摘要: Degeneration of the cochlear spiral ganglion neurons (SGNs) is one of the major causes of sensorineural hearing loss and significantly impacts the outcomes of cochlear implantation. Functional regeneration of SGNs holds great promise for treating sensorineural hearing loss. In this study, we systematically screened 33 transcriptional regulators implicated in neuronal and SGN fate. Using gene expression array and principal component analyses, we identified a sequential combination of Ascl1, Pou4f1 and Myt1l (APM) in promoting functional reprogramming of SGNs. The neurons induced by APM expressed mature neuronal and SGN lineage-specific markers, displayed mature SGN-like electrophysiological characteristics and exhibited single-cell transcriptomes resembling the endogenous SGNs. Thus, transcription factors APM may serve as novel candidates for direct reprogramming of SGNs and hearing recovery due to SGN damages.

摘要: Collagenase digestion (d) and cellular outgrowth (og) are the current modalities of meniscus fibrochondrocytes (MFC) isolation for bioengineering and mechanobiology-related studies. However, the impact of these modalities on study outcomes is unknown. Here, we show that og- and d-isolated MFC have distinct proliferative capacities, transcriptomic profiles via RNA sequencing (RNAseq), extracellular matrix (ECM)-forming, and migratory capacities. Our data indicate that microtissue pellet models developed from og-isolated MFC display a contractile phenotype with higher expressions of alpha-smooth muscle actin (ACTA2) and transgelin (TAGLN) and are mechanically stiffer than their counterparts from d-MFC. Moreover, we introduce a novel method of MFC isolation designated digestion-after-outgrowth (dog). The transcriptomic profile of dog-MFC is distinct from d- and og-MFC, including a higher expression of mechanosensing caveolae-associated caveolin-1 (CAV1). Additionally, dog-MFC were superior chondrogenically and generated larger-size microtissue pellet models containing a higher frequency of smaller collagen fibre diameters. Thus, we demonstrate that the modalities of MFC isolation influence the downstream outcomes of bioengineering and mechanobiology-related studies.

摘要: The vocal fold is an architecturally complex organ comprising a heterogeneous mixture of various layers of individual epithelial and mesenchymal cell lineages. Here we performed single-cell RNA sequencing profiling of 5836 cells from the vocal folds of adult Sprague-Dawley rats. Combined with immunostaining, we generated a spatial and transcriptional map of the vocal fold cells and characterized the subpopulations of epithelial cells, mesenchymal cells, endothelial cells, and immune cells. We also identified a novel epithelial-to-mesenchymal transition-associated epithelial cell subset that was mainly found in the basal epithelial layers. We further confirmed that this subset acts as intermediate cells with similar genetic features to epithelial-to-mesenchymal transition in head and neck squamous cell carcinoma. Finally, we present the complex intracellular communication network involved homeostasis using CellChat analysis. These studies define the cellular and molecular framework of the biology and pathology of the VF mucosa and reveal the functional importance of developmental pathways in pathological states in cancer. Dimensionality reduction and clustering analysis of sc-RNA seq data from healthy SD rats' vocal folds identified five cell populations. Notably, a Krt15+/Col3a1+ cell type with epithelial-mesenchymal features may play a crucial role in head and neck squamous cell carcinoma.image

摘要: N-6-methyladenosine (m(6)A) exerts essential roles in early embryos, especially in the maternal-to-zygotic transition stage. However, the landscape and roles of RNA m(6)A modification during the transition between pluripotent stem cells and 2-cell-like (2C-like) cells remain elusive. Here, we utilised ultralow-input RNA m(6)A immunoprecipitation to depict the dynamic picture of transcriptome-wide m(6)A modifications during 2C-like transitions. We found that RNA m(6)A modification was preferentially enriched in zygotic genome activation (ZGA) transcripts and MERVL with high expression levels in 2C-like cells. During the exit of the 2C-like state, m(6)A facilitated the silencing of ZGA genes and MERVL. Notably, inhibition of m(6)A methyltransferase METTL3 and m(6)A reader protein IGF2BP2 is capable of significantly delaying 2C-like state exit and expanding 2C-like cells population. Together, our study reveals the critical roles of RNA m(6)A modification in the transition between 2C-like and pluripotent states, facilitating the study of totipotency and cell fate decision in the future.

摘要: The study of neurogenesis is essential to understanding fundamental developmental processes and for the development of cell replacement therapies for central nervous system disorders. Here, we designed an in vivo drug screening protocol in developing zebrafish to find new molecules and signalling pathways regulating neurogenesis in the ventral spinal cord. This unbiased drug screen revealed that 4 cyclooxygenase (COX) inhibitors reduced the generation of serotonergic interneurons in the developing spinal cord. These results fitted very nicely with available single-cell RNAseq data revealing that floor plate cells show differential expression of 1 of the 2 COX2 zebrafish genes (ptgs2a). Indeed, several selective COX2 inhibitors and two different morpholinos against ptgs2a reduced the number of serotonergic neurons in the ventral spinal cord and led to locomotor deficits. Single-cell RNAseq data and different pharmacological manipulations further revealed that COX2-floor plate-derived prostaglandin D-2 promotes neurogenesis in the developing spinal cord by promoting mitotic activity in progenitor cells. Rescue experiments using a phosphodiesterase-4 inhibitor suggest that intracellular changes in cAMP levels underlie the effects of COX inhibitors on neurogenesis and locomotion. Our study provides compelling in vivo evidence showing that prostaglandin signalling promotes neurogenesis in the ventral spinal cord.

摘要: Apical-out intestinal organoids are a relatively simple method of gaining access to the apical cell surface and have faced increasing scientific interest over the last few years. Apical-out organoids can thus be used for disease modelling to compare differing effects on the basolateral versus the apical cell surface. However, these 'inside-out' organoids die relatively quickly and cannot be propagated as long as their basal-out counterparts. Here, we show that apical-out organoids have drastically reduced proliferative potential, as evidenced by immunohistochemical staining and the incorporation of the thymidine analogue EdU. At the same time, cell death levels are increased. Nevertheless, these phenomena cannot be explained by an induction of differentiation, as the gene expression of key marker genes for various cell types does not change over time.

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摘要: Glutaminase-1 (GLS1) has garnered considerable interest as a metabolic target in cancer due to its heightened involvement and activity. However, the precise fate of glutaminolysis catalysed by GLS1 in cancer cells remains elusive. We found that GLS1 knockout led to significant suppression of cancer cell proliferation, which can be reversed or partially restored by supplementation of glutamate or non-essential amino acids that can be converted into glutamate. The addition of spliceosomal KGA or GAC ameliorates cancer cell growth in vitro and in vivo, providing both simultaneously completely reverse the effect. The primary metabolic fate of glutamate produced through glutaminolysis in cancer cells is mainly used to produce glutathione (GSH) for redox homeostasis, not entering the tricarboxylic acid cycle or synthesising nucleotides. GSH monoethyl ester (GSH-MEE) effectively rescues the inhibition of cancer cell proliferation caused by GLS1 knockout. Deletion of GLS1 results in an elevation of reactive oxygen species (ROS) and malondialdehyde (MDA), a reduction of NADPH/NADP+ ratio, and an augmented susceptibility of cells to ferroptosis. Glutathione Peroxidase 4 (GPX4) and GPX1 exhibit complementary roles in redox regulation, with GLS1 knockout promoting GPX4 degradation. Pharmacological inhibition of GLS1 synergises with GPX4 inhibitor to suppress tumour growth. Dual targeting of GPX4 and GPX1 presents a potent anti-cancer strategy. This metabolic mechanism facilitates a deeper comprehension of the abnormal glutamine metabolism in cancer cells, establishing a theoretical basis for the potential clinical utilisation of GLS1 inhibitors and presenting novel perspectives for advancing combinatorial therapeutic approaches.

摘要: Mitochondria perform multiple functions within the cell, including the production of ATP and a great deal of metabolic intermediates, while also contributing to the cellular stress response. The majority of mitochondrial proteins are encoded by nuclear genomes, highlighting the importance of mitonuclear communication for sustaining mitochondrial homeostasis and functional. As a crucial part of the intracellular signalling network, mitochondria can impact stem cell fate determinations. Considering the essential function of stem cells in tissue maintenance, regeneration and aging, it is important to understand how mitochondria influence stem cell fate. This review explores the significant roles of mitonuclear communication and mitochondrial proteostasis, highlighting their influence on stem cells. We also examine how mitonuclear interactions contribute to cellular homeostasis, stem cell therapies, and the potential for extending lifespan.

摘要: G protein-coupled receptor-associated sorting protein 2 (GPRASP2) has been identified as the causative gene for X-linked recessive syndromic hearing loss (SHL) in our previous study. However, the role of GPRASP2 in auditory function remains unclear. The present study demonstrated that Gprasp2 overexpression in mouse organoids promoted the proliferation of supporting cells (SCs), which was mainly mediated by the Hedgehog signalling pathway. Meanwhile, GPRASP2 promoted hair cell (HC) formation from SCs via beta-catenin signalling. In addition, GPRASP2 deficiency resulted in increased lysosomal degradation of SMO protein, leading to decreased expression of beta-catenin and the Hedgehog pathway transcription factor GLI1. In neomycin-treated mouse cochlear explant, the smoothened agonist (SAG) recured the HC loss and further facilitated AAV-ie-Gprasp2 to promote the proliferation of SCs and formation of HCs. Our results suggested that GPRASP2 could be a potential candidate for gene therapy in the regeneration of HCs.