Spiral ganglia neurons (SGNs) impairment can cause deafness. One important therapeutic approach involves utilizing stem cells to restore impaired auditory circuitry. Nevertheless, the inadequate implementation of research methodologies poses a challenge in accurately assessing the functionality of derived cells within the circuit. Here, we describe a novel method for converting human embryonic stem cells (hESCs) into otic neurons (ONs) and assess their functional connectivity using an optogenetic approach with cells or an organotypic slice of rat cochlear nucleus (CN) in coculture. Embryonic stem cell-derived otic neurons (eONs) exhibited SGN marker expression and generated functional synaptic connection when cocultured with cochlear nucleus neurons (CNNs). Synapsin 1 and VGLUT expression are found in the cochlear nucleus of brain slices, where eONs projected processes during the coculture of eONs and CN brain slices. Action potential spikes and INa+/IK+ of CNNs increased in tandem with light stimulations to eONs. These findings provide further evidence that eONs may be a candidate source to treat SGN-deafness.

OBJECTIVE: Meibomian glands (MGs) are crucial for maintaining tear film stability and ocular surface health. Here, we aim to establish a novel organotypic culture model of MGs and explore the risk factors of MG dysfunction (MGD).
METHODS: We developed a novel organotypic culture model for MGs at the air-liquid interface. The viability and cell proliferation of MGs were assessed using CCK-8, immunofluorescence, and qPCR. Lipid accumulation was evaluated by Nile red staining and microscopic examination. Protein expression levels were evaluated by immunofluorescence and Western blot assay. EdU assay was employed to track the proliferation of acinar cells. The validity of the model was confirmed through culturing MGs from mice of different ages and incorporating certain drugs (Dex) into the culture system.
RESULTS: Utilizing the novel culture model, the MG tissue exhibited sustained viability, cellular division, and continuous production of lipids for a duration of 7 days. Lipid droplets formed were directly visualized using light field microscopy. Through the cultivation of aged mice\'s MGs, it was discovered that aging resulted in diminished proliferation and lipid synthesis, along with an aberrant increase in Krt10 expression. Further application of this model showed that Dex treatment diminished MG\'s proliferation and lipid synthesis. Finally, an in vivo study was conducted to provide additional confirmation of the phenomenon of Dex-induced abnormalities.
CONCLUSIONS: In this study, a stable organotypic culture model of the MGs was established. The organotypic culture model offers a valuable tool to investigate the pathophysiological mechanisms and facilitate drug screening for MG-related diseases.

Meibomian gland dysfunction (MGD) is a group of disorders linked by functional abnormalities of the meibomian glands. Current studies on MGD pathogenesis focus on meibomian gland cells, providing information on a single cell\'s response to experimental manipulation, and do not maintain the architecture of an intact meibomian gland acinus and the acinar epithelial cells\' secretion state in vivo. In this study, rat meibomian gland explants were cultured by a Transwell chamber-assisted method under an air-liquid interface (airlift) in vitro for 96 h. Analyses for tissue viability, histology, biomarker expression, and lipid accumulation were performed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and TUNEL assays, hematoxylin and eosin (H&E) staining, immunofluorescence, Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), transmission electron microscopy (TEM), and western blotting (WB). MTT, TUNEL, and H&E staining indicated better tissue viability and morphology than the submerged conditions used in previous studies. Levels of MGD biomarkers, including keratin 1 (KRT1) and 14 (KRT14) and peroxisome proliferator-activated receptor-gamma (PPAR-γ), along with oxidative stress markers, including reactive oxygen species, malondialdehyde, and 4-hydroxy-2-nonenal, gradually increased over culture time. The MGD pathophysiological changes and biomarker expression of meibomian gland explants cultured under airlift conditions were similar to those reported by previous studies, indicating that abnormal acinar cell differentiation and glandular epithelial cell hyperkeratosis may contribute to obstructive MGD occurrence.

Cisplatin is a platinum-containing drug with ototoxicity commonly used clinically and has significant efficacy against a variety of solid tumors. One of the most important mechanisms of ototoxicity is that cisplatin induces apoptosis of hair cells. According to relevant literature, X-linked inhibitor of apoptosis protein (XIAP, anti-apoptotic protein) could inhibit the apoptotic pathway. We hypothesized that this protein might protect cochlear hair cells from cisplatin-induced injury. To figure it out, we treated cochlea of normal mice with various concentrations of cisplatin to observe the response and morphology of hair cells and determine a reasonable concentration. Next, Western Blot and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) experiments were conducted to make an investigation about the expression of XIAP protein and mRNA. In addition, we constructed and identified XIAP overexpressing mice. Finally, we treated cochlear tissues of normal and overexpressing mice with cisplatin to investigate the cyto-protection of XIAP on hair cells, respectively. It was found that 50 μmol/L cisplatin resulted in significant loss and disorganization of hair cells, while simultaneously downregulating the protein and mRNA of XIAP. In XIAP overexpressing mice, the loss and disorganization of hair cells were significantly lessened. These results showed that XIAP can lessen cisplatin-induced hair cell loss and play a role in otoprotection.

Epidemiological evidence has shown that smoking is associated with an increased risk of hearing loss. However, the underlying mechanisms regarding the impact of nicotine on the cochlea remain unclear. This study aimed to investigate the cytotoxic effects of nicotine on cochlear cells using cultured cochlear basilar membranes. Cochlear basilar membranes were isolated from newborn rats, cultured, and treated with 1-100 ng/mL nicotine for 48 h. Cuticular plates and stereocilia bundle staining were used to evaluate hair cell (HC) loss. Spiral ganglion neuron and acoustic nerve fiber staining were assessed to evaluate cochlear neural injury. Scanning electron microscopy and transmission electron microscopy imaging were employed to examine cochlear ultrastructural changes. Our results showed that compared to spiral ganglia and nerve fibers, HCs are more susceptible to nicotine-induced toxicity. HC loss was more severe in the basal turn than in the middle and apical turns, while nerve fibers and spiral ganglion cells were morphologically maintained. Ultrastructural changes revealed disordered and damaged stereocilia, swelling and decreased mitochondrial density, swelling, and degranulation of the endoplasmic reticulum. Our results suggest that nicotine causes HCs\' degeneration and loss and may have implications for smoking-related hearing loss.

Skeletal muscle decellularization allows the generation of natural scaffolds that retain the extracellular matrix (ECM) mechanical integrity, biological activity, and three-dimensional (3D) architecture of the native tissue. Recent reports showed that in vivo implantation of decellularized muscles supports muscle regeneration in volumetric muscle loss models, including nervous system and neuromuscular junctional homing. Since the nervous system plays pivotal roles during skeletal muscle regeneration and in tissue homeostasis, support of reinnervation is a crucial aspect to be considered. However, the effect of decellularized muscles on reinnervation and on neuronal axon growth has been poorly investigated. Here, we characterized residual protein composition of decellularized muscles by mass spectrometry and we show that scaffolds preserve structural proteins of the ECM of both skeletal muscle and peripheral nervous system. To investigate whether decellularized scaffolds could per se attract neural axons, organotypic sections of spinal cord were cultured three dimensionally in vitro, in presence or in absence of decellularized muscles. We found that neural axons extended from the spinal cord are attracted by the decellularized muscles and penetrate inside the scaffolds upon 3D coculture. These results demonstrate that decellularized scaffolds possess intrinsic neurotrophic properties, supporting their potential use for the treatment of clinical cases where extensive functional regeneration of the muscle is required.

Investigating the pathophysiological mechanisms underlying brain disorders is a priority if novel therapeutic strategies are to be developed. In vivo studies of animal models and in vitro studies of cell lines/primary cell cultures may provide useful tools to study certain aspects of brain disorders. However, discrepancies among these studies or unsuccessful translation from animal/cell studies to human/clinical studies often occur, because these models generally represent only some symptoms of a neuropsychiatric disorder rather than the complete disorder. Human brain slice cultures from postmortem tissue or resected tissue from operations have shown that, in vitro, neurons and glia can stay alive for long periods of time, while their morphological and physiological characteristics, and their ability to respond to experimental manipulations are maintained. Human brain slices can thus provide a close representation of neuronal networks in vivo, be a valuable tool for investigation of the basis of neuropsychiatric disorders, and provide a platform for the evaluation of novel pharmacological treatments of human brain diseases. A brain bank needs to provide the necessary infrastructure to bring together donors, hospitals, and researchers who want to investigate human brain slices in cultures of clinically and neuropathologically well-documented material.

Human mesenchymal stromal stem cells (hMSCs) hold regenerative medicine potential due to their availability, in vitro expansion readiness, and autologous feasibility. For neural repair, hMSCs show translational value in research on stroke, spinal cord injury (SCI), and traumatic brain injury. It is pivotal to establish multimodal in vitro systems to investigate molecular mechanisms underlying neural actions of hMSCs. Here, we describe a platform protocol on how to set up organotypic co-cultures of hMSCs (alone or polymer-scaffolded) with explanted adult rat dorsal root ganglia (DRGs) to determine neural injury and recovery events for designing implants to counteract neurotrauma sequelae. We emphasize in vitro hMSC propagation, polymer scaffolding, hMSC stemness maintenance, hMSC-DRG interaction profiling, and analytical formulas of neuroinflammation, trophic factor expression, DRG neurite outgrowth and tropic tracking, and in vivo verification of tailored implants in rodent models of SCI. © 2018 by John Wiley & Sons, Inc.

OBJECTIVE: A challenge in studying human salivary glands is to maintain the cells ex vivo in their three-dimensional (3D) morphology with an intact native extracellular matrix (ECM) environment. This paper established a human salivary 3D organotypic slice culture model that could maintain its physiological functions as well as allowing a direct visualization of the cells.
METHODS: Human salivary biopsies from six patients were embedded in agarose and submerged in cold buffer for thin (50 μm) sectioning using a vibratome. \'Salivary slices\' were mechanically supported by a porous membrane insert that allowed an air-liquid interface and cultured in serum-free culture media. Cell viability, proliferation, apoptosis, physiological functions, and gene expression were assessed during 14 days of culture.
RESULTS: Human salivary slices maintained cell survival (70-40%) and proliferation (6-17%) for 14 days ex vivo. The protein secretory (amylase) function decreased, but fluid (intracellular calcium mobilization) function was maintained. Acinar, ductal, and myoepithelial cell populations survived and maintained their 3D organization within the slice culture model.
CONCLUSIONS: The human salivary slice culture model kept cells alive ex vivo for 14 days as well as maintaining their 3D morphology and physiological functions.

BACKGROUND: Human amniotic epithelial cells (hAECs) is a desirable reserve of stem cells. Human de-epidermized dermis (DED) retains basic tissue structure and parts of the basement membrane (BM) components at the acelluIar dermal surface, and provides a potential tool for skin regeneration.
OBJECTIVE: To evaluate the potential role of hAECs in skin regeneration, we used DED to perform organotypic culture of hAECs to develop organotypic skin.
METHODS: HAECs were isolated and cultured. Biological characteristics of hAECs were determined by immunocytochemistry and flow cytometry. To prepare DED, the epidermis was removed and then repeated freeze-thaw cycles. HAECs and fibroblast were seeded onto DED to perform the submerged culture for 3 days and then to be maintained at the air-liquid interface for 14 days to form organotypic culture. To identify whether the obtained DED retain the BM structure and components, the histological characteristics of DED and the BM were detected by immunohistochemistry. To evaluate whether the organotypic skin has similar histological characteristics with normal human skin, the marks of epidermal proliferation and differentiation and basement membrane component were detected by immunohistochemistry. Moreover, cell ultrastructure, cell-cell contact and ultrastructure of BM were examined under the transmission electron microscopy.
RESULTS: HAECs has stem-cell characteristics with strong pluripotent Oct-4 and embryonic marker SSEA-4 expression. DED has effectively cleansed the cell components and continuous distributions of laminin and collagen IV. The histological appearance of tissue-engineered skin in vitro has 4 to 9 continuous layers of stratified epithelium and is similar to normal human skin in morphology. Immunohistochemical studies revealed that proliferation and differentiation markers such as Ki67, CK19, CK14, CK10, filaggrin but not CK18 expressed similar pattern characteristics to normal human epidermis. In addition, Periodic acid-Schiff stain showed that a uniform red staining strip located at the epidermal-dermal junction. BM component proteins (type IV collagen and laminin) and cell adhesion protein (desmoglein) were detected by immunohistochemistry in organotypic skin. Ultrastructurally, desmosomes, hemidesmosomes and BM zone (BMZ) were observed in organotypic skin.
CONCLUSIONS: Our studies indicate that the hAECs is a promising stem cell source for tissue-engineered skin, and DED with hAECs is a potential application prospects in regenerative medicine.