Progress in Modeling and Targeting Inner Ear Disorders with Pluripotent Stem Cells

Sensorineural hearing loss and vestibular dysfunction are caused by damage to neurons and mechanosensitive hair cells, which do not regenerate to any clinically relevant extent in humans. Several protocols have been devised to direct pluripotent stem cells (PSCs) into inner ear hair cells and neurons, which display many properties of their native counterparts. The efficiency, reproducibility, and scalability of these protocols are enhanced by incorporating knowledge of inner ear development. Modeling human diseases in vitro through genetic manipulation of PSCs is already feasible, thereby permitting the elucidation of mechanistic understandings of a wide array of disease etiologies. Early studies on transplantation of PSC-derived otic progenitors have been successful in certain animal models, yet restoration of function and long-term cell survival remain unrealized. Through further research, PSC-based approaches will continue to revolutionize our understanding of inner ear biology and contribute to the development of therapeutic treatments for inner ear disorders

Improved autologous cortical bone harvest and viability with 2Flute otologic burs

Objectives To determine if 2Flute (Stryker Corporation, Kalamazoo, MI) otologic burs improve the size, cellular content, and bone healing of autologous cortical bone grafts harvested during canal wall reconstruction (CWR) tympanomastoidectomy with mastoid obliteration. Study Design Institutional review board-approved prospective cohort study. Methods Human autologous cortical bone chips were harvested using various burs (4 and 6 mm diameter; multiflute, and 2Flute [Stryker Corporation]) from patients undergoing CWR tympanomastoidectomy for the treatment of chronic otitis media with cholesteatoma. Bone chip size, cell counts, cellular gene expression, and new bone formation were quantified. Results Bone chips were significantly larger when harvested with 2Flute (Stryker Corporation) bur compared to multiflute burs at both 6 mm diameter (113 ± 14 μm2 vs. 66 ± 8 μm2; P < 0.05) and 4 mm diameter (70 ± 8 μm2 vs. 50 ± 3 μm2; P < 0.05). After 2 weeks in culture, cell numbers were significantly higher when harvested with 2Flute (Stryker Corporation) bur compared to multiflute burs at both 6 mm diameter (48.7 ± 3 vs. 31.8 ± 3 cells/μg bone; P < 0.05) and 4 mm diameter (27.6 ± 1.2 vs. 8.8 ± 1.2 cells/μg bone; P < 0.05). Bone-derived cells express osteoblast markers (alkaline phosphatase, osteocalcin). Cultured cells are able to form new bone in culture, and bone formation is facilitated by the presence of bone chips. Conclusion Use of 2Flute (Stryker Corporation) otologic burs for human autologous cortical bone harvest results in more viable bone fragments, with larger bone chips and more osteoblasts. Future studies are needed to determine if this leads to improved bone healing

Defective Tmprss3-Associated Hair Cell Degeneration in Inner Ear Organoids

Mutations in the gene encoding the type II transmembrane protease 3 (TMPRSS3) cause human hearing loss, although the underlying mechanisms that result in TMPRSS3-related hearing loss are still unclear. We combined the use of stem cell-derived inner ear organoids with single-cell RNA sequencing to investigate the role of TMPRSS3. Defective Tmprss3 leads to hair cell apoptosis without altering the development of hair cells and the formation of the mechanotransduction apparatus. Prior to degeneration, Tmprss3-KO hair cells demonstrate reduced numbers of BK channels and lower expressions of genes encoding calcium ion-binding proteins, suggesting a disruption in intracellular homeostasis. A proteolytically active TMPRSS3 was detected on cell membranes in addition to ER of cells in inner ear organoids. Our in vitro model recapitulated salient features of genetically associated inner ear abnormalities and will serve as a powerful tool for studying inner ear disorders

Assessing the Impacts of Experimentally Elevated Temperature on the Biological Composition and Molecular Chaperone Gene Expression of a Reef Coral

Due to the potential for increasing ocean temperatures to detrimentally impact reef-building corals, there is an urgent need to better understand not only the coral thermal stress response, but also natural variation in their sub-cellular composition. To address this issue, while simultaneously developing a molecular platform for studying one of the most common Taiwanese reef corals, Seriatopora hystrix, 1,092 cDNA clones were sequenced and characterized. Subsequently, RNA, DNA and protein were extracted sequentially from colonies exposed to elevated (30°C) temperature for 48 hours. From the RNA phase, a heat shock protein-70 (hsp70)-like gene, deemed hsp/c, was identified in the coral host, and expression of this gene was measured with real-time quantitative PCR (qPCR) in both the host anthozoan and endosymbiotic dinoflagellates (genus Symbiodinium). While mRNA levels were not affected by temperature in either member, hsp/c expression was temporally variable in both and co-varied within biopsies. From the DNA phase, host and Symbiodinium hsp/c genome copy proportions (GCPs) were calculated to track changes in the biological composition of the holobiont during the experiment. While there was no temperature effect on either host or Symbiodinium GCP, both demonstrated significant temporal variation. Finally, total soluble protein was responsive to neither temperature nor exposure time, though the protein/DNA ratio varied significantly over time. Collectively, it appears that time, and not temperature, is a more important driver of the variation in these parameters, highlighting the need to consider natural variation in both gene expression and the molecular make-up of coral holobionts when conducting manipulative studies. This represents the first study to survey multiple macromolecules from both compartments of an endosymbiotic organism with methodologies that reflect their dual-compartmental nature, ideally generating a framework for assessing molecular-level changes within corals and other endosymbioses exposed to changes in their environment

Development of Gene Expression Markers of Acute Heat-Light Stress in Reef-Building Corals of the Genus Porites

Coral reefs are declining worldwide due to increased incidence of climate-induced coral bleaching, which will have widespread biodiversity and economic impacts. A simple method to measure the sub-bleaching level of heat-light stress experienced by corals would greatly inform reef management practices by making it possible to assess the distribution of bleaching risks among individual reef sites. Gene expression analysis based on quantitative PCR (qPCR) can be used as a diagnostic tool to determine coral condition in situ. We evaluated the expression of 13 candidate genes during heat-light stress in a common Caribbean coral Porites astreoides, and observed strong and consistent changes in gene expression in two independent experiments. Furthermore, we found that the apparent return to baseline expression levels during a recovery phase was rapid, despite visible signs of colony bleaching. We show that the response to acute heat-light stress in P. astreoides can be monitored by measuring the difference in expression of only two genes: Hsp16 and actin. We demonstrate that this assay discriminates between corals sampled from two field sites experiencing different temperatures. We also show that the assay is applicable to an Indo-Pacific congener, P. lobata, and therefore could potentially be used to diagnose acute heat-light stress on coral reefs worldwide

Cadherin-23 may be dynamic in hair bundles of the model sea anemone Nematostella vectensis.

Cadherin 23 (CDH23), a component of tip links in hair cells of vertebrate animals, is essential to mechanotransduction by hair cells in the inner ear. A homolog of CDH23 occurs in hair bundles of sea anemones. Anemone hair bundles are located on the tentacles where they detect the swimming movements of nearby prey. The anemone CDH23 is predicted to be a large polypeptide featuring a short exoplasmic C-terminal domain that is unique to sea anemones. Experimentally masking this domain with antibodies or mimicking this domain with free peptide rapidly disrupts mechanotransduction and morphology of anemone hair bundles. The loss of normal morphology is accompanied, or followed by a decrease in F-actin in stereocilia of the hair bundles. These effects were observed at very low concentrations of the reagents, 0.1-10 nM, and within minutes of exposure. The results presented herein suggest that: (1) the interaction between CDH23 and molecular partners on stereocilia of hair bundles is dynamic and; (2) the interaction is crucial for normal mechanotransduction and morphology of hair bundles

CDH23 immunocytochemistry and effects of CDH23 antibodies on vibration sensitivity in <i>Nematostella vectensis</i>.

<p>(A) Affinity purified CDH23 antibodies label stereocilia of a hair bundle (HB) with punctate fluorescence. The tip (t) and base (b) of the hair bundle are indicated. The merged image of a hair bundle is shown in transmitted light by oblique contrast (assigned green) and epifluorescence microscopy for CDH23 immunocytochemistry (assigned red). Scale bar  = 2 µm. (B) Vibration sensitivity was evaluated by means of a bioassay based on counting nematocysts discharged into test probes touched to tentacles in the presence of nearby vibrations at a key frequency. Vibration sensitivity was tested at intervals after adding 0.1 nM CDH23 antibodies (final concentration) to the seawater containing intact anemones. The mean number of nematocysts counted per field of view at 400× (±SEM, n = 10–15) is plotted (open circles). Data also are plotted for healthy, vibrating controls (closed squares) and non-vibrating controls (closed diamond) for which the animals were not exposed to exogenously supplied antibodies. (C) Vibration sensitivity was tested at intervals after adding 0.1 nM TRPA1 antibodies (final concentration) as an antibody-loading control. The mean number of nematocysts counted per field of view at 400× (±SEM, n = 5–6) is plotted (open circles). Data also are plotted for healthy, vibrating controls (solid squares). ^aSignificant difference in the mean number of nematocysts discharged between vibrating controls and 0.1 nM CDH23 antibody-treated specimens and ^bsignificant difference between the mean number of nematocysts discharged between the vibrating controls and non-vibrating control (<i>p</i><0.05).</p

CDH23 peptide cytochemistry and effects of CDH23 peptides on vibration sensitivity in <i>Nematostella vectensis</i>.

<p>(A) Fluorescently tagged CDH23 peptides label stereocilia near the base of a hair bundle with punctate fluorescence. The upper micrograph depicts a merged image of a hair bundle shown in transmitted light using oblique contrast (assigned green) and epifluorescence microscopy for CDH23 peptide-FITC cytochemistry (assigned red). The tip (t) and base (b) of the hair bundle (HB) are indicated. The lower micrograph depicts immunocytochemistry for TRPN1. It is intended to show the morphology of stereocilia in the hair bundle. White arrowheads indicate the height of the tips of several small diameter stereocilia. Scale bar  = 2 µm. (B) Vibration sensitivity was tested at intervals after adding 0.1 nM CDH23 peptide (open triangles, final concentration) or 10 nM CDH23 peptide (open circles, final concentration) to the seawater containing intact anemones. The mean number of nematocysts counted per field of view (±SEM, n = 6) is plotted for the experimental animals as well as for untreated, vibrating controls (closed squares) and non-vibrating controls (closed diamond). ^aSignificant difference between mean nematocyst discharge for the 10 nM CDH23 peptide treated animals and untreated, vibrating controls. ^bSignificant difference between mean nematocyst discharge for the 0.1 nM CDH23 peptide treated animals and untreated, vibrating controls. ^cSignificant difference between non-vibrating controls and the other three treatments at time 0. (C) Vibration sensitivity was tested at intervals after adding 10 nM harmonin peptide (open circles, final concentration) as a peptide-loading control. The mean number of nematocysts discharged is plotted (±SEM, n = 6–8) for untreated vibrating controls (closed squares) and for animals exposed to the harmonin peptide (open circles). Asterisks indicate significant differences in mean nematocyst discharge (<i>p</i><0.05).</p

Effects of CDH23 peptides on the mechanoelectric responses induced by deflecting hair bundles.

<p>Hair bundles were deflected by pressure jets delivered by a nearby puffer pipette. Recording pipettes were attached to the plasma membrane adjacent to the base of the hair bundles. The TTL signal depicting the opening and closing the solenoid valve controlling pressure delivered to a puffer pipet is shown above representative traces depicting membrane current transients induced by hair bundle deflection. (A) Recordings are shown for a perfusion control before (left) and after perfusion of potassium-enriched seawater (right). (B) Recordings are shown before (left) and 3 min after the perfusion of potassium-enriched seawater containing 10 nM CDH23 peptide (right). (C) Recordings are shown before (left) and 5 min after the perfusion of potassium-enriched seawater containing 10 nM CDH23 peptide (right). (D) The mean peak membrane current induced by deflecting hair bundles was calculated and plotted for before and after perfusion (±SEM, n = 10). Perfusion controls (empty bar) had perfusion of potassium-enriched seawater only. The CDH23 peptide-treated specimens (hatched bar) were exposed to potassium-enriched seawater containing 10 nM CDH23 peptide (final concentration). The peak membrane current induced by deflecting hair bundles was measured at two sampling times after the perfusion, 3 min and 5 min, in the CDH23 peptide-treated specimens. Asterisks indicate significant differences in mean peak membrane current between before and after perfusion (<i>p</i><0.05).</p

Effects of CDH23 antibodies on morphology and abundance of hair bundles.

<p>Morphology was assayed by measuring diameter of hair bundles at their bases (b) and tips (t) and then determining the tip/base ratio for each hair bundle. Hair bundle abundance was measured by the number of hair bundles at the tip of tentacles in a field of view (∼260 µm of tentacle length). (A) The mean tip/base ratio (±SEM, n = 8) is plotted for hair bundles imaged in untreated, healthy controls (closed squares) and in animals exposed to 0.1 nM CDH23 antibodies (open circles). Insets show images of an extremely splayed hair bundle (top) and a normal hair bundle (bottom). Scale bar  = 2 µm. (B) The mean abundance of hair bundles (±SEM, n = 3) on the tentacle epithelium is plotted for untreated, healthy controls (closed squares) and for animals exposed to 0.1 nM CDH23 antibody (open circles). Asterisks indicate significant differences based on time-adjusted pairwise comparisons between controls and experimentals (<i>p</i><0.05).</p