Ken Kojima

Alpha-Klotho as a regulator of calcium homeostasis

α-klotho was identified as a gene associated with premature aging–like phenotypes characterized by short lifespan. In mice, we found the molecular association of α-Klotho (α-Kl) and Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) and provide evidence for an increase of abundance of Na+,K+-ATPase at the plasma membrane. Low concentrations of extracellular free calcium ([Ca2+]e) rapidly induce regulated parathyroid hormone (PTH) secretion in an α-Kl- and Na+,K+-ATPase–dependent manner. The increased Na+ gradient created by Na+,K+-ATPase activity might drive the transepithelial transport of Ca2+ in cooperation with ion channels and transporters in the choroid plexus and the kidney. Our findings reveal fundamental roles of α-Kl in the regulation of calcium metabolism.

Survival of Neural Stem Cells in the Cochlea

Adult rat hippocampus-derived neural stem cells (NSCs) have been reported to have been successfully grafted in several brain regions. To evaluate the possibility of treatment of sensorineural hearing loss using NSCs, survival of NSCs in the cochlea was estimated. NSCs were grafted into newborn rat cochleas. Within 2-4 weeks of grafting to the cochlea, some NSCs survived in the cochlear cavity. Some of them had adopted the morphologies and positions of hair cells. This suggests that NSCs can adapt to the environment of the cochlea and gives hope for treatment of the damaged cochlea and sensorineural hearing loss.Adult rat hippocampus-derived neural stem cells (NSCs) have been reported to have been successfully grafted in several brain regions. To evaluate the possibility of treatment of sensorineural hearing loss using NSCs, survival of NSCs in the cochlea was estimated. NSCs were grafted into newborn rat cochleas. Within 2-4 weeks of grafting to the cochlea, some NSCs survived in the cochlear cavity. Some of them had adopted the morphologies and positions of hair cells. This suggests that NSCs can adapt to the environment of the cochlea and gives hope for treatment of the damaged cochlea and sensorineural hearing loss.

Transplantation of neural stem cells into the modiolus of mouse cochleae injured by cisplatin.

This study aimed to examine the possibility of restoration of spiral ganglion neurons, which transmit sound stimulation to the brain, by transplantation of fetal neural stem cells (NSCs) into the modiolus of cochleae. Fetal mouse NSCs expressing green fluorescence were injected into the modiolus of cisplatin-treated cochleae of mice. The temporal bones were collected 14 days after transplantation, and provided histological examination. The cell fate of transplants was determined by immunohistochemistry for a neural or glial cell-marker. Histological analysis 2 weeks after transplantation revealed robust survival of transplant-derived cells in the modiolus of the cochlea. NSCs injected in the basal portion of cochleae migrated as far as the apical end of the modiolus Grafted NSCs expressing a neural cell marker were identified, but the majority of grafted NSCs differentiated into glial cells. These findings suggest the possible use of NSCs in cell therapy for restoration of spiral ganglion neurons. However, further treatments are required to increase the number of NSC-derived neurons in the modiolus to realize functional recovery.

Potential of embryonic stem cell-derived neurons for synapse formation with auditory hair cells.

Recent studies have indicated that embryonic stem cells (ESCs) can be a source for the replacement of spiral ganglion neurons (SGNs), auditory primary neurons, and neurite projections from ESC‐derived neurons to auditory sensory epithelia. However, the potential of ESC‐derived neurons for synapse formation with auditory hair cells (HCs) has not been elucidated. The present study therefore aimed to examine the ability of ESC‐derived neurons to form synaptic connections with HCs in vitro. Mouse ESC‐derived neural progenitors expressing enhanced green fluorescence protein (EGFP) were cocultured with explants of cochlea sensory epithelia obtained from postnatal day 3 mice. After a 7‐day culture, neurites of ESC‐derived neurons predominantly elongated toward inner hair cells (IHCs), which play a crucial role in sound transmission to SGNs. Immunohistochemical analyses revealed the expression of synapsin 1 and synaptophysin in the nerve endings of ESC‐derived neurons adjacent to IHCs, indicating the formation of synaptic connections. Transmission electron microscopy demonstrated synaptic contacts between nerve endings of ESC‐derived neurons and IHCs. The present findings show that ESC‐derived neurons can make synaptic connections with IHCs.

Silencing p27 reverses post-mitotic state of supporting cells in neonatal mouse cochleae

The post-natal cochlear mammalian epithelium have no capacity to proliferate in tissue, however, dissociated supporting cells exhibit the ability to divide and trans-differentiate into new hair cells in vitro, with this process found to be correlated with the downregulation of the cyclin-dependent kinase inhibitor p27(kip1). Here we show that knockdown of p27(kip1) with short hairpin RNA-expressing vectors results in the cell-cycle reentry of post-mitotic supporting cells in the post-natal mouse cochleae ex vivo. The p27(kip1)-knockdown cells incorporated BrdU, and then divided into two daughter cells. However, there was also activation of the apoptotic pathway in some supporting cells. These results indicate that the use of RNA interference to target p27(kip1) is an effective strategy for inducing cell-cycle reentry in post-mitotic supporting cells in the post-natal mammalian cochleae, although additional manipulations of the supporting cells are required to achieve hair cell regeneration.

Surgical techniques for cell transplantation into the mouse cochlea.

This study investigated surgical procedures for cell transplantation into the mouse inner ear. Female C57BL/6 mice were used as recipient animals. Fetal mouse neural stem cells expressing green fluorescence were used as donor cells. Two methods, an injection of transplants from the lateral semicircular canal (LSCC) and from the cochlear lateral wall (CLW), were examined. Two weeks after transplantation, the distribution of transplant-derived cells in the cochlea was examined. Effects on auditory function were assessed by measurement of auditory brain stem responses (ABRs). Cochleae receiving cell transplantation from the LSCC exhibited robust survival of transplant-derived cells mainly in the scala vestibuli and scala tympani. Transplantation from the LSCC caused elevation of ABR thresholds by less than 10 dB SPL. However, transplantation from the CLW resulted in considerable hearing loss, even though transplant-derived cells settled in the scala media. These findings demonstrate that an approach from the LSCC can be utilized for cell transplantation into the perilymph without causing apparent auditory disorder, while an approach from the CLW delivers cells to the endolymph but appears to cause auditory dysfunction.

Survival of fetal rat otocyst cells grafted into the damaged inner ear

Hair cell loss induced by aging, ototoxic drugs and noise leads to irreversible hearing loss and balance disorders in mammals due to the failure of hair cells to regenerate. To investigate the possibility of transplantation therapy to repair damaged inner ear, we have examined whether grafted fetal otocyst cells could survive and migrate into injured sensory organs. We obtained otocyst cells from green fluorescein protein (GFP)-transgenic rats on embryonic day 12.5, then transplanted these cells into the inner ears of young rats previously exposed to intense sound. One month after transplantation, the grafted inner ear sensory organs were examined immunohistochemically. Grafted otocyst cells had survived and demonstrated special morphological features in the host organs; cells that migrated into the organ of Corti were similar to supporting cells. These results indicate that injured sensory organs express some kind of scaffolding that plays important roles in the survival and differentiation of the grafted otocyst cells

Generation of inner ear hair cell immunophenotypes from neurospheres obtained from fetal rat central nervous system in vitro.

Neural stem cells are suggested to possess a highly plastic ability to differentiate into several specific cell types, not only neuronal lineages but also other germ layer tissue-specific cell lineages. To examine whether hair cell immunophenotypes could be derived from the central nervous system (CNS), we established cell cultures from embryonic day 16.5 fetal rat brain tissues, and analyzed changes in immunohistochemical features of the CNS cell cultures by induction of differentiation. The results of this study showed that neural progenitors obtained from fetal rat CNS generated hair cell immunophenotypes with expression of both epitopes of hair cell marker proteins Brn-3c and myosin VIIa in vitro. These findings indicate that immature neural progenitors possess the potential to differentiate into hair cell phenotypes. Immature neural progenitors may be useful as materials for cell transplantation therapy for replacement of damaged inner ear hair cells.

Selective vulnerability of adult cochlear nucleus neurons to de-afferentation by mechanical compression

It is well established that the cochlear nucleus (CN) of developing species is susceptible to loss of synaptic connections from the auditory periphery. Less information is known about how de-afferentation affects the adult auditory system. We investigated the effects of de-afferentation to the adult CN by mechanical compression. This experimental model is quantifiable and highly reproducible. Five weeks after mechanical compression to the axons of the auditory neurons, the total number of neurons in the CN was evaluated using un-biased stereological methods. A region-specific degeneration of neurons in the dorsal cochlear nucleus (DCN) and posteroventral cochlear nucleus (PVCN) by 50% was found. Degeneration of neurons in the anteroventral cochlear nucleus (AVCN) was not found. An imbalance between excitatory and inhibitory synaptic transmission after de-afferentation may have played a crucial role in the development of neuronal cell demise in the CN. The occurrence of a region-specific loss of adult CN neurons illustrates the importance of evaluating all regions of the CN to investigate the effects of de-afferentation. Thus, this experimental model may be promising to obtain not only the basic knowledge on auditory nerve/CN degeneration but also the information relevant to the application of cochlear or auditory brainstem implants.

Nestin expression in the developing Rat cochlea sensory epithelia

An intermediate filament (IF), nestin, is used as an immature cell marker because nestin occurs in neural progenitors during early development. Recent cell culture studies have indicated that proliferating otic progenitor cells express nestin in vitro. However, localization of nestin in the developing inner ear has not yet been clarified. In this study, the ontogenetical expression of nestin epitopes in the rat cochlea was examined immunohistochemically. Sensory epithelial cells in the rat Corti organ (e.g. hair cells and support cells) transiently demonstrated immunoreactivity for nestin during the late embryonic period. After birth, nestin expression in the sensory epithelia disappeared gradually. The findings of this study indicate that the expression of nestin epitopes in the developing cochlea is linked with the plasticities of sensory epithelial cells, such as proliferation or differentiation.