Abnormalities in alternative splicing are a hallmark of cancer formation. In this study, we investigated the role of the splicing factor PHD finger protein 5A (PHF5A) in melanoma. Malignant melanoma is the deadliest form of skin cancer, and patients with a high PHF5A expression show poor overall survival. Our data revealed that an siRNA-mediated downregulation of PHF5A in different melanoma cell lines leads to massive splicing defects of different tumour-relevant genes. The loss of PHF5A results in an increased rate of apoptosis by triggering Fas- and unfolded protein response (UPR)-mediated apoptosis pathways in melanoma cells. These findings are tumour-specific because we did not observe this regulation in fibroblasts. Our study identifies a crucial role of PHF5A as driver for melanoma malignancy and the described underlying splicing network provides an interesting basis for the development of new therapeutic targets for this aggressive form of skin cancer.
The extracellular microenvironment encompasses the extracellular matrix, neighbouring cells, cytokines, and fluid components. Anomalies in the microenvironment can trigger aging and a decreased differentiation capacity in mesenchymal stem cells (MSCs). MSCs can perceive variations in the firmness of the extracellular matrix and respond by regulating mitochondrial function. Diminished mitochondrial function is intricately linked to cellular aging, and studies have shown that mitochondria-lysosome contacts (M-L contacts) can regulate mitochondrial function to sustain cellular equilibrium. Nonetheless, the influence of M-L contacts on MSC aging under varying matrix stiffness remains unclear. In this study, utilizing single-cell RNA sequencing and atomic force microscopy, we further demonstrate that reduced matrix stiffness in older individuals leads to MSC aging and subsequent decline in osteogenic ability. Mechanistically, augmented M-L contacts under low matrix stiffness exacerbate MSC aging by escalating mitochondrial oxidative stress and peripheral division. Moreover, under soft matrix stiffness, cytoskeleton reorganization facilitates rapid movement of lysosomes. The M-L contacts inhibitor ML282 ameliorates MSC aging by reinstating mitochondrial network and function. Overall, our findings confirm that MSC aging is instigated by disruption of the mitochondrial network and function induced by matrix stiffness, while also elucidating the potential mechanism by which M-L Contact regulates mitochondrial homeostasis. Crucially, this presents promise for cellular anti-aging strategies centred on mitochondria, particularly in the realm of stem cell therapy.
Osteoporosis, a condition marked by the deterioration of bone microarchitecture and increased facture risk, arises from a disruption in bone metabolism, with osteoclasts surpassing osteoblasts in bone resorption versus formation. The Wnt signalling pathway, a key regulator of bone maintenance, remains partially understood in osteoporosis. Our research delves into the role of Wnt-related molecules in this disease. In osteoporotic adipose-derived stem cells (OP-ASCs), we detected a significant decrease in Ctnnb1 and Frizzled-6 (Fzd6), contrasted by an increase in Gsk-3β and Wnt5a. Activation of the Wnt pathway by LiCl resulted in elevated Ctnnb1 and Fzd6, but decreased Gsk-3β and Wnt5a levels, promoting OP-ASCs’ bone-formation capacity. In contrast, inhibition of this pathway by DKK-1 led to diminished Ctnnb1 and Fzd6, and increased Gsk-3β and Wnt5a, adversely affecting osteogenesis. Furthermore, our findings show that overexpressing Wnt5a impedes, while silencing it enhances the bone-forming capability of OP-ASCs. In a cranial bone defect model, the implantation of Wnt5a-silenced OP-ASCs with biphasic calcium phosphate scaffolds significantly promoted new bone formation. These observations indicated a repression of the canonical Wnt pathway and a stimulation of the non-canonical pathway in OP-ASCs. Silencing Wnt5a increased the osteogenic and regenerative abilities of OP-ASCs. Our study suggests targeting Wnt5a could be a promising strategy for enhancing bone regeneration in post-menopausal osteoporosis.
The coronavirus disease 2019 (COVID-19) pandemic increases the risk of adverse fetal outcomes during pregnancy. Maternal infection during pregnancy, particularly with cytomegalovirus (CMV), hepatitis B and C virus, and human immunodeficiency virus can have detrimental effects on both mother and fetus, potentially leading to adverse outcomes such as spontaneous abortion or neonatal infection. However, the impact of severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection on the maternal–fetal interface remains poorly understood. In this study, we initially utilised immunofluorescence and immunohistochemical to investigate placental samples from pregnant women who were infected with SARS-CoV-2 during the first trimester. Our data indicate that infection in the first trimester induces an upregulation of hypoxia inducible factor (HIF) levels at the maternal–fetal interface. Subsequently, single-cell RNA sequencing and metabolomics sequencing analyses reveal alterations in maternal–fetal interface. Remarkably, immune cells exhibited low expression levels of HIF possibly associated with immune activation. Furthermore, our findings demonstrate a gradual reduction in transcriptome and metabolic changes as gestation progressed beyond 12–16 weeks compared to samples obtained at 6–8 weeks gestation. Overall, our study suggests that early-stage SARS-CoV-2 infection during the first trimester leads to severe hypoxia and aberrant cell metabolism at the maternal–fetal interface which gradually resolves as pregnancy progresses. Nevertheless, these abnormal changes may have long-term implications for maternal–fetal interface development.
Transmembrane protein 184b (Tmem184b) has been implicated in axon degeneration and neuromuscular junction dysfunction. Notably, Tmem184b exhibits high expression levels in the retina; however, its specific function within this tissue remains poorly understood. To elucidate the role of Tmem184b in the mammalian visual system, we developed a Tmem184b knockout (KO) model for further investigation. Loss of Tmem184b led to significant decreases in both a and b wave amplitudes of scotopic electroretinogram (ERG) and reduced b wave amplitudes of photopic ERG, respectively, reflecting damage to both the photoreceptors and secondary neuronal cells of the retina. Histologic analyses showed a progressive retinal thinning accompanied by the significantly loss of retinal cells including cone, rod, bipolar, horizontal and retinal ganglion cells. The expression levels of photo-transduction-related proteins were down-regulated in KO retina. TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated Uridine-5’-triphosphate [UTP] nick end labelling) and glial fibrillary acidic protein (GFAP)-labelling results suggested the increased cell death and inflammation in the KO mice. RNA-sequencing analysis and GO enrichment analysis revealed that Tmem184b deletion resulted in down-regulated genes involved in various biological processes such as visual perception, response to hypoxia, regulation of transmembrane transporter activity. Taken together, our study revealed essential roles of Tmem184b in the mammalian retina and confirmed the underlying mechanisms including cell death, inflammation and hypoxia pathway in the absence of Tmem184b, providing a potential target for therapeutic and diagnostic development.
Pathological observations show that cancer cells frequently invade the surrounding normal tissue in collective rather than individual cell migration. However, general principles governing collective cell migration remain to be discovered. Different from individual cell migration, we demonstrated that the Notch-1-activation reduced collective cells speed and distances. In particular, Notch-1-activation induced cellular cytoskeletal remodelling, strengthened the intercellular junctions and cell-matrix adhesions. Mechanistically, Notch-1 activation prevented the phosphorylation of GSK-3β and the translocation of cytoplasmic free β-catenin to the nucleus, which increased E-cadherin expression and tight intercellular junctions. Moreover, Notch-1 signalling also activated the RhoA/ROCK pathway, promoting reorganization of F-actin and contractile forces produced by myosin. Further, Notch-1 activation increased cell adhesion to the extracellular substrate, which inhibited collective cell migration. These findings highlight that cell adhesions and cell–cell junctions contribute to collective cell migration and provide new insights into mechanisms of the modulation of Notch-1 signalling pathway on cancer cell malignancy.
The transition from fetal primordial germ cells (PGCs) to spermatogonia (SPG) is critical for male germ cell development; however, the detailed transcriptomic dynamics and regulation underlying this transition remain poorly understood. Here by interrogating the comprehensive transcriptome atlas dataset of mouse male germ cells and gonadal cells development, we elucidated the regulatory networks underlying this transition. Our single-cell transcriptome analysis revealed that the transition from PGCs to SPG was characterized by global hypertranscription. A total of 315 highly active regulators were identified to be potentially involved in this transition, among which a non-transcription factor (TF) regulator TAGLN2 was validated to be essential for spermatogonial stem cells (SSCs) maintenance and differentiation. Metabolism profiling analysis also revealed dynamic changes in metabolism-related gene expression during PGC to SPG transition. Furthermore, we uncovered that intricate cell–cell communication exerted potential functions in the regulation of hypertranscription in germ cells by collaborating with stage-specific active regulators. Collectively, our work extends the understanding of molecular mechanisms underlying male germ cell development, offering insights into the recapitulation of germ cell generation in vitro.
How to improve the neurogenic potential of mesenchymal stem cells (MSCs) and develop biological agent based on the underlying epigenetic mechanism remains a challenge. Here, we investigated the effect of histone demethylase Lysine (K)-specific demethylase 2B (KDM2B) on neurogenic differentiation and nerve injury repair by using MSCs from dental apical papilla (SCAP). We found that KDM2B promoted the neurogenic indicators expression and neural spheres formation in SCAP, and modified the Histone H3K4 trimethylation (H3K4me3) methylation on neurogenesis-related genes. KDM2B improved the SCAP mediated recovery of motor ability at the early healing stage of spinal cord injury rats. Meanwhile, KDM2B acted as a negative regulator to its partner EZH2 during neurogenic differentiation, enhancer of zeste homologue 2 (EZH2) suppressed the neurogenic ability of SCAP. Further, the protein interaction between KDM2B and EZH2 was identified which decreased during neurogenic differentiation. On this basis, we revealed seven key protein binding sequences of KDM2B to EZH2, and synthesized KDM2B-peptides based on these sequences. By the usage of KDM2B-peptides, EZH2 function was effectively intervened and the neurogenic ability of SCAP was promoted. More, KDM2B-peptides significantly improved the SCAP mediated functional recovery at SCI early phase. Our study revealed that KDM2B acted as a promotor to neurogenic differentiation ability of dental MSCs through binding and negatively regulating EZH2, and provided the KDM2B-peptides as candidate agents for improving the neurogenic ability of MSCs and nerve injury repair.
Intestinal stem cells differentiate into absorptive enterocytes, characterised by increased brush border enzymes such as intestinal alkaline phosphatase (IAP), making up the majority (95%) of the terminally differentiated cells in the villus. Loss of integrity of the intestinal epithelium plays a key role in inflammatory diseases and gastrointestinal infection. Here, we show that the intestinal microRNA (miR)-27a-3p is an important regulator of intestinal epithelial cell proliferation and enterocyte differentiation. Repression of endogenous miR-27a-3p leads to increased enterocyte differentiation and decreased intestinal epithelial cell proliferation in mouse and human small intestinal organoids. Mechanistically, miR-27a-3p regulates intestinal cell differentiation and proliferation at least in part through the regulation of retinoic acid receptor α (RXRα), a modulator of Wnt/β-catenin signalling. Repression of miR-27a-3p increases the expression of RXRα and concomitantly, decreases the expression of active β-catenin and cyclin D1. In contrast, overexpression of miR-27a-3p mimic decreases the expression of RXRα and increases the expression of active β-catenin and cyclin D1. Moreover, overexpression of the miR-27a-3p mimic results in impaired enterocyte differentiation and increases intestinal epithelial cell proliferation. These alterations were attenuated or blocked by Wnt inhibition. Our study demonstrates an miR-27a-3p/RXRα/Wnt/β-catenin pathway that is important for the maintenance of enterocyte homeostasis in the small intestine.
Mucosal healing is critical to maintain and restore intestinal homeostasis in inflammation. Previous data provide evidence that glial cell line-derived neurotrophic factor (GDNF) restores epithelial integrity by largely undefined mechanisms. Here, we assessed the role of GDNF for mucosal healing. In dextran sodium sulphate (DSS)-induced colitis in mice application of GDNF enhanced recovery as revealed by reduced disease activity index and histological inflammation scores. In biopsy-based wounding experiments GDNF application in mice improved healing of the intestinal mucosa. GDNF-induced epithelial recovery was also evident in wound assays from intestinal organoids and Caco2 cells. These observations were accompanied by an increased number of Ki67-positive cells in vivo after GDNF treatment, which were present along elongated proliferative areas within the crypts. In addition, the intestinal stem cell marker and R-spondin receptor LGR5 was significantly upregulated following GDNF treatment in all experimental models. The effects of GDNF on cell proliferation, LGR5 and Ki67 upregulation were blocked using the RET-specific inhibitor BLU-667. Downstream of RET-phosphorylation, activation of Src kinase was involved to mediate GDNF effects. GDNF promotes intestinal wound healing by promoting cell proliferation. This is mediated by RET-dependent activation of Src kinase with consecutive LGR5 upregulation, indicating activation of the stem cell niche.
Breast cancer is associated with high morbidity and mortality, which are closely influenced by protein post-translational modifications (PTMs). Lysine crotonylation (Kcr) serves as a newly identified PTM type that plays a role in various biological processes; however, its involvement in breast cancer progression remains unclear. Minichromosome maintenance 6 (MCM6) is a critical component of DNA replication and has been previous confirmed to exhibit a significant role in tumorigenesis. Despite this, a comprehensive analysis of MCM6, particularly regarding its modifications in breast cancer is lacking. In this study, we found MCM6 is upregulated in breast invasive carcinoma (BRCA) and is associated with poorer overall survival by regulating the DNA damage repair mechanisms. Furthermore, MCM6-knockdown resulted in decreased cell proliferation and inhibited the DNA replication, leading to DNA replication stress and sustained DNA damage, thereby enhancing the chemotherapeutic sensitivity of breast cancer. Additionally, SIRT7-mediated crotonylation of MCM6 at K599 (MCM6-K599cr) was significantly upregulated in response to DNA replication stress, primarily due to the disassemebly of the MCM2-7 complex and regulated by RNF8-mediated ubiquitination. Concurrently, kaempferol, which acts as a regulator of SIRT7, was found to enhance the Kcr level of MCM6, reducing tumour weight, particular when combined with paclitaxel, highlighting its potential chemotherapeutic target for BRCA therapy.
As major somatic cells in the testis, Sertoli cell development is precisely regulated by numerous factors, and aberrant development of these cells is associated with male reproductive diseases. JNK signalling is evolutionarily conserved and involved in multiple critical biological processes. Here, we found that the double knockout of Jnk1 and Jnk2 resulted in aberrant localisation of Sertoli cells at early developmental stages, with most Sertoli cells being lost at later stages. Further studies revealed that the inactivation of JNK signalling caused polarity loss in Sertoli cells. In vitro-cultured Jnk1/2-DKO Sertoli cells exhibited a senescence-associated phenotype. Mechanistic studies demonstrate that JNK signalling is likely involved in establishing Sertoli cell polarity by regulating the expression of TGF-β2, mediated by c-Jun. The senescence of Sertoli cells in JNKs-deficient mice is caused by aberrant proteolysis of P27KIP1, mediated by c-Myc. This study demonstrates the role of JNK signalling in Sertoli cell development and functional maintenance, which may also represent an aetiology of male infertility in humans.
Extraembryonic mesoderm cells (EXMCs) are involved in the development of multiple embryonic lineages and umbilical cord formation, where they subsequently develop into mesenchymal stem cells (MSCs). Although EXMCs can be generated from human naïve embryonic stem cells (ESCs), it is unclear whether human primed ESCs (hpESCs) can differentiate into EXMCs that subsequently produce MSCs. The present report described a three-dimensional differentiation protocol to induce hpESCs into EXMCs by activating the Wnt pathway using CHIR99021. Single-cell transcriptome and immunostaining analyses revealed that the EXMC characteristics were similar to those of post-implantation embryonic EXMCs. Cell sorting was used to purify and expand the EXMCs. Importantly, these EXMCs secreted extracellular matrix proteins, including COL3A1 and differentiated into MSCs. Inconsistent with other MSC types, these MSCs exhibited a strong differentiation potential for chondrogenic and osteogenic cells and lacked adipocyte differentiation. Together, these findings provided a protocol to generate EXMCs and subsequent MSCs from hpESCs.
As crucial phagocytes of the innate immune system, macrophages (Mϕs) protect mammalian hosts, maintain tissue homeostasis and influence disease pathogenesis. Nonetheless, Mϕs are susceptible to various pathogens, including bacteria, viruses and parasites, which cause various infectious diseases, necessitating a deeper understanding of pathogen–Mϕ interactions and therapeutic insights. Pluripotent stem cells (PSCs) have been efficiently differentiated into PSC-derived Mϕs (PSCdMϕs) resembling primary Mϕs, advancing the modelling and cell therapy of infectious diseases. However, the mass production of PSCdMϕs, which lack proliferative capacity, relies on large-scale expansions of PSCs, thereby increasing both costs and culture cycles. Notably, Mϕs deficient in the MafB/c-Maf genes have been reported to re-enter the cell cycle with the stimulation of specific growth factor cocktails, turning into self-renewing Mϕs (SRMϕs). This review summarizes the applications of PSCdMϕs in the modelling and cell therapy of infectious diseases and strategies for establishing SRMϕs. Most importantly, we innovatively propose that PSCs can serve as a gene editing platform to creating PSC-derived SRMϕs (termed PSRMϕs), addressing the resistance of Mϕs against genetic manipulation. We discuss the challenges and possible solutions in creating PSRMϕs. In conclusion, this review provides novel insights into the development of physiologically relevant and expandable Mϕ models, highlighting the enormous potential of PSRMϕs as a promising avenue for the modelling and cell therapy of infectious diseases.
The developing human foetal brain is sensitive to thermal stimulation during pregnancy. However, the mechanisms by which heat exposure affects human foetal brain development remain unclear, largely due to the lack of appropriate research models for studying thermal stimulation. To address this, we have developed a periodic heating model based on brain organoids derived from human pluripotent stem cells. The model recapitulated neurodevelopmental disruptions under prenatal heat exposure at the early stages, providing a paradigm for studying the altered neurodevelopment under environmental stimulation. Our study found that periodic heat exposure led to decreased size and impaired neural tube development in the brain organoids. Bulk RNA-seq analysis revealed that the abnormal WNT signalling pathway and the reduction of G2/M progenitor cells might be involved in heat stimulation. Further investigation revealed increased neural differentiation and decreased proliferation under heat stimulation, indicating that periodic heat exposure might lead to abnormal brain development by altering key developmental processes. Hence, our model of periodically heating brain organoids provides a platform for modelling the effects of maternal fever on foetal brain development and could be extended to applications in neurodevelopmental disorders intervention.