摘要: Human midbrain organoids with functional dopaminergic (DA) neurons are invaluable for the therapeutic development of Parkinson's disease (PD). However, current methods face significant limitations, including challenges in generating pint-sized organoids enriched with DA neurons and the lack of robust functional assays for efficiently evaluating neural networks over extended periods. Here we present an innovative approach that combines developmental patterning with mechanical cutting to produce small midbrain organoids, with diameters less than 300 mu m, suitable for long-term evaluation, along with a comprehensive functional assay system consisting of calcium transient assay, neurite extension assay, and multielectrode array (MEA) assay. Radial cutting of organoids into four to eight portions according to their sizes at the appropriate developmental stage significantly increases the yield of viable organoids while reducing necrotic cell regions. Using the functional assay system, we demonstrate that DA neurons within the organoids extend long projections, respond to dopamine stimulation, and form neural networks characterised by giant depolarising potential-like events. Our approach supports the generation of midbrain organoids and PD models that can be used for long-term functional testing.

摘要: CTCF plays a vital role in shaping chromatin structure and regulating gene expression. Clinical studies have associated CTCF mutations with congenital developmental abnormalities, including congenital cardiomyopathy. In this study, we investigated the impact of the homozygous CTCF-R567W (CtcfR567W/R567W) mutation on cardiac tissue morphogenesis during mouse embryonic development. Our results reveal significant impairments in heart development, characterised by ventricular muscle trabecular hyperplasia and reduced ventricular cavity sizes. We also observe a marked downregulation of genes involved in sarcomere assembly, calcium ion transport, and mitochondrial function in heart tissues from homozygous mice. Furthermore, the CtcfR567W/R567W mutation disrupts CTCF's interaction with chromatin, resulting in alterations to topologically associating domain (TAD) structure within specific genomic regions and diminishing crucial promoter-enhancer interactions necessary for cardiac development. Additionally, we find that the heterozygous CTCF-R567W (Ctcf+/R567W) mutation significantly compromises cardiac contractility in 8-week-old mice. This study elucidates the mechanism by which the CTCF-R567W mutation hampers cardiac development, underscoring the essential role of CTCF-R567 in embryonic heart development and maturation.

摘要: Current therapeutic drug exploring targeting at myocardial ischemia/reperfusion (I/R) injury is limited due to the lack of humanized cardiac models that resemble myocardial damage and inflammatory response. Herein, we develop ventricular cardiac organoids from human induced pluripotent stem cells (hiPSCs) and simulate I/R injury by hypoxia/reoxygenation (H/R), which results in increased cardiomyocytes apoptosis, elevated oxidative stress, disrupted morphological structure and decreased beat amplitude. RNA-seq reveals a potential role of type I interferon (IFN-I) in this I/R injury model. We then introduce THP-1 cells and reveal inflammatory responses between monocytes/macrophages and H/R-induced ventricular cardiac organoids. Furthermore, we demonstrate Anifrolumab, an FDA approved antagonist of IFN-I receptor, effectively decreases IFN-I secretion and related gene expression, attenuates H/R-induced inflammation and oxidative stress in the co-culture system. This study advances the modelling of myocardial I/R injury with inflammatory response in human cardiac organoids, which provides a reliable platform for preclinical study and drug screening.

摘要: Bone metastasis (BM) is a mortality-related event of late-stage cancer, with non-small cell lung cancer (NSCLC) being a common origin for BM. However, the detailed molecular profiling of the metastatic bone ecosystem is not fully understood, hindering the development of effective therapies for advanced patients. In this study, we examined the cellular heterogeneity between primary tumours and BM from tissues and peripheral blood by single-cell transcriptomic analysis, which was verified using multiplex immunofluorescence staining and public datasets. Our results demonstrate a senescent microenvironment in BM tissues of NSCLC. BM has a significantly higher infiltration of malignant cells with senescent characteristics relative to primary tumours, accompanied by aggravated metastatic properties. The endothelial-mesenchymal transition involved with SOX18 activation is related to the cellular senescence of vascular endothelial cells from BM. CD4Tstr cells, with pronounced stress and senescence states, are preferentially infiltrated in BM, indicating stress-related dysfunction contributing to the immunocompromised environment during tumour metastasis to bone. Moreover, we identify the SPP1 pathway-induced cellular crosstalk among T cells, vascular ECs and malignant cells in BM, which activates SOX18 and deteriorates patient survival. Our findings highlight the roles of cellular senescence in modulating the microenvironment of BM and implicate anti-senescence therapy for advanced NSCLC patients. The NSCLC bone metastases demonstrate a senescent microenvironment characterized by aggravated metastatic properties of cancer cells, vascular ECs undergoing EndMT and increased infiltration of CD4Tstr cells. The SPP1 pathway may induce cellular communication among T cells, vascular ECs and malignant cells, shaping a pro-angiogenic microenvironment that contributes to bone metastasis.image

摘要: The search for effective strategies to target tumour angiogenesis remains a critical goal of cancer research. We present a pioneering approach using alternating electric fields to inhibit tumour angiogenesis and enhance the therapeutic efficacy of bevacizumab. Chicken chorioallantoic membrane, cell viability and in vitro endothelial tube formation assays revealed that electric fields with a frequency of 1000 kHz and an electric intensity of 0.6 V/cm inhibited the growth of vascular endothelial cells and suppressed tumour-induced angiogenesis. In an animal U87MG glioma model, 1000 kHz electric fields inhibited tumour angiogenesis and suppressed tumour growth. As demonstrated by 3D vessel analysis, tumour vasculature in the control group was a stout, interwoven network. However, electric fields transformed it into slim, parallel capillaries that were strictly perpendicular to the electric field direction. This architectural transformation was accompanied by apoptosis of vascular endothelial cells and a notable reduction in tumour vessel number. Additionally, we found that the anti-angiogenesis and tumour-suppression effects of electric fields synergised with bevacizumab. The anti-angiogenic mechanisms of electric fields include disrupting spindle formation during endothelial cell division and downregulating environmental angiogenesis-related cytokines, such as interleukin-6, CXCL-1, 2, 3, 5 and 8, and matrix metalloproteinases. In summary, our findings demonstrate the potential of alternating electric fields (AEFs) as a therapeutic modality to impede angiogenesis and restrain cancer growth. The application of 1000 kHz alternating electric fields effectively suppresses tumour vasculature, transforming it into orderly, parallel straight capillaries. This not only halts the growth of xenograft tumours but also synergistically enhances the therapeutic effects of bevacizumab, offering a promising approach in cancer treatment.image

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摘要: DDB1-Cullin-4-associated factor-2 (DCAF2, also known as DTL or CDT2), a conserved substrate recognition protein of Cullin-RING E3 ligase 4 (CRL4), recognizes and degrades several substrate proteins during the S phase to maintain cell cycle progression and genome stability. Dcaf2 mainly expressed in germ cells of human and mouse. Our study found that Dcaf2 was expressed in mouse spermatogonia and spermatocyte. The depletion of Dcaf2 in germ cells by crossing Dcaf2fl/fl mice with stimulated by retinoic acid gene 8(Stra8)-Cre mice caused a reduction in progenitor spermatogonia and differentiating spermatogonia, eventually leading to the failure of meiosis initiation and male infertility. Further studies showed that depletion of Dcaf2 in germ cells caused abnormal accumulation of the substrate proteins, cyclin-dependent kinase inhibitor 1A (p21) and thymine DNA glycosylase (TDG), decreasing of cell proliferation, increasing of DNA damage and apoptosis. Overexpression of p21 or TDG attenuates proliferation and increases DNA damage and apoptosis in GC-1 cells, which is exacerbated by co-overexpression of p21 and TDG. The findings indicate that DCAF2 maintains the proliferation and differentiation of progenitor spermatogonia by targeting the substrate proteins p21 and TDG during the S phase.

摘要: Nuclear receptor coactive 4 (NCOA4), which functions as a selective cargo receptor, is a critical regulator of the particularly autophagic degradation of ferritin, a process known as ferritinophagy. Mechanistically, NCOA4-mediated ferritinophagy performs an increasingly vital role in the maintenance of intracellular iron homeostasis by promoting ferritin transport and iron release as needed. Ferritinophagy is not only involved in iron-dependent responses but also in the pathogenesis and progression of various human diseases, including metabolism-related, neurodegenerative, cardiovascular and infectious diseases. Therefore, ferritinophagy is of great importance in maintaining cell viability and function and represents a potential therapeutic target. Recent studies indicated that ferritinophagy regulates the signalling pathway associated with ferroptosis, a newly discovered type of cell death characterised by iron-dependent lipid peroxidation. Although accumulating evidence clearly demonstrates the importance of the interplay between dysfunction in iron metabolism and ferroptosis, a deeper understanding of the double-edged sword effect of ferritinophagy in ferroptosis has remained elusive. Details of the mechanisms underlying the ferritinophagy-ferroptosis axis in regulating relevant human diseases remain to be elucidated. In this review, we discuss the latest research findings regarding the mechanisms that regulate the biological function of NCOA4-mediated ferritinophagy and its contribution to the pathophysiology of ferroptosis. The important role of the ferritinophagy-ferroptosis axis in human diseases will be discussed in detail, highlighting the great potential of targeting ferritinophagy in the treatment of diseases. Nuclear receptor coactive 4 (NCOA4), served as a selective cargo receptor, is critically responsible for the mediation of autophagic degradation of ferritin that is defined as ferrinnophagy. Mechanically, NCOA4-mediated ferritinophagy is considered as a pre-requisite for maintenance of intracellular iron homeostasis by promoting ferritin transport and iron release in accordance with requirements. Therefore, ferritinophagy is of great importance in safeguarding the viability and functionality of cells, and might be a potential therapeutic target for various diseases. In this review, we conclude the latest research findings and regulated mechanisms covering the biological function of NCOA4-mediated ferritinophagy and its contribution to the pathophysiology of ferroptosis. Moreover, the pivotal role of ferritinophagy-ferroptosis axis in human diseases will be discussed in details, proposing to attract attention on the great potential of ferritinophagy in the treatment of various diseases. image

摘要: Osteoarthritis (OA) is the most prevalent disorder of synovial joint affecting multiple joints. In the past decade, we have witnessed conceptual switch of OA pathogenesis from a 'wear and tear' disease to a disease affecting entire joint. Extensive studies have been conducted to understand the underlying mechanisms of OA using genetic mouse models and ex vivo joint tissues derived from individuals with OA. These studies revealed that multiple signalling pathways are involved in OA development, including the canonical Wnt/beta-catenin signalling and its interaction with other signalling pathways, such as transforming growth factor beta (TGF-beta), bone morphogenic protein (BMP), Indian Hedgehog (Ihh), nuclear factor kappa B (NF-kappa B), fibroblast growth factor (FGF), and Notch. The identification of signalling interaction and underlying mechanisms are currently underway and the specific molecule(s) and key signalling pathway(s) playing a decisive role in OA development need to be evaluated. This review will focus on recent progresses in understanding of the critical role of Wnt/beta-catenin signalling in OA pathogenesis and interaction of beta-catenin with other pathways, such as TGF-beta, BMP, Notch, Ihh, NF-kappa B, and FGF. Understanding of these novel insights into the interaction of beta-catenin with other pathways and its integration into a complex gene regulatory network during OA development will help us identify the key signalling pathway of OA pathogenesis leading to the discovery of novel therapeutic strategies for OA intervention. The Wnt/beta-catenin signalling pathway plays critical roles in OA pathogenesis. Abnormal beta-catenin signalling in chondrocytes, synovial fibroblast and osteocytes leads to OA-like structural changes, such as progressive cartilage lesion, synovitis and sclerosis of subchondral bone and osteophyte formation.image

摘要: Human granulosa cells in different stages are essential for maintaining normal ovarian function, and granulosa cell defect is the main cause of ovarian dysfunction. To address this problem, it is necessary to induce functional granulosa cells at different stages in vitro. In this study, we established a reprogramming method to induce early- and late-stage granulosa cells with different steroidogenic abilities. We used an AMH-fluorescence-reporter system to screen candidate factors for cellular reprogramming and generated human induced granulosa-like cells (hiGC) by overexpressing FOXL2 and NR5A1. AMH-EGFP(+) hiGC resembled human cumulus cells in transcriptome profiling and secreted high levels of oestrogen and progesterone, similar to late-stage granulosa cells at antral or preovulatory stage. Moreover, we identified CD55 as a cell surface marker that can be used to isolate early-stage granulosa cells. CD55(+) AMH-EGFP(-) hiGC secreted high levels of oestrogen but low levels of progesterone, and their transcriptome profiles were more similar to early-stage granulosa cells. More importantly, CD55(+) hiGC transplantation alleviated polycystic ovary syndrome (PCOS) in a mouse model. Therefore, hiGC provides a cellular model to study the developmental program of human granulosa cells and has potential to treat PCOS.