Summary The mammalian hair follicle arises during embryonic development from coordinated interactions between the epidermis and dermis. It is currently unclear how to recapitulate hair follicle induction in pluripotent stem cell cultures for use in basic research studies or in vitro drug testing. To date, generation of hair follicles in vitro has only been possible using primary cells isolated from embryonic skin, cultured alone or in a co-culture with stem cell-derived cells, combined with in vivo transplantation. Here, we describe the derivation of skin organoids, constituting epidermal and dermal layers, from a homogeneous population of mouse pluripotent stem cells in a 3D culture. We show that skin organoids spontaneously produce de novo hair follicles in a process that mimics normal embryonic hair folliculogenesis. This in vitro model of skin development will be useful for studying mechanisms of hair follicle induction, evaluating hair growth or inhibitory drugs, and modeling skin diseases.

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.

Summary 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.

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.

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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 6 14 lm 2 vs. 66 6 8 lm 2 ; P < 0.05) and 4 mm diameter (70 6 8 lm 2 vs. 50 6 3 lm 2 ; 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 6 3 vs. 31.8 6 3 cells/lg bone; P < 0.05) and 4 mm diameter (27.6 6 1.2 vs. 8.8 6 1.2 cells/lg 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.

Tzeng, C-H., Chen, C-S., Tang, P-C., and Chiu, T-S. 2009. Microsatellite and mitochondrial haplotype differentiation in blue mackerel (Scomber australasicus) from the western North Pacific. – ICES Journal of Marine Science, 66: 816–825. Blue (Scomber australasicus) and chub mackerel (Scomber japonicus) occur sympatrically in the western North Pacific. Blue mackerel were previously classified as a subspecies of S. japonicus based on morphological similarities. The practical management unit for blue mackerel is contentious owing to incomplete resolution by biological analyses. We used rapidly evolving microsatellites and slow-changing mitochondrial cytochrome b (mtCyt-b) markers to examine the phylogeographic relationships of the two species across four major hydrographic regions of the western North Pacific. Genetic variability was high in each hydrographic region and in the putative species unit, and Hardy–Weinberg equilibrium tests confirmed that blue and chub mackerel are separate genetic components. Population genetic and multiple-dimensional scaling analyses of the genotypes indicated clear genetic differences, and phylogenetic analyses of the mtCyt-b haplotypes showed a level of genetic divergence (FST = 0.038, p < 0.001) consistent with separation of the two species. Based on the coalescence theory, the difference in mtCyt-b genes was small, indicating incipient speciation between blue and chub mackerel, with a diversification time of 1.9 million years ago (mya) during the Pleistocene when the East China Sea (ECS) and the South China Sea (SCS) were separated physically. Significant differences between populations of blue mackerel in the ECS and SCS were also found. Our findings confirm that blue mackerel is a valid biological species and that its populations in the ECS and SCS should be considered separate fishery stocks and conservation units for management.

Embryonic stem (ES) cells are pluripotent cells from the inner cell mass (ICM) of the blastocyst. They are capable of differentiating to various cell types, such as neural cells, cardiocytes, hepatic cells, and germ cells. The aim of this study was to establish rabbit ES cell lines as an animal model for human diseases. Blastocysts were collected from New Zealand White rabbits during Days 4 to 5 after breeding. After removal of the mucin coat and the zona pellucida by pronase, the embryos were directly cultured in ES cell medium on mitomycin C-treated mouse embryonic fibroblast (MEF) or STO feeder layers. In Experiment 1, the efficiencies of 2 different feeder layers, MEF and STO, in generating rabbit ES cell lines were compared. Six blastocysts were used for each STO and MEF feeder group. The primary ICM colonies were formed in 67% (4/6) of the cultures on the STO and 83% (5/6) on the MEF. Sixty percent of those primary colonies (3/5) were successfully grown into ES-like cell lines in the MEF feeder group. However, no cell lines were established on the STO feeder. In Experiment 2, whole blastocysts or ICMs isolated by immunosurgery were cultured to establish ES cell lines. A total of 21 blastocysts were recovered from 2 does. Eighteen whole blastocysts and 3 isolated ICMs were cultured on the MEF feeders. Twelve (67%) of the cultured whole blastocysts formed primary ICM colonies, of which 5 (42%) of the cultures continuously propagated and formed ES-like cell lines. In the immunosurgical group, 2 of the 3 isolated ICMs formed primary colonies but only 1 ES-like cell line was established. A total of 9 ES-like cell lines maintained morphological undifferentiation after 14 passages and expressed alkaline phosphatase activity. Seven of the 9 ES-like cells expressed Oct-4 and the stage-specific embryonic antigen-4 (SSEA-4) as detected by immunocytochemical staining. Two cell lines were further induced to differentiate into embryoid bodies in suspension culture. Another 3 cell lines were injected into SCID mice and one of them formed a teratoma. The competence of generating chimeric rabbits and the teratogenicity of the established ES-like cell lines are under evaluation. In conclusion, rabbit ES-like cells were efficiently generated and whole-blastocyst culturing on the MEF feeder appeared to be a preferred method for the isolation and maintenance of rabbit ES-like cell lines.