Sensorineural correlates of failed functional recovery after natural regeneration of vestibular hair cells in adult mice.

Vestibular hair cells (HCs) are mechanoreceptors that sense head motions by modulating the firing rate of vestibular ganglion neurons (VGNs), whose central processes project to vestibular nucleus neurons (VNNs) and cerebellar neurons. We explored vestibular function after HC destruction in adult Pou4f3+/DTR (DTR) mice, in which injections of high-dose (50 ng/g) diphtheria toxin (DT) destroyed most vestibular HCs within 2 weeks. At that time, DTR mice had lost the horizontal vestibulo-ocular reflex (aVORH), and their VNNs failed to upregulate nuclear cFos expression in response to a vestibular stimulus (centrifugation). Five months later, 21 and 14% of HCs were regenerated in utricles and horizontal ampullae, respectively. The vast majority of HCs present were type II. This degree of HC regeneration did not restore the aVORH or centrifugation-evoked cFos expression in VNNs. The failure to regain vestibular pathway function was not due to degeneration of VGNs or VNNs because normal neuron numbers were maintained after HC destruction. Furthermore, sinusoidal galvanic stimulation at the mastoid process evoked cFos protein expression in VNNs, indicating that VGNs were able to regulate VNN activity after HC loss. aVORH and cFos responses in VNNs were robust after low-dose (25 ng/g) DT, which compared to high-dose DT resulted in a similar degree of type II HC death and regeneration but spared more type I HCs in both organs. These findings demonstrate that having more type I HCs is correlated with stronger responses to vestibular stimulation and suggest that regenerating type I HCs may improve vestibular function after HC loss.

A Steadier Hand: The First Human Clinical Trial of a Single-Use Robotic-Assisted Surgical Device for Cochlear Implant Electrode Array Insertion.

OBJECTIVE: To evaluate the safety and utility of an investigational robotic-assisted cochlear implant insertion system. STUDY DESIGN: Prospective, single-arm, open-label study under abbreviated Investigational Device Exemption requirements. SETTING: All procedures were performed, and all data were collected, at a single tertiary referral center. PATIENTS: Twenty-one postlingually deafened adult subjects that met Food and Drug Administration indication criteria for cochlear implantation. INTERVENTION: All patients underwent standard-of-care surgery for unilateral cochlear implantation with the addition of a single-use robotic-assisted insertion device during cochlear electrode insertion. MAIN OUTCOME MEASURES: Successful insertion of cochlear implant electrode array, electrode array insertion time, postoperative implant function. RESULTS: Successful robotic-assisted insertion of lateral wall cochlear implant electrode arrays was achieved in 20 (95.2%) of 21 patients. One insertion was unable to be achieved by either robotic-assisted or manual insertion methods, and the patient was retrospectively found to have a preexisting cochlear fracture. Mean intracochlear electrode array insertion time was 3 minutes 15 seconds. All implants with successful robotic-assisted electrode array insertion (n = 20) had normal impedance and neural response telemetry measures for up to 6 months after surgery. CONCLUSIONS: Here we report the first human trial of a single-use robotic-assisted surgical device for cochlear implant electrode array insertion. This device successfully and safely inserted lateral wall cochlear implant electrode arrays from the three device manufacturers with devices approved but he Food and Drug Administration.

Vesicular Glutamatergic Transmission in Noise-Induced Loss and Repair of Cochlear Ribbon Synapses.

Noise-induced excitotoxicity is thought to depend on glutamate. However, the excitotoxic mechanisms are unknown, and the necessity of glutamate for synapse loss or regeneration is unclear. Despite absence of glutamatergic transmission from cochlear inner hair cells in mice lacking the vesicular glutamate transporter-3 (Vglut3KO ), at 9-11 weeks, approximately half the number of synapses found in Vglut3WT were maintained as postsynaptic AMPA receptors juxtaposed with presynaptic ribbons and voltage-gated calcium channels (CaV1.3). Synapses were larger in Vglut3KO than Vglut3WT In Vglut3WT and Vglut3+/- mice, 8-16 kHz octave-band noise exposure at 100 dB sound pressure level caused a threshold shift (∼40 dB) and a loss of synapses (>50%) at 24 h after exposure. Hearing threshold and synapse number partially recovered by 2 weeks after exposure as ribbons became larger, whereas recovery was significantly better in Vglut3WT Noise exposure at 94 dB sound pressure level caused auditory threshold shifts that fully recovered in 2 weeks, whereas suprathreshold hearing recovered faster in Vglut3WT than Vglut3+/- These results, from mice of both sexes, suggest that spontaneous repair of synapses after noise depends on the level of Vglut3 protein or the level of glutamate release during the recovery period. Noise-induced loss of presynaptic ribbons or postsynaptic AMPA receptors was not observed in Vglut3KO , demonstrating its dependence on vesicular glutamate release. In Vglut3WT and Vglut3+/-, noise exposure caused unpairing of presynaptic ribbons and presynaptic CaV1.3, but not in Vglut3KO where CaV1.3 remained clustered with ribbons at presynaptic active zones. These results suggest that, without glutamate release, noise-induced presynaptic Ca2+ influx was insufficient to disassemble the active zone. However, synapse volume increased by 2 weeks after exposure in Vglut3KO , suggesting glutamate-independent mechanisms.SIGNIFICANCE STATEMENT Hearing depends on glutamatergic transmission mediated by Vglut3, but the role of glutamate in synapse loss and repair is unclear. Here, using mice of both sexes, we show that one copy of the Vglut3 gene is sufficient for noise-induced threshold shift and loss of ribbon synapses, but both copies are required for normal recovery of hearing function and ribbon synapse number. Impairment of the recovery process in mice having only one functional copy suggests that glutamate release may promote synapse regeneration. At least one copy of the Vglut3 gene is necessary for noise-induced synapse loss. Although the excitotoxic mechanism remains unknown, these findings are consistent with the presumption that glutamate is the key mediator of noise-induced synaptopathy.

Pluripotent stem cell derived cardiomyocytes for cardiac repair.

OPINION STATEMENT: The adult mammalian heart has limited capacity for regeneration, and any major injury such as a myocardial infarction results in the permanent loss of up to 1 billion cardiomyocytes. The field of cardiac cell therapy aims to replace these lost contractile units with de novo cardiomyocytes to restore lost systolic function and prevent progression to heart failure. Arguably, the ideal cell for this application is the human cardiomyocyte itself, which can electromechanically couple with host myocardium and contribute active systolic force. Pluripotent stem cells from human embryonic or induced pluripotent lineages are attractive sources for cardiomyocytes, and preclinical investigation of these cells is in progress. Recent work has focused on the efficient generation and purification of cardiomyocytes, tissue engineering efforts, and examining the consequences of cell transplantation from mechanical, vascular, and electrical standpoints. Here we discuss historical and contemporary aspects of pluripotent stem cell-based cardiac cell therapy, with an emphasis on recent preclinical studies with translational goals.

Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

Targeted genomic integration of a selectable floxed dual fluorescence reporter in human embryonic stem cells.

The differentiation of pluripotent stem cells involves transition through a series of specific cell states. To understand these cell fate decisions, the field needs improved genetic tools for the labeling, lineage tracing and selection of specific cell types from heterogeneous differentiating populations, particularly in the human embryonic stem cell (hESC) system. We used zinc finger nuclease technology to stably insert a unique, selectable, floxed dual-fluorescence reporter transgene into the AAVS1 locus of RUES2 hESCs. This "stoplight" transgene, mTmG-2a-Puro, strongly expresses membrane-localized tdTomato red fluorescent protein until Cre-dependent recombination causes a switch to expression of membrane-localized enhanced green fluorescent protein (eGFP) and puromycin resistance. First, to validate this system in undifferentiated cells, we transduced transgenic hESCs with a lentiviral vector driving constitutive expression of Cre and observed the expected phenotypic switch. Next, to demonstrate its utility in lineage-specific selection, we transduced differentiated cultures with a lentiviral vector in which the striated muscle-specific CK7 promoter drives Cre expression. This yielded near-homogenous populations of eGFP(+) hESC-derived cardiomyocytes. The mTmg-2a-Puro hESC line described here represents a useful new tool for both in vitro fate mapping studies and the selection of useful differentiated cell types.