell loss is irreversible and leads to permanent hearing loss. To restore hearing physiologically, it is necessary to generate new functional hair cells either from endogenous cells or from exogenously transplanted hair cells/progenitors. Previous studies suggest that cochlear greater epithelial ridge (GER) and lesser epithelial ridge (LER) cells are capable of differentiating into hair cells. While it was recently possible to obtain and culture pure LER progenitors, isolation of pure GER progenitors has not been reported. Here we describe a method that allows isolation of pure GER cells from neonatal rat cochleae. The cochlear epithelial sheet (CES) containing GER progenitor cells was mechanically separated from the underlying mesenchymal tissue after digestion with thermolysin. The GER area could then be dissected following mechanical removal of organ of Corti as well as all the lateral area. The isolated GER cells showed significant proliferation and expressed markers for GER cells but not markers for hair cells or LER. When the GER cells were cultured in serum-free medium containing epidermal growth factor, spheres were formed where they continued to proliferate. Furthermore, when GER cells were induced to express Hath1 or co-cultured with mesenchymal cells prepared from neonate rat cochleae, they showed the potential to differentiate into hair cell-like cells. Successful isolation, culture and differentiation of GER hair cell progenitors will shed additional light on the mechanism of hair cell differentiation and potential hair cell replacement.

[Effects of noise on antioxidant enzymes of cochlea in guinea pigs].

OBJECTIVE: To investigate the effect of noise on the antioxidant enzymes of cochleae. METHODS: 16 male pigmented guinea pigs (250 - 300 g) were randomly divided into 2 groups, control group and noise group. Each group had 8 animals. The animals in noise group were performed auditory evoked brainstem responses (ABR) recording before and after exposure to a continuous noise (4 kHz, octave band, 100 dB, SPL) 8 h/d for 3 consecutive days. Immediately at the end of the third day's noise exposure after ABR recording, guinea pigs were decapitated. Both the right and the left cochlea with the bony capsule removed were homogenized, and the supernatants were prepared for assays. Reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were measured. RESULTS: ROS level of the noise group [(281.2 +/- 3.5) U/mg pro] was significantly higher than that of the control group [(273.0 +/- 3.2) U/mg pro, P < 0.05] and SOD, CAT and GSH-Px activities of the noise group [(206.5 +/- 5.1) NU/mg pro, (47.0 +/- 9.0) U/g pro, (14.1 +/- 2.5) U/mg pro respectively] were significantly lower than that of the control group [(221.8 +/- 4.8) NU/mg pro, (60.8 +/- 9.9) U/g pro, (21.1 +/- 3.1) U/mg pro respectively, P < 0.05]. CONCLUSION: Noise may damage the defensive system of antioxidant enzymes in cochlea.

Chondrocyte-specific Smad4 gene conditional knockout results in hearing loss and inner ear malformation in mice.

Smad4 is the central intracellular mediator of transforming growth factor-beta (TGF-beta) signaling, which plays crucial roles in tissue regeneration, cell differentiation, embryonic development, and regulation of the immune system. Conventional Smad4 gene knockout results in embryonic lethality, precluding its use in studies of the role of Smad4 in inner ear development. We used chondrocyte-specific Smad4 knockout mice (Smad4Co/Co) to investigate the function of Smad4 in inner ear development. Smad4Co/Co mice were characterized by a smaller cochlear volume, bone malformation, and abnormalities of the osseous spiral lamina and basilar membrane. The development of the hair cells was also abnormal, as evidenced by the disorganized stereocilia and reduced density of the neuronal processes beneath the hair cells. Auditory function tests revealed the homozygous Smad4Co/Co mice suffered from severe sensorineural hearing loss. Our results suggest that Smad4 is required for inner ear development and normal auditory function in mammals.

Smad5 haploinsufficiency leads to hair cell and hearing loss.

The Smads are a group of related intracellular proteins critical for transmitting the signals to the nucleus from the transforming growth factor-beta superfamily at the cell surface. Knockout of the Smad5 is embryonic lethal. However, the Smad5 knockout of single allele (+/-) could survive. We used Smad5 heterozygous knockout (+/-) to determine the role of Smad5 in the development of inner ear morphology and function. In situ hybridization showed that Smad5 was expressed predominantly in hair cells, spiral ganglion, and supporting cells. Measurements of hearing thresholds using auditory brainstem response showed that Smad5 defect resulted in progressive hearing loss between 4 and 24 weeks after birth. Morphological examination revealed apoptosis in the inner ear, with significant loss of outer hair cells in adult Smad5 mutant mice. Our results indicated that deficiency in the Smad5-mediated signaling resulted in apoptosis of hair cells, suggesting Smad5 is a gene that may be related with presbycusis.

[Overexpression of Hath1 induces production of hair cell-like cells in greater epithelial ridge cell cultures from postnatal rat cochlea].

OBJECTIVE: To investigate the effect of Hath1 (human atonal homolog 1) overexpression on greater epithelial ridge (GER) cells from postnatal rat cochlea in vitro. METHODS: GER cells were isolated by using a combinatorial approach of enzymatic digestion and mechanical separation from P1 rat cochlear. The GER cell cultures were infected by adenovirus containing Hath1 and enhanced green fluorescent protein (ad-Hath1-EGFP), while transfecting EGFP(ad-EGFP) was as controls. Immunostaining were performed at different time points after adenovirus infection. RESULTS: Some of the infected GER cells became myosin VIIa-positive following ad-Hath1-EGFP infection. The earliest time point to see induction of hair cell differentiation (hair cell marker expression) by ad-Hathl was 5 days post-infection. In contrast, infection of the GER sheet cultures with ad-EGFP control virus did not show any myosin VIIa-positive cells at 3-12 days post-infection in all cultures examined. CONCLUSIONS: GER cells may potentially serve as hair cell progenitors and they are capable of differentiating hair cell-like cells when forced to express Hath1.

[Relevance between clinical behavior and bionomical findings of acoustic neuromas].

OBJECTIVE: To evaluate the relationship between labeling index (LI) Ki-67, proliferating cell nuclear antigen (PCNA) and transforming growth factor-beta1 (TGF-beta1) with the clinical behavior of acoustic neuroma. METHODS: Expression of Ki-67, PCNA and TGF-beta1 was detected by immunohistochemistry in 53 specimens of acoustic neuromas. The relationship among tumor proliferation, histological representation, size of tumor, clinical proliferation index of tumor and tumor proliferation activity were analyzed. RESULTS: In all 53 cases, the positive rate of Ki-67 was 77.4% (41/53) but the positive rate of PCNA was 84.9% (45/53). There was significant difference between the proliferate index, clinic growth rate and course of disease (t = 2.14, t = 2.70; P < 0.05). The positive rate of TGF-beta1 was 83.0% (44/53). The correlation of TGF-beta1 with LI (Ki-67) was significant difference (r = 0.36, P < 0.05). Cystic degeneration often occurred in large-size tumor (Z = 4.44, P < 0.05). There was no significant relationship between the expression of LI (Ki-67), LI (PCNA) and TGF-beta1 and the course of disease as well as between the cystic degeneration and the non-cystic degeneration. Although clinic growth rate of cystic degeneration was bigger than that of non-cystic degeneration, there was not statistically significant. CONCLUSIONS: Ki-67 and PCNA are reflected proliferation activities of tumor cells in acoustic neuromas. Cell proliferation-labeling index LI (PCNA) was related with clinical growth rates. TGF-beta1 might participate in the biological behavior of acoustic neuroma. Cystic degeneration was one of special pattern of acoustic neuroma, however, tumor enlargement might due to the volume of the cystic but unrelated to fast proliferation of parenchyma cell.

[In vitro culture of greater epithelial ridge cells from rat cochleae].

OBJECTIVE: To establish in vitro culture systems of greater epithelial ridge (GER) cells from rat cochlear and to investigate the characterization, growth pattern and ultrastructure of GER cells. METHODS: Using a combinatorial approach of enzymatic digestion and mechanical separation to allow isolation and culture of GER cells from P1 rat cochleae. The dissociated GER cells were cultured in serum-free or 10% fetal bovine serum DMEM respectively. BrdU, phalloidin, ZO1, calretinin and myosin VIIa immunostaining and scanning electron microscope observation were performed in GER cell cultures. RESULTS: The dissociated GER cell cultures showed positive to ZO1, phalloidin and BrdU staining, but negative to myosin VIIa and calretinin. They assumed a polygonal morphology which was similar to epithelial cells and grew in islands-like patches in medium containing 10% fetal bovine serum while forming spheres in serum-free medium. The GER cells presented significant ability to proliferate in both conditions. Scanning electron microscope showed that there was microvillus and centre bodies but not hair cell specific stereociliary bundles on the surface of GER cultures. CONCLUSIONS: The GER cell cultures showed significant ability to proliferate and grew in islands-like patches in medium containing 10% fetal bovine serum while forming spheres in serum-free medium. The dissociated GER cells expressed epithelial cell specific marker but not marker of hair cells.

[Expression of three kinds of transcription factors in greater epithelial ridge cells of rat cochlear].

OBJECTIVE: To detect the expression of Math1, Hes1 and Hes5 in greater epithelial ridge (GER) cells of rat cochlear and explore their influence on hair cell differentiation. METHODS: Postnatal day 0 (P0), day 1 (P1) , day 3 (P3) day 4 (P4) and day 5 (P5) rat cochlear were dissected respectively and then pure GER cells were separated by a combinatorial approach of attachment and mechanical separation. The total RNA of GER cells was extracted by Trizol one step method and the expression of Math1, Hes1 and Hes5 in GER cells was detected with reverse transcription polymerase chain reaction. RESULTS: Math1 was expressed in P0 - P5 rat GER cells and Hes1 was expressed only in PO - P3 rat GER cells, while there was no expression of Hes5 in P0 - P5 rat GER cells. CONCLUSIONS: Probably only when the expression of Math1 reaches a certain level can it induce GER cells to differentiate into hair cells. Meanwhile this process might controlled by Hes1 to some extent.

[Construction of bicistronic eukaryotic vector containing basic fibroblast growth factor and study of their functions in gene therapy for hearing impairment].

OBJECTIVE: To construct basic fibroblast growth factor(bFGR) and enhance green fluorescence protein(EGFP) fusion gene eukaryotic expression vector internal ribosome entry site (pIRES)-bFGF-GFP and to evaluate the effect of transduction bFGF gene on noise induced hearing-loss in inner ear hair cells of guinea pigs. METHODS: Human bFGF cDNA was inserted into mammalian expressed plasmid pIRES-EGFP. The recombinant expression plasmid pIRES-bFGF-EGFP was transfected into inner ear of guinea pigs, using lipofectin method. The transduced bFGF gene was mediated by SA lipidsome. IRES- bFGF-GFP was administered into the round window as rescue agent at the same time of noise exposure or as a protective agent 7 days before. RESULTS: SA liposome-mediated bFGF expressed at a high level in the cochlea of guinea pigs, and in the rescue group, a significant lower hearing thresholds was displayed. pIRES- bFGF-EGFP could protect hair cells. It demonstrated that pIRES- bFGF-GFP could protect the inner ear both structurally and functionally. bFGF/EGFP gene could be transcripted and translated into inner ear hair cells of guinea pig. bFGF/EGFP gene could express a specific protein. The recombinant bFGF/EGFP had significant protective effect as well as manifestation of autonomous fluorescence. CONCLUSION: bFGF/EGFP fusion protein not only expressed in hair cells of guinea pig but also showed significant bFGF activity and autonomous fluorescence. IRES induced exogenous gene could enter the hair cells.