Recent reports on ES cell differentiation have suggested the possibility that information on in vivo neurogenesis might be systematically linked to stem cell technology (3, 4). However, it remains to be known whether ES cell-derived neural precursors generated in vitro can produce the full dorsal-ventral range of neuroectodermal derivatives in response to embryonic positional information. To address this question, we have tested in this study whether SDIA-treated ES cells have the competence of differentiating into the dorsal-(neural crest) and ventralmost (floor plate) cells under embryologically relevant conditions. Materials and MethodsCell Culture and Treatment with Patterning Factors. Mouse ES cells (EB5), primate ES cells (cynomolgus monkey-derived; purchased from Asahi Technoglass, Funabashi, Japan), and PA6 cells were maintained and used for induction as described (1, 2, 5). Human bone morphogenetic protein (BMP)4 and mouse were purchased from R&D Systems and freshly added at each medium change. The day on which ES cells are seeded on PA6 is defined as day 0.Immunocytochemistry, Statistics, and RT-PCR. Cells were fixed with 4% paraformaldehyde, and immunostaining was performed with secondary antibodies conjugated with FITC, cy3, or cy5. For statistics, Ϸ100 colonies were observed in each experiment, and three or more experiments were performed. P values for statistical significance (t test) are described in the corresponding figure legends. The values shown in graphs represent the mean Ϯ SD. RT-PCR was performed with ES cell colonies detached from feeder cells as described (1). The primary antibodies and primers used are described in Supporting Materials and Methods, which are published as supporting information on the PNAS web site, www.pnas.org.Colony Isolation and Axon Guidance Assays. The 3D collagen gel assay for axon guidance was performed by using isolated ES cell colonies (day 8; ref. 1) and the cerebellar plate region excised from embryonic day 13 Wistar rats as a responder (6). Results Positional Identity of Neural Tissues Induced from ES Cells by SDIA.We first examined the expression of rostral-caudal CNS markers by RT-PCR (Fig. 1A). SDIA-treated mouse ES cells express the forebrain markers Otx2 and Six3, the ventral diencephalon marker Rx, the ventral forebrain marker Nkx2.1, the midbrainhindbrain border marker En2, and the hindbrain marker Gbx2, but not the spinal cord markers Hoxb4 and b9 (lane 2). These results show that a majority of neural cells induced by SDIA express rostral neural markers. This idea is consistent with our previous report that dopaminergic neurons generated by the SDIA method are those of the midbrain type (1).We next attempted to alter the rostral-caudal identity of SDIA-induced neural cells by the caudalizing factor retinoic acid (RA; ref. 7). RA treatment (0.2 M all-trans RA; Fig. 1 A, lane 3) suppressed the forebrain markers Otx2, Six3, Rx, and Nkx2.1, whereas it induced the hindbrain marker Gbx2 and the spinal cord markers Hoxb4 and b9. RA treatment did not sign...

Cochlear hair cells convert sound vibration into electrical potential, and loss of these cells diminishes auditory function. In response to mechanical stimuli, piezoelectric materials generate electricity, suggesting that they could be used in place of hair cells to create an artificial cochlear epithelium. Here, we report that a piezoelectric membrane generated electrical potentials in response to sound stimuli that were able to induce auditory brainstem responses in deafened guinea pigs, indicating its capacity to mimic basilar membrane function. In addition, sound stimuli were transmitted through the external auditory canal to a piezoelectric membrane implanted in the cochlea, inducing it to vibrate. The application of sound to the middle ear ossicle induced voltage output from the implanted piezoelectric membrane. These findings establish the fundamental principles for the development of hearing devices using piezoelectric materials, although there are many problems to be overcome before practical application.cochlear implant | hearing loss | mechanoelectrical transduction | traveling wave | regeneration T he cochlea is responsible for auditory signal transduction in the auditory system. It responds to sound-induced vibrations and converts these mechanical signals into electrical impulses, which stimulate the auditory primary neurons. The human cochlea operates over a three-decade frequency band from 20 Hz to 20 kHz, covers a 120-dB dynamic range, and can distinguish tones that differ by <0.5% in frequency (1). It is relatively small, occupying a volume of <1 cm 3 , and it requires ∼14 μW power to function (2). The mammalian ear is composed of three parts: the outer, middle, and inner ears (Fig. 1A) (3). The outer ear collects sound and funnels it through the external auditory canal to the tympanic membrane. The cochlea consists of three compartments: scala vestibuli and scala tympani, which are filled with perilymph fluid, and scala media, which is filled with endolymph fluid (Fig. 1C). The scala vestibuli and scala tympani form a continuous duct that opens onto the middle ear through the oval and round windows. The stapes, an ossicle in the middle ear, is directly coupled to the oval window. Sound vibration is transmitted from the ossicles to the cochlear fluids through the oval window as a pressure wave that travels from the base to the apex of the scala vestibuli through the scala tympani and finally to the round window (Fig. 1B). The scala media are membranous ducts that are separated from the scala vestibuli by Reissner's membrane and separated from the scala tympani by the basilar membrane. The pressure wave propagated by the vibration of the stapes footplate causes oscillatory motion of the basilar membrane, where the organ of Corti is located. The organ of Corti contains the sensory cells of the auditory system; they are known as hair cells, because tufts of stereocilia protrude from their apical surfaces (Fig. 1D). The oscillatory motion of the basilar membrane results in the shear motion of the st...

This study aimed to evaluate the potential of embryonic stem cell-derived neural progenitors for use as transplants for the replacement of the auditory primary neurons, spiral ganglion neurons. Mouse embryonic stem cell-derived neural progenitors were implanted into the base of the cochlear modiolus of normal or deafened guinea pigs, which contains spiral ganglion neurons and cochlear nerve fibers. Histological analysis demonstrated the survival and neural differentiation of transplants in the cochlear modiolus and active neurite outgrowth of transplants toward host peripheral or central auditory systems. Functional assessments indicated the potential of transplanted embryonic stem cell-derived neural progenitors to elicit the functional recovery of damaged cochleae. These findings support the hypothesis that transplantation of embryonic stem cell-derived neural progenitors can contribute to the functional restoration of spiral ganglion neurons.

BackgroundSudden sensorineural hearing loss (SSHL) is a common condition in which patients lose the hearing in one ear within 3 days. Systemic glucocorticoid treatments have been used as standard therapy for SSHL; however, about 20% of patients do not respond. We tested the safety and efficacy of topical insulin-like growth factor 1 (IGF1) application using gelatin hydrogels as a treatment for SSHL.MethodsPatients with SSHL that showed no recovery to systemic glucocorticoid administration were recruited. We applied gelatin hydrogels, impregnated with recombinant human IGF1, into the middle ear. The primary outcome measure was the proportion of patients showing hearing improvement 12 weeks after the test treatment. The secondary outcome measures were the proportion of patients showing improvement at 24 weeks and the incidence of adverse events. The null hypothesis was that 33% of patients would show hearing improvement, as was reported for a historical control after hyperbaric oxygen therapy.ResultsIn total, 25 patients received the test treatment at a median of 23 days (range 15-32) after the onset of SSHL, between 2007 and 2009. At 12 weeks after the test treatment, 48% (95% CI 28% to 69%; P = 0.086) of patients showed hearing improvement, and the proportion increased to 56% (95% CI 35% to 76%; P = 0.015) at 24 weeks. No serious adverse events were observed.ConclusionsTopical IGF1 application using gelatin hydrogels is well tolerated and may be efficacious for hearing recovery in patients with SSHL that is resistant to systemic glucocorticoids.