The genetic correction of induced pluripotent stem cells (iPSCs) induced from somatic cells of patients with sensorineural hearing loss (caused by hereditary factors) is a promising method for its treatment. The correction of gene mutations in iPSCs could restore the normal function of cells and provide a rich source of cells for transplantation. In the present study, iPSCs were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G>A and c.4118C>T; P-iPSCs), the asymptomatic father of the patient (MYO7A c.1184G>A mutation; CF-iPSCs), and a normal donor (MYO7A WT/WT ; C-iPSCs). One of MYO7A mutation sites (c.4118C>T) in the P-iPSCs was corrected using CRISPR/Cas9. The corrected iPSCs (CP-iPSCs) retained cell pluripotency and normal karyotypes. Hair cell-like cells induced from CP-iPSCs showed restored organization of stereocilia-like protrusions; moreover, the electrophysiological function of these cells was similar to that of cells induced from C-iPSCs and CF-iPSCs. These results might facilitate the development of iPSC-based gene therapy for genetic disorders. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:561-571 SIGNIFICANCEInduced pluripotent stem cells (iPSCs) were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G.A and c.4118C.T). One of the MYO7A mutation sites (c.4118C.T) in the iPSCs was corrected using CRISPR/Cas9. The genetic correction of MYO7A mutation resulted in morphologic and functional recovery of hair cell-like cells derived from iPSCs. These findings confirm the hypothesis that MYO7A plays an important role in the assembly of stereocilia into stereociliary bundles. Thus, the present study might provide further insight into the pathogenesis of sensorineural hearing loss and facilitate the development of therapeutic strategies against monogenic disease through the genetic repair of patient-specific iPSCs.

Peripheral nerve injury causes neuropathic pain and microglia activation. P2Y12 receptors on microglia are thought to be a key player in the surveillance of the local environment, but whether or how these receptors are engaged in the cross-talk between microglia and neurons of the dorsal horn remain ambiguous. Using a rodent model of nerve injury-induced pain, we investigated the roles of P2Y12 in microglia activation, excitatory synaptic transmission, and nociceptive allodynia. We found that spinal nerve ligation (SNL) significantly increased the level of P2Y12 receptors specifically in the microglia of the ipsilateral dorsal horn. Injections of P2Y12 antagonists (MRS2395 or clopidogrel) attenuated microglia activation and increased the paw withdrawal latency in response to thermal stimuli on the ipsilateral side without affecting the basal threshold on the contralateral side. These effects on pain behaviors were replicated in P2Y12 knockout mice. Patch-clamp recordings further revealed that partial sciatic nerve ligation (PSNL)-induced excessive miniature excitatory postsynaptic currents (mEPSCs) were significantly attenuated in P2Y12 knockout mice. Moreover, we found that SNL activates the GTP-RhoA/ROCK2 signaling pathway and elevates the level of phosphorylated p38 mitogen-activated protein kinase (MAPK), which was inhibited by the P2Y12 antagonist. The phosphorylation of p38 MAPK was inhibited by a ROCK inhibitor, but not vice versa, suggesting that p38 MAPK is downstream of ROCK activation. Our findings suggest that nerve injury engages the P2Y12 receptor-dependent GTP-RhoA/ROCK2 signaling pathway to upregulate excitatory synaptic transmission in the dorsal horn. This cross-talk ultimately participates in the manifestation of nociceptive allodynia, implicating P2Y12 receptor as a potential target for alleviating neuropathic pain.

Zebrafish (Danio rerio) is an important model organism in hearing research. However, data on the hearing sensitivity of zebrafish vary across different reports. In the present study, the hearing sensitivity of zebrafish was examined by analysing the auditory evoked potentials (AEPs) over a range of total lengths (TLs) from 12 to 46 mm. Morphological changes in the hair cells (HCs) of the saccule (the main auditory end organ) and their synapses with primary auditory neurons were investigated. The AEPs were detected up to a much higher frequency limit (12 kHz) than previously reported. No significant difference in the frequency response range was observed across the TL range examined. However, the AEP thresholds demonstrated both developmental improvement and age-related loss of hearing sensitivity. The changes in hearing sensitivity were roughly consistent with the morphological changes in the saccule including (1) the number and density of HCs, (2) the organization of stereocilia, and (3) the quantity of a main ribbon protein, Ribeye b. The results of this study established a clear baseline for the hearing ability of zebrafish and revealed that the changes in the saccule contribute to the observed changes in TL (age)-related hearing sensitivity.