Isolation and Characterization of Mammalian Otic Progenitor Cells that Can Differentiate into Both Sensory Epithelial and Neuronal Cell Lineages.

The mammalian inner ear mediates hearing and balance and during development generates both cochleo-vestibular ganglion neurons and sensory epithelial receptor cells, that is, hair cells and support cells. Cell marking experiments have shown that both hair cells and support cells can originate from a common progenitor. Here, we demonstrate the lineage potential of individual otic epithelial cell clones using three cell lines established by a combination of limiting dilution and gene-marking techniques from an embryonic day 12 (E12) rat otocyst. Cell-type specific marker analyses of these clonal lines under proliferation and differentiation culture conditions demonstrate that during differentiation immature cell markers (Nanog and Nestin) were downregulated and hair cell (Myosin VIIa and Math1), support cell (p27Kip1 and cytokeratin) and neuronal cell (NF-H and NeuroD) markers were upregulated. Our results suggest that the otic epithelium of the E12 mammalian inner ear possess multipotent progenitor cells able to generate cell types of both sensory epithelial and neural cell lineages when cultured under a differentiation culture condition. Understanding the molecular mechanisms of proliferation and differentiation of multipotent otic progenitor cells may provide insights that could contribute to the development of a novel cell therapy with a potential to initiate or stimulate the sensorineural repair of damaged inner ear sensory receptors. Anat Rec, 303:451-460, 2020. © 2019 American Association for Anatomy.

n-Propyl gallate activates hypoxia-inducible factor 1 by modulating intracellular oxygen-sensing systems.

HIF-1 (hypoxia-inducible factor 1) is a master regulator of cellular adaptive responses to hypoxia. The expression and transcriptional activity of the HIF-1alpha subunit is stringently controlled by intracellular oxygen tension through the action of prolyl and asparaginyl hydroxylases. In the present study we demonstrate that PG (n-propyl gallate) activates HIF-1 and expression of its downstream target genes under normoxic conditions in cultured cells and in mice. The stability and transcriptional activity of HIF-1alpha are increased by PG. PG treatment inhibits the interaction between HIF-1alpha and VHL (von Hippel-Lindau protein) and promotes the interaction between HIF-1alpha and p300, indicating that PG inhibits the activity of both prolyl and asparaginyl HIF-1alpha hydroxylases. We conclude that PG activates HIF-1 and enhances the resultant gene expression by directly affecting the intracellular oxygen sensing system in vitro and in vivo and that PG represents a lead compound for the development of a non-toxic activator of HIF-1.

alpha-Klotho as a regulator of calcium homeostasis.

alpha-klotho was identified as a gene associated with premature aging-like phenotypes characterized by short lifespan. In mice, we found the molecular association of alpha-Klotho (alpha-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 alpha-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 alpha-Kl in the regulation of calcium metabolism.

Autocorrelogram sorting: a novel method for evaluating negative-feedback regulation of spike firing.

Spontaneously firing units were recorded extracellularly from the hippocampus of anesthetized rats, and autocorrelograms were conventionally constructed. These conventional autocorrelograms were sorted into frequency-specific autocorrelograms according to the instantaneous firing frequency of the spike at Deltat=0, which was calculated based on the interval between the spike at Deltat=0 and the preceding spike. In this fashion, we found that autocorrelogram values were negatively correlated with instantaneous firing frequency during the 4-12 ms post-spike time period. The negative correlation during the 4-6 ms post-spike period could not have been due to the refractory period or GABAergic inhibition, and thus represented a third type of feedback regulation of spike firing completed within single neurons. Application of acetylcholine significantly enhanced this feedback regulation. Our 'autocorrelogram sorting' method thus proved to be successful in detecting cholinergically enhanced feedback regulation of spike firing intrinsic to single neurons.

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.