Stem cells, both normal somatic (SSC) and cancer stem cells (CSC) exist in minimally two states, i.e., quiescent and activated. Regulation of these two states, including their reliance on different metabolic processes, i.e., FAO and glycolysis in quiescent versus activated stem cells respectively, involves the analysis of a complex array of factors (nutrient and oxygen levels, adhesion molecules, cytokines, etc.) to initiate the epigenetic changes to either depart or enter quiescence. Quiescence is a critical feature of SSC that is required to maintain the genomic integrity of the stem cell pool, particularly in long lived complex organisms. Quiescence in CSC, whether they are derived from mutations arising in SSC, aberrant microenvironmental regulation, or via dedifferentiation of more committed progenitors, is a critical component of therapy resistance and disease latency and relapse. At the beginning of vertebrate evolution, approximately 450 million years ago, a gene duplication generated the two members of the Kat3 family, CREBBP (CBP) and EP300 (p300). Despite their very high degree of homology, these two Kat3 coactivators play critical and non-redundant roles at enhancers and super-enhancers via acetylation of H3K27, thereby controlling stem cell quiescence versus activation and the cells metabolic requirements. In this review/perspective, we discuss the unique regulatory roles of CBP and p300 and how specifically targeting the CBP/β-catenin interaction utilizing small molecule antagonists, can correct lineage infidelity and safely eliminate quiescent CSC.

Takifugu rubripes is an economically valuable fish species in Asia. The implementation of all-male culture for T. rubripes is highly anticipated in aquaculture. Aromatase inhibitor (AI, letrozole) treatment was found to be an efficient method to induced masculinization in T. rubripes, as reported in our previous study. Here, to further explore the underlying regulation mechanism of AI-induced masculinization, a whole-transcriptome analysis comparing was conducted between AI-induced masculinized XX (AI-XX) gonads and control (Con) gonads in T. rubripes.

In Con-XX/Con-XY comparison, 1,172 differential expression (DE) mRNAs, 129 DEmiRNAs, 210 DElncRNAs, and 4 DEcircRNAs were identified. In the Con-XX/AI-XX comparison, 1,329 DEmRNAs, 174 DEmiRNAs, 6 DEcircRNAs and 280 DElncRNAs were found. Con-XX/Con-XY and Con-XX/AI-XX comparisons shared 690 DEmRNAs, 50 DEmiRNAs, 3 DEcircRNAs, and 105 DElncRNAs. The analyses of protein-protein interaction (PPI) and competitive endogenous RNA (ceRNA) network identified interactions among these shared DERNAs. Kcnh2b, trim27, cnnm2b, reln, cckbra, pkd1l2, steap4, gsg1l, hamp, and foxg1c were predicted as the top ten of hub genes. miRNAs included miRNA-27 family and miRNA-489 family showed targeting relationship with hub genes. GO and KEGG functional enrichment analysis showed that the targeted genes were mainly enriched in GO:0065008 regulation of biological quality and TGF-beta signaling pathway. qPCR validation confirmed the differential expression of selected mRNAs, and ncRNAs.

This research comprehensively reveals the potential regulatory effects of ncRNAs on cellular motility, fate regulation, and hormonal regulation during gonadal masculinization in T. rubripes. It may provide significant insights into the regulation mechanisms underlying sex reversal in fish.

Human amniotic epithelial cells (hAECs) have shown excellent efficacy in clinical research and have prospective applications in the treatment of many diseases. However, the properties of the hAECs and their proliferative mechanisms remain unclear. Here, single-cell RNA sequencing (scRNA-seq) is performed on hAECs obtained from amniotic tissues at different gestational ages and passages during in vitro culture. The results showed that the proliferation of hAECs is associated with epithelial-mesenchymal plasticity (EMP) during amniogenesis. Freshly isolated, full-term hAECs are identified as mature epithelial cells. Once cultured in vitro, they are observed to rapidly undergo epithelial-mesenchymal transition (EMT) and enter a partial epithelial-mesenchymal transition (pEMT) state to regain their EMP properties and proliferation capacities. With the continuous development of EMT, hAECs eventually enter a senescent state. The addition of SB431542 and microcarrier screening enabled the effective 3D expansion of hAECs by 50 fold while maintaining the EMP status in hAECs for further proliferation. This study not only elucidated the central proliferation mechanism of hAECs during development and expansion but also optimized the in vitro culture system so that it is sufficient to generate hAECs for 50 patients from a single donor amniotic membrane.

During ocular trauma, excessive proliferation and transdifferentiation of corneal stromal fibroblasts cause haze/fibrosis in the cornea. Transforming growth factor β (TGFβ) plays a key role in corneal fibrosis through the Smad signaling pathway. The aberrant activity of TGFβ signaling during ocular trauma (viz. mechanical, infectious, chemical, or surgically altered TGFβ/Smad signaling) leads to regulating the predominant expression of myogenic proteins and the extracellular matrix (ECM). We sought to investigate the functional role of Smad3 in corneal wound repair and stromal ECM assembly using Smad3+/+ wild-type and Smad3-/- deficient mice.

Corneal injury was introduced with the topical application of an alkali-soaked 2-mm filter disc on the central cornea in the Smad3+/+ (C57BL/6J) and Smad3-/- (129-Smad3tm1Par/J) mouse strains. Slit-lamp and stereo microscopy were used for clinical assessment and corneal haze grading in live animals. Hematoxylin and eosin and Masson's trichrome staining were used to study comparative morphology and collagen level alterations between the groups. Real-time qRT-PCR, western blot, and immunohistochemistry were used to measure changes in profibrotic genes at the mRNA and protein levels.

Slit-lamp clinical exams and stereo microscopy detected notably less opaque cornea in the eyes of Smad3-/- compared with Smad3+/+ mice at 3 weeks (p<0.01) in live animals. Corneal tissue sections of Smad3-/- mice showed significantly fewer α-smooth muscle actin-positive cells compared with those of the Smad3+/+ animals (p<0.05). The corneas of the Smad3-/- mice showed significantly lower mRNA levels of pro-fibrotic genes, α-smooth muscle actin, fibronectin, and collagen I (p<0.05, p<0.01, and p<0.001). In addition, the matrix metalloproteinase and tissue inhibitors of metalloproteinase levels were significantly increased (p<0.001) in the corneal tissue during alkali injury in both Smad3+/+ wild-type and Smad3-/- deficient mice.

The significant changes in profibrotic genes and stromal ECM proteins revealed a direct role of Smad3 in stromal ECM proteins and TGFβ/Smad-driven wound healing. Smad3 appears to be an attractive molecular target for limiting abnormal stroma wound healing to treat corneal fibrosis in vivo.

Embryonic development during the preimplantation stages is highly sensitive and critically dependent on the reception of signaling cues. The precise coordination of diverse pathways and signaling factors is essential for successful embryonic progression. Even minor disruptions in these factors can result in physiological dysfunction, fetal malformations, or embryonic arrest. This issue is particularly evident in assisted reproductive technologies, such as in vitro fertilization, where embryonic arrest is frequently observed. A detailed understanding of these pathways enhances insight into the fundamental mechanisms underlying cellular processes and their contributions to embryonic development. The significance of elucidating signaling pathways and their regulatory factors in preimplantation development cannot be overstated. The application of this knowledge in laboratory settings has the potential to support strategies for modeling developmental stages and diseases, drug screening, therapeutic discovery, and reducing embryonic arrest. Furthermore, using various factors, small molecules, and pharmacological agents can enable the development or optimization of culture media for enhanced embryonic viability. While numerous pathways influence preimplantation development, this study examines several critical signaling pathways in this contex.

Ion channels are essential for mineral ion homeostasis in mammalian cells, and these are activated or inhibited by environmental stimuli such as heat, cold, mechanical, acidic, or basic stresses. These expressions and functions are quite diverse between cell types. The function and importance of ion channels are well-studied in neurons and cardiac cells, while those functions in pluripotent stem cells (PSCs) were not fully understood. Some sodium, potassium, chloride, calcium, transient receptor potential channels and mechanosensitive Piezo channels are found to be expressed and implicated in pluripotency and self-renewal capacity in PSCs. This review summarizes present and previous reports about ion channels and their response to environmental stimuli in PSCs. Furthermore, we compare the expressions and roles between PSCs and their differentiated embryoid bodies. We then discuss those contributions to pluripotency and differentiation.

Spermatogonial stem cells (SSCs) play crucial roles in the preservation of male fertility. However, successful ex vivo expansion of authentic human SSCs remains elusive due to the inadequate understanding of SSC homeostasis regulation. Using rhesus monkeys (Macaca mulatta) as a representative model, we characterized SSCs and progenitor subsets through single-cell RNA sequencing using a cell-specific network approach. We also profiled chromatin status and major histone modifications (H3K4me1, H3K4me3, H3K27ac, H3K27me3 and H3K9me3), and subsequently mapped promoters and active enhancers in TSPAN33+ putative SSCs. Comparing the epigenetic changes between fresh TSPAN33+ cells and cultured TSPAN33+ cells (resembling progenitors), we identified the regulatory elements with higher activity in SSCs, and the potential transcription factors and signaling pathways implicated in SSC regulation. Specifically, TGF-β signaling is activated in monkey putative SSCs. We provided evidence supporting its role in promoting self-renewal of monkey SSCs in culture. Overall, this study outlines the epigenetic landscapes of monkey SSCs and provides clues for optimization of the culture condition for primate SSCs expansion.

The asymmetric cell division determines cell diversity and distinct sibling cell fates by mechanisms linked to mitosis. Many adult stem cells divide asymmetrically to balance self-renewal and differentiation. The process of asymmetric cell division involves an axis of polarity and, second, the localization of cell fate determinants at the cell poles. Asymmetric division of stem cells is achieved by intrinsic and extrinsic fate determinants such as signaling molecules, epigenetics factors, molecules regulating gene expression, and polarized organelles. At least some stem cells perform asymmetric and symmetric cell divisions during development. Asymmetric division ensures that the number of stem cells remains constant throughout life. The asymmetric division of stem cells plays an important role in biological events such as embryogenesis, tissue regeneration and carcinogenesis. This review summarizes recent advances in the regulation of asymmetric stem cell division in model organisms.

Human stem cells are undifferentiated cells with the capacity for self-renewal and differentiation into distinct cell lineages, playing important role in the development and maintenance of diverse tissues and organs. The microenvironment of stem cell provides crucial factors and components that exert significant influence over the determination of cell fate. Among these factors, cytokines from the transforming growth factor β (TGFβ) superfamily, including TGFβ, bone morphogenic protein (BMP), Activin and Nodal, have been identified as important regulators governing stem cell maintenance and differentiation. In this review, we present a comprehensive overview of the pivotal roles played by TGFβ superfamily signaling in governing human embryonic stem cells, somatic stem cells, induced pluripotent stem cells, and cancer stem cells. Furthermore, we summarize the latest research and advancements of TGFβ family in various cancer stem cells and stem cell-based therapy, discussing their potential clinical applications in cancer therapy and regeneration medicine.

Accumulating evidence indicates that long noncoding RNAs (lncRNAs) play important roles in molecular and cellular biology. Although many algorithms have been developed to reveal their associations with complex diseases by using downstream targets, the upstream (epi)genetic regulatory information has not been sufficiently leveraged to predict the function of lncRNAs in various biological processes. Therefore, we present FunlncModel, a machine learning-based interpretable computational framework, which aims to screen out functional lncRNAs by integrating a large number of (epi)genetic features and functional genomic features from their upstream/downstream multi-omic regulatory networks. We adopted the random forest method to mine nearly 60 features in three categories from >2000 datasets across 11 data types, including transcription factors (TFs), histone modifications, typical enhancers, super-enhancers, methylation sites, and mRNAs. FunlncModel outperformed alternative methods for classification performance in human embryonic stem cell (hESC) (0.95 Area Under Curve (AUROC) and 0.97 Area Under the Precision-Recall Curve (AUPRC)). It could not only infer the most known lncRNAs that influence the states of stem cells, but also discover novel high-confidence functional lncRNAs. We extensively validated FunlncModel's efficacy by up to 27 cancer-related functional prediction tasks, which involved multiple cancer cell growth processes and cancer hallmarks. Meanwhile, we have also found that (epi)genetic regulatory features, such as TFs and histone modifications, serve as strong predictors for revealing the function of lncRNAs. Overall, FunlncModel is a strong and stable prediction model for identifying functional lncRNAs in specific cellular contexts. FunlncModel is available as a web server at https://bio.liclab.net/FunlncModel/.

Cell and Gene therapy are referred to as advanced therapies that represent overlapping fields of regenerative medicine. They have similar therapeutic goals such as to modify cellular identity, improve cell function, or fight a disease. These two therapeutic avenues, however, possess major differences. While cell therapy involves introduction of new cells, gene therapy entails introduction or modification of genes. Furthermore, the aim of cell therapy is often to replace, or repair damaged tissue, whereas gene therapy is used typically as a preventive approach. Diabetes mellitus severely affects the quality of life of afflicted individuals and has various side effects including cardiovascular, ophthalmic disorders, and neuropathy while putting enormous economic pressure on both the healthcare system and the patient. In recent years, great effort has been made to develop cutting-edge therapeutic interventions for diabetes treatment, among which cell and gene therapies stand out. This review aims to highlight various cell- and gene-based therapeutic approaches leading to the generation of new insulin-producing cells as a topmost "panacea" for treating diabetes, while deliberately avoiding a detailed molecular description of these approaches. By doing so, we aim to target readers who are new to the field and wish to get a broad helicopter overview of the historical and current trends of cell- and gene-based approaches in β-cell regeneration.

The α-Klotho protein (hereafter Klotho) is an obligate coreceptor for fibroblast growth factor 23 (FGF23). It is produced in the kidneys, brain and other sites. Klotho insufficiency causes hyperphosphatemia and other anomalies. Importantly, it is associated with chronic pathologies (often age-related) that have an inflammatory component. This includes atherosclerosis, diabetes and Alzheimer's disease. Its mode of action in these diseases is not well understood, but it inhibits or regulates multiple major pathways. Klotho has a membrane form and a soluble form (s-Klotho). Cytosolic Klotho is postulated but not well characterized. s-Klotho has endocrine properties that are incompletely elucidated. It binds to the FGF receptor 1c (FGFR1c) that is widely expressed (including endothelial cells). It also attaches to soluble FGF23, and FGF23/Klotho binds to FGFRs. Thus, s-Klotho might be a roaming FGF23 coreceptor, but it has other functions. Notably, Klotho (cell-bound or soluble) counteracts inflammation and appears to mitigate related aging (inflammaging). It inhibits NF-κB and the NLRP3 inflammasome. This inflammasome requires priming by NF-κB and produces active IL-1β, membrane pores and cell death (pyroptosis). In accord, Klotho countered inflammation and cell injury induced by toxins, damage-associated molecular patterns (DAMPs), cytokines, and reactive oxygen species (ROS). s-Klotho also blocks the TGF-β receptor and Wnt ligands, which lessens fibrotic disease. Low Klotho is associated with loss of muscle mass (sarcopenia), as occurs in aging and chronic diseases. s-Klotho counters the inhibitory effects of myostatin and TGF-β on muscle, reduces inflammation, and improves muscle repair following injury. The inhibition of TGF-β and other factors may also be protective in diabetic retinopathy and age-related macular degeneration (AMD). This review examines Klotho functions especially as related to inflammation and potential applications.

The establishment of stable porcine embryonic stem cells (pESCs) can contribute to basic and biomedical research, including comparative developmental biology, as well as assessing the safety of stem cell-based therapies. Despite these advantages, most pESCs obtained from in vitro blastocysts require complex media and feeder layers, making routine use, genetic modification, and differentiation into specific cell types difficult. We aimed to establish pESCs with a single cell-passage ability, high proliferative potency, and stable in long-term culture from in vitro-derived blastocysts using a simplified serum-free medium.

We evaluated the establishment efficiency of pESCs from in vitro blastocysts using various basal media (DMEM/F10 (1:1), DMEM/F12, and a-MEM) and factors (FGF2, IWR-1, CHIR99021, and WH-4-023). The pluripotency and self-renewal capacity of the established pESCs were analyzed under feeder or feeder-free conditions. Ultimately, we developed a simplified culture medium (FIW) composed of FGF2, IWR-1, and WH-4-023 under serum-free conditions.

The pESC-FIW lines were capable of single-cell passaging with short cell doubling times and expressed the pluripotency markers POU5F1, SOX2, and NANOG, as well as cell surface markers SSEA1, SSEA4, and TRA-1-60. pESC-FIW showed a stable proliferation rate and normal karyotype, even after 50 passages. Transcriptome analysis revealed that pESC-FIW were similar to reported pESC maintained in complex media and showed gastrulating epiblast cell characteristics. pESC-FIW were maintained for multiple passages under feeder-free conditions on fibronectin-coated plates using mTeSR™, a commercial medium used for feeder-free culture, exhibiting characteristics similar to those observed under feeder conditions.

These results indicated that inhibition of WNT and SRC was sufficient to establish pESCs capable of single-cell passaging and feeder-free expansion under serum-free conditions. The easy maintenance of pESCs facilitates their application in gene editing technology for agriculture and biomedicine, as well as lineage commitment studies.

The integrity of the intestinal mucosal barrier is crucial for protecting the intestinal epithelium against invasion by commensal bacteria and pathogens, thereby combating colitis. The investigation revealed that the absence of TSP50 compromised the integrity of the intestinal mucosal barrier in murine subjects. This disruption facilitated direct contact between intestinal bacteria and the intestinal epithelium, thereby increasing susceptibility to colitis. Mechanistic analysis indicated that TSP50 deficiency in intestinal stem cells (ISCs) triggered aberrant activation of the TGF-β signaling pathway and impeded the differentiation of goblet cells in mice, leading to impairment of mucosal permeability. By inhibiting the TGF-β pathway, the functionality of the intestinal mucosal barrier is successfully restored and mitigated colitis in TSP50-deficient mice. In conclusion, TSP50 played a crucial role in maintaining the intestinal mucosal barrier function and exhibited the preventive effect against the development of colitis by regulating the TGF-β signaling pathway.

Cherax quadricarinatus exhibit sexual dimorphism, with males outpacing females in size specification and growth rate. However, there is limited understanding of the molecular mechanisms underlying sex determination and sex differentiation in crustaceans. To study the differences between intersex individuals and normal individuals, this study counted the proportion of intersex individuals in the natural population, collected the proportion of 7 different phenotypes in 200 intersex individuals, and observed the differences in tissue sections. RNA-seq was used to study the different changes in the transcriptome of normal and intersex gonads. The results showed that: the percentage of intersex in the natural population was 1.5 %, and the percentage of different types of intersex ranged from 0.5 % to 22.5 %; the sections revealed that the development of normal ovaries was stagnant at the primary oocyte stage when intersex individuals with ovaries were present; We screened for pathways and genes that may be associated with gonadal development and sex, including ovarian steroid synthesis, estrogen signaling pathway, oocyte meiosis, progesterone-mediated oocyte maturation, etc. Relevant genes including tra2a, dmrta2, ccnb2, foxl2, and smad4. This study provides an important molecular basis for sex determination, sex-controlled breeding, and unisex breeding in red crayfish.

Asymmetric division is a fundamental process for generating cell diversity and maintaining the stem cell population. During asymmetric division, proteins, organelles, and even RNA are distributed unequally between the two daughter cells, determining their distinct cell fates. The mechanisms orchestrating this process are extremely complex. Dysregulation of asymmetric division can potentially trigger cancer progression. Cancer stem cells, in particular, undergo asymmetric division, leading to intra-tumoral heterogeneity, which contributes to treatment refractoriness. In this review, we delve into the cellular and molecular mechanisms that govern asymmetric division and explore its relevance to tumorigenesis.

Interferon regulatory factors (IRF-1 and IRF-2) are transcription factors widely implicated in various cellular processes, including regulation of inflammatory responses to pathogens, cell proliferation, oncogenesis, differentiation, autophagy, and apoptosis.

We have studied the expression of IRF-1, IRF-2 mRNAs by RT-PCR, cellular localization of the proteins by immunofluorescence, and expression of mRNAs of genes regulated by IRF-1, IRF-2 by RT-PCR in mouse bone marrow cells (BMCs) and mesenchymal stem cells (MSCs).

Higher level of IRF-1 mRNA was observed in BMCs and MSCs compared to that of IRF-2. Similarly, differential expression of IRF-1 and IRF-2 proteins was observed in BMCs and MSCs. IRF-1 was predominantly localized in the cytoplasm, whereas IRF-2 was localized in the nuclei of BMCs. MSCs showed nucleo-cytoplasmic distribution of IRF-1 and nuclear localization of IRF-2. Constitutive expression of IRF-1 and IRF-2 target genes: monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), and caspase-1 was observed in both BMCs and MSCs. MSCs showed constitutive expression of the pluripotency-associated factors, Oct3/4 and Sox-2. Lipopolysaccharide (LPS)-treatment of MSCs induced prominent cellular localization of IRF-1 and IRF-2.

Our results suggest that IRF-1 and IRF-2 exhibit differential expression of their mRNAs and subcellular localization of the proteins in BMCs and MSCs. These cells also show differential levels of constitutive expression of IRF-1 and IRF-2 target genes. This may regulate immune-responsive properties of BMCs and MSCs through IRF-1, IRF-2-dependent gene expression and protein-protein interaction. Regulating IRF-1 and IRF-2 may be helpful for immunomodulatory functions of MSCs for cell therapy and regenerative medicine.

Human embryonic stem cells (hESCs) cultured in media containing bone morphogenic protein 4 (BMP4; B) differentiate into trophoblast-like cells. Supplementing media with inhibitors of activin/nodal signaling (A83-01) and of fibroblast growth factor 2 (PD173074) suppresses mesoderm and endoderm formation and improves specification of trophoblast-like lineages, but with variable effectiveness. We compared differentiation in four BMP4-containing media: mTeSR1-BMP4 only, mTeSR1-BAP, basal medium with BAP (basal-BAP), and a newly defined medium, E7-BAP. These media variably drive early differentiation towards trophoblast-like lineages with upregulation of early trophoblast markers CDX2 and KRT7 and downregulation of pluripotency markers (OCT4 and NANOG). As expected, based on differences between media in FGF2 and its inhibitors, downregulation of mesendoderm marker EOMES was variable between media. By day 7, only hESCs grown in E7-BAP or basal-BAP expressed HLA-G protein, indicating the presence of cells with extravillous trophoblast characteristics. Expression of HLA-G and other differentiation markers (hCG, KRT7, and GCM1) was highest in basal-BAP, suggesting a faster differentiation in this medium, but those cultures were more inhomogeneous and still expressed some endodermal and pluripotency markers. In E7-BAP, HLA-G expression increased later and was lower. There was also a low but maintained expression of some C19MC miRNAs, with more CpG hypomethylation of the ELF5 promoter, suggesting that E7-BAP cultures differentiate slower along the trophoblast lineage. We conclude that while all protocols drive differentiation into trophoblast lineages with varying efficiency, they have advantages and disadvantages that must be considered when selecting a protocol for specific experiments.

TGFβ signaling and the DNA damage response (DDR) are two cellular toolboxes with a strong impact on cancer biology. While TGFβ as a pleiotropic cytokine affects essentially all hallmarks of cancer, the multifunctional DDR mostly orchestrates cell cycle progression, DNA repair, chromatin remodeling and cell death. One oncogenic effect of TGFβ is the partial activation of epithelial-to-mesenchymal transition (EMT), conferring invasiveness, cellular plasticity and resistance to various noxae. Several reports show that both individual networks as well as their interface affect chemo-/radiotherapies. However, the underlying mechanisms remain poorly resolved. EMT often correlates with TGFβ-induced slowing of proliferation, yet numerous studies demonstrate that particularly the co-activated EMT transcription factors counteract anti-proliferative signaling in a partially non-redundant manner. Collectively, evidence piled up over decades underscore a multifaceted, reciprocal inter-connection of TGFβ signaling / EMT with the DDR / cell cycle progression, which we will discuss here. Altogether, we conclude that full cell cycle arrest is barely compatible with the propagation of oncogenic EMT traits and further propose that 'EMT-linked DDR plasticity' is a crucial, yet intricate facet of malignancy, decisively affecting metastasis formation and therapy resistance.

The discovery of Helicobacter pylori (Hp) infection of gastric mucosa leading to active chronic gastritis, gastroduodenal ulcers, and MALT lymphoma laid the groundwork for understanding of the general relationship between chronic infection, inflammation, and cancer. Nevertheless, this sequence of events is still far from full understanding with new players and mediators being constantly identified. Originally, the Hp virulence factors affecting mainly gastric epithelium were proposed to contribute considerably to gastric inflammation, ulceration, and cancer. Furthermore, it has been shown that Hp possesses the ability to penetrate the mucus layer and directly interact with stroma components including fibroblasts and myofibroblasts. These cells, which are the source of biophysical and biochemical signals providing the proper balance between cell proliferation and differentiation within gastric epithelial stem cell compartment, when exposed to Hp, can convert into cancer-associated fibroblast (CAF) phenotype. The crosstalk between fibroblasts and myofibroblasts with gastric epithelial cells including stem/progenitor cell niche involves several pathways mediated by non-coding RNAs, Wnt, BMP, TGF-β, and Notch signaling ligands. The current review concentrates on the consequences of Hp-induced increase in gastric fibroblast and myofibroblast number, and their activation towards CAFs with the emphasis to the altered communication between mesenchymal and epithelial cell compartment, which may lead to inflammation, epithelial stem cell overproliferation, disturbed differentiation, and gradual gastric cancer development. Thus, Hp-activated fibroblasts may constitute the target for anti-cancer treatment and, importantly, for the pharmacotherapies diminishing their activation particularly at the early stages of Hp infection.

Skeletal muscle is a complex heterogeneous tissue and characterizing its cellular heterogeneity and transcriptional and epigenetic signatures are important for understanding the details of its ontogeny. In our study, we applied scRNA-seq and scATAC-seq to investigate the cell types, molecular features, transcriptional and epigenetic regulation, and patterns of developing bovine skeletal muscle from gestational, lactational and adult stages. Detailed molecular analyses were used to dissect cellular heterogeneity, and we deduced the differentiation trajectory of myogenic cells and uncovered their dynamic gene expression profiles. SCENIC analysis was performed to demonstrate key regulons during cell fate decisions. We explored the future expression states of these heterogeneous cells by RNA velocity analysis and found extensive networks of intercellular communication using the toolkit CellChat. Moreover, the transcriptomic and chromatin accessibility modalities were confirmed to be highly concordant, and integrative analysis of chromatin accessibility and gene expression revealed key transcriptional regulators acting during myogenesis. In bovine skeletal muscle, by scRNA-seq and scATAC-seq analysis, different cell types such as adipocytes, endothelial cells, fibroblasts, lymphocytes, monocytes, pericyte cells and eight skeletal myogenic subpopulations were identified at the three developmental stages. The pseudotime trajectory exhibited a distinct sequential ordering for these myogenic subpopulations and eight distinct gene clusters were observed according to their expression pattern. Moreover, specifically expressed TFs (such as MSC, MYF5, MYOD1, FOXP3, ESRRA, BACH1, SIX2 and ATF4) associated with muscle development were predicted, and likely future transcriptional states of individual cells and the developmental dynamics of differentiation among neighbouring cells were predicted. CellChat analysis on the scRNA-seq data set then classified many ligand-receptor pairs among these cell clusters, which were further categorized into significant signalling pathways, including BMP, IGF, WNT, MSTN, ANGPTL, TGFB, TNF, VEGF and FGF. Finally, scRNA-seq and scATAC-seq results were successfully integrated to reveal a series of specifically expressed TFs that are likely to be candidates for the promotion of cell fate transition during bovine skeletal muscle development. Overall, our results outline a single-cell dynamic chromatin/transcriptional landscape for normal bovine skeletal muscle development; these provide an important resource for understanding the structure and function of mammalian skeletal muscle, which will promote research into its biology.

The multi-kinase inhibitor dasatinib has been implicated to be effective in pre-B-cell receptor (pre-BCR)-positive acute lymphoblastic leukemia (ALL) expressing the E2A-PBX1 fusion oncoprotein. The TGFβ signaling pathway is involved in a wide variety of cellular processes, including embryonic development and cell homeostasis, and it can have dual roles in cancer: suppressing tumor growth at early stages and mediating tumor progression at later stages. In this study, we identified the upregulation of the TGFβ signaling pathway in our previously generated human dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells using global transcriptomic analysis. We confirm the upregulation of the TGFβ pathway member SMAD3 at the transcriptional and translational levels in dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells. Hence, dasatinib blocks, at least partially, TGFβ-induced SMAD3 phosphorylation in several B-cell precursor (BCP) ALL cell lines as well as in dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells. Activation of the TGFβ signaling pathway by TGF-β1 leads to growth inhibition by cell cycle arrest at the G0/G1 stage, increase in apoptosis and transcriptional changes of SMAD-targeted genes, e.g. c-MYC downregulation, in pre-BCR+/E2A-PBX1+ ALL cells. These results provide a better understanding about the role that the TGFβ signaling pathway plays in leukemogenesis of BCP-ALL as well as in secondary drug resistance to dasatinib.

The ability of neural stem/progenitor cells (NSPCs) to proliferate and differentiate is required through different stages of neurogenesis. Disturbance in the regulation of neurogenesis causes many neurological diseases, such as intellectual disability, autism, and schizophrenia. However, the intrinsic mechanisms of this regulation in neurogenesis remain poorly understood. Here, we report that Ash2l (Absent, small or homeotic discs-like 2), one core component of a multimeric histone methyltransferase complex, is essential for NSPC fate determination during postnatal neurogenesis. Deletion of Ash2l in NSPCs impairs their capacity for proliferation and differentiation, leading to simplified dendritic arbors in adult-born hippocampal neurons and deficits in cognitive abilities. RNA sequencing data reveal that Ash2l primarily regulates cell fate specification and neuron commitment. Furthermore, we identified Onecut2, a major downstream target of ASH2L characterized by bivalent histone modifications, and demonstrated that constitutive expression of Onecut2 restores defective proliferation and differentiation of NSPCs in adult Ash2l-deficient mice. Importantly, we identified that Onecut2 modulates TGF-β signaling in NSPCs and that treatment with a TGF-β inhibitor rectifies the phenotype of Ash2l-deficient NSPCs. Collectively, our findings reveal the ASH2L-Onecut2-TGF-β signaling axis that mediates postnatal neurogenesis to maintain proper forebrain function.

To explore the protective effect and related mechanism of miR-140-5p on liver fibrosis by interfering with TGF-β/Smad signaling pathway.

Liver fibrosis mice models were established by intraperitoneal injection of CCL4. Hematoxylin and eosin (HE) staining was used to detect the structural and morphological changes of the liver. Masson staining was used to detect collagen deposition. Human hepatic stellate cells (HSCs, LX-2) were transfected with miR-140-5p mimic or inhibitor then treated with TGF-β1. The qRT-PCR and Western blotting was used to detect the expression of related molecules. The luciferase reporter assay was used to identify the target of miR-140-5p.

Our results indicated that miR-140-5p expression was downregulated in fibrotic liver tissues of model mice and LX-2 cells treated with TGF-β1. The overexpression of miR-140-5p decreased the expression of collagen1(COL1) and α-smooth muscle actin(α-SMA), inhibited the phosphorylation of Smad-2/3 (pSmad-2/3) in LX-2 cells. Conversely, the knockdown of miR-140-5p upregulated COL1 and α-SMA expression, increased Smad-2/3 phosphorylation. A dual-luciferase reporter assay showed that TGFβR1 was a target gene of miR-140-5p. The overexpression of miR-140-5p suppressed TGFβR1 expression in LX-2 cells. Additionally, knockdown of TGFβR1 decreased the expression of COL1 and α-SMA. Conversely, the overexpression of TGFβR1 reversed the inhibitory effect of miR-140-5p upregulation on expression of COL1 and α-SMA.

miR-140-5p bound to TGFβR1 mRNA 3'-untranslated region(3'UTR) and inhibited the expression of TGFβR1, pSmad-2/3, COL1 and α-SMA, thereby exerting a potential therapeutic effect on hepatic fibrosis.

Three-dimensional (3D) human brain spheroids are instrumental to study central nervous system (CNS) development and (dys)function. Yet, in current brain spheroid models the limited variety of cell types hampers an integrated exploration of CNS (disease) mechanisms.

Here we report a 5-month culture protocol that reproducibly generates H9 embryonic stem cell-derived human cortical spheroids (hCSs) with a large cell-type variety.

We established the presence of not only neuroectoderm-derived neural progenitor populations, mature excitatory and inhibitory neurons, astrocytes and oligodendrocyte (precursor) cells, but also mesoderm-derived microglia and endothelial cell populations in the hCSs via RNA-sequencing, qPCR, immunocytochemistry and transmission electron microscopy. Transcriptomic analysis revealed resemblance between the 5-months-old hCSs and dorsal frontal rather than inferior regions of human fetal brains of 19-26 weeks of gestational age. Pro-inflammatory stimulation of the generated hCSs induced a neuroinflammatory response, offering a proof-of-principle of the applicability of the spheroids.

Our protocol provides a 3D human brain cell model containing a wide variety of innately developing neuroectoderm- as well as mesoderm-derived cell types, furnishing a versatile platform for comprehensive examination of intercellular CNS communication and neurological disease mechanisms.

Diabetic nephropathy is one of the most common microvascular complications of diabetes mellitus that finally leads to complete loss of kidney function. Therefore, this study aimed to evaluate the effect of crocin and losartan on TGF-β gene expression and histopathology of kidney tissue in a rat model of diabetic nephropathy.

Forty male Wistar rats were randomly divided into five groups (n=8): Untreated control, Diabetic (D), D + crocin, D + losartan, and D + losartan + crocin. Induction of diabetes was performed using streptozotocin (50 mg/kg/ Intraperitoneal injection). At the end of the eight-week period, the rats were sacrificed. Spectrophotometry measured serum glucose, urea, creatinine, and uric acid levels. Microalbumin and creatinine levels were measured in 24-hour urine. Real-time PCR was used to determine the relative expression of the TGF-β gene in kidney tissue. Renal tissue histopathology was also examined.

The results showed that hyperglycemia increased biochemical factors associated with diabetes, TGF-β gene expression, and kidney damage. Separate treatment with crocin and losartan led to a decrease in renal function factors and TGF-β gene expression and improved kidney damage.

Our results showed that crocin could improve kidney function in diabetic conditions. In addition, we showed that crocin increases the effectiveness of losartan. Consequently, we suggest that crocin in combination with chemical drugs can be a potential therapeutic agent for diabetes and its complications. Nonetheless, human studies are needed to make firm findings.

Transforming growth factor-beta (TGF-β) superfamily members orchestrate a wide breadth of biological processes. Through Sma and Mad (Smad)-related dependent or noncanonical pathways, TGF-β members involve in the occurrence and development of many diseases such as cancers, fibrosis, autoimmune diseases, cardiovascular diseases and brain diseases. Glycosylation is one kind of the most common posttranslational modifications on proteins or lipids. Abnormal protein glycosylation can lead to protein malfunction and biological process disorder, thereby causing serious diseases. Previously, researchers commonly make comprehensive systematic overviews on the roles of TGF-β signaling in a specific disease or biological process. In recent years, more and more evidences associate glycosylation modification with TGF-β signaling pathway, and we can no longer disengage and ignore the roles of glycosylation from TGF-β signaling to make investigation. In this review, we provide an overview of current findings involved in glycosylation within TGF-βs and theirs receptors, and the interaction effects between glycosylation and TGF-β subfamily signaling, concluding that there is an intricate mutual regulation between glycosylation and TGF-β signaling, hoping to present the glycosylation regulatory patterns that concealed in TGF-βs signaling pathways.

The generation of midbrain dopaminergic neurons (mDAs) from pluripotent stem cells (hPSC) holds much promise for both disease modelling studies and as a cell therapy for Parkinson's disease (PD). Generally, dopaminergic neuron differentiation paradigms rely on inhibition of smad signalling for neural induction followed by hedgehog signalling and an elevation of β-catenin to drive dopaminergic differentiation. Post-patterning, differentiating dopaminergic neuron cultures are permitted time for maturation after which the success of these differentiation paradigms is usually defined by expression of tyrosine hydroxylase (TH), the rate limiting enzyme in the synthesis of dopamine. However, during maturation, culture media is often supplemented with additives to promote neuron survival and or promote cell differentiation. These additives include dibutyryl cyclic adenosine monophosphate (dbcAMP), transforming growth factor β3 (TGFβ3) and or the γ-secretase inhibitor (DAPT). While these factors are routinely added to cultures, their impact upon pluripotent stem cell-derived mDA phenotype is largely unclear. In this study, we differentiate pluripotent stem cells toward a dopaminergic phenotype and investigate how the omission of dbcAMP, TGFβ3 or DAPT, late in maturation, affects the regulation of multiple dopaminergic neuron phenotype markers. We now show that the removal of dbcAMP or TGFβ3 significantly and distinctly impacts multiple markers of the mDA phenotype (FOXA2, EN1, EN2, FOXA2, SOX6), while commonly increasing both MSX2 and NEUROD1 and reducing expression of both tyrosine hydroxylase and WNT5A. Removing DAPT significantly impacted MSX2, OTX2, EN1, and KCNJ6. In the absence of any stressful stimuli, we suggest that these culture additives should be viewed as mDA phenotype-modifying, rather than neuroprotective. We also suggest that their addition to cultures is likely to confound the interpretation of both transplantation and disease modelling studies.

Maintenance of stem cells requires the concerted actions of niche-derived signals and stem cell-intrinsic factors. Although Decapentaplegic (Dpp), a Drosophila bone morphogenetic protein (BMP) molecule, can act as a long-range morphogen, its function is spatially limited to the germline stem cell niche in the germarium. We show here that Integrator, a complex known to be involved in RNA polymerase II (RNAPII)-mediated transcriptional regulation in the nucleus, promotes germline differentiation by restricting niche-derived Dpp/BMP activity in the cytoplasm. Further results show that Integrator works in various developmental contexts to desensitize the cellular response to Dpp/BMP signaling during Drosophila development. Mechanistically, our results show that Integrator forms a multi-subunit complex with the type I receptor Thickveins (Tkv) and other Dpp/BMP signaling components and acts in a negative feedback loop to promote Tkv turnover independent of its transcriptional activity. Similarly, human Integrator subunits bind transforming growth factor β (TGF-β)/BMP signaling components and antagonize their activity, suggesting a conserved role of Integrator across metazoans.

The outbreak of mass mortality of M. salmoides occurred in an aquaculture farm in Jiangsu province of China, showing signs of skin ulceration and haemorrhages. The bacteria were isolated from diseased largemouth bass, and identified as Plesiomonas shigelloides based on morphological, physiological and biochemical features, as well as 16S rRNA gene sequence analysis. The pathogenicity of P. shigelloides was determined by challenge experiments, and the median lethal dosage (LD₅₀) of the isolate NJS1 for M. salmoides was calculated as 1.6 × 10⁵ CFU/mL at 7 d post-infection. Histopathological analysis revealed that extensive necrosis, vacuolization and inflammation were presented in the kidney, liver and gill of the diseased fish. Detection of virulence-related genes showed that P. shigelloides NJS1 was positive for astA, astB, astD, astE, actP and 6 ahpA. Additionally, the host defensive response of M. salmoides infected by P. shigelloides was analyzed by quantitive real-time PCR (qRT-PCR), and the results showed that the expression levels of Cas3, Hep1, HIF, IgM, IL15 and TGF were significantly up-regulated in head kidney, liver and spleen in different hours post-infection, which revealed varying expression profiles and clear transcriptional activation of immune related genes. The results suggested that P. shigelloides was an etiological element in the mass mortalities of M. salmoides and this study provided deeper insights for the pathogenesis and host defensive system in P. shigelloides invasion.

Transforming Growth Factor beta (TGF-β) is a multifunctional cytokine that regulates cell proliferation, differentiation, and apoptosis. Dysregulation of the TGF-β signaling is one of the major mechanisms underlying tumor progression. We have previously reported that anaplastic lymphoma kinase (ALK) phosphorylates Smad4 at Tyr95, which compromises the DNA-binding ability of Smad4 and thus renders ALK-positive cancer cells resistant to TGF-β tumor-suppressive action. In this study, we demonstrated that tyrosine phosphatase PTPN2 positively regulated TGF-β signaling through dephosphorylating Smad4 at the Tyr95 site. Both in vitro and cell-based assays revealed that PTPN2 bound to and dephosphorylated Smad4, thereby preserving the DNA-binding ability of Smad4. Furthermore, overexpression of PTPN2 restored TGF-β transcriptional and growth inhibitory responses in ALK-positive cancer cells. Consistently, Spermidine, an activator of PTPN2, also promoted TGF-β-induced gene expression, apoptosis, and anti-proliferation effect. Taken together, we revealed that PTPN2 functioned as a tumor suppressor to antagonize the inhibitory effect of tyrosine phosphorylation of Smad4 and to ensure the proper TGF-β growth inhibitory signaling in cancer cells.

In the current research context of precision treatment of malignant tumors, the advantages of immunotherapy are unmatched by conventional antitumor therapy, which can prolong progression-free survival and overall survival. The search for new targets and novel combination therapies can improve the efficacy of immunotherapy and reduce adverse effects. Since current research targets for immunotherapy mainly focus on lymphocytes, little research has been done on erythrocytes. Nucleated erythroid precursor stem cells have been discovered to play an essential role in tumor progression. Researchers are exploring new targets and therapeutic approaches for immunotherapy from the perspective of erythroid progenitor cells (EPCs). Recent studies have shown that different subtypes of EPCs have specific surface markers and distinct biological roles in tumor immunity. CD45⁺ EPCs are potent myeloid-derived suppressor cell-like immunosuppressants that reduce the patient's antitumor immune response. CD45^- EPCs promote tumor invasion and metastasis by secreting artemin. A specific type of EPC also promotes angiogenesis and provides radiation protection. Therefore, EPCs may be involved in tumor growth, infiltration, and metastasis. It may also be an important cause of anti-angiogenesis and immunotherapy resistance. This review summarizes recent research advances in erythropoiesis, EPC features, and their impacts and processes on tumors.

A genetic hallmark of malignant germ cell tumours (GCTs) is isochromosome 12p, but oncogenes located in 12p that are specifically expressed in GCT have not yet been identified. SIN3-HDAC complex-associated factor (SINHCAF) is a subunit of the Sin3/histone deacetylase (HDAC) complex, and it defines a Sin3a-Hdac complex variant that is required for the self-renewal of mouse embryonic stem cells. This study demonstrated that SINHCAF is expressed in a vast majority of malignant GCTs and is rarely expressed in somatic malignancy. Fluorescence in situ hybridisation revealed SINHCAF amplification in malignant GCTs. SINHCAF silencing using shRNA reduced anchorage-dependent cell proliferation and tumoursphere formation and inhibited tumour cell migration and invasion in GCT cell lines. Moreover, in the GCT cell line NTERA2/D1, SINHCAF silencing inhibited the expression of genes associated with embryonic stem cells and induced the expression of genes associated with neuronal and white fat cell differentiation. Compared with somatic cell lines, GCT cell lines were more susceptible to HDAC inhibitor treatment. Thus, we identified SINHCAF to be a potential oncogene located in the amplicon of chromosome 12p and showed that SINHCAF was specifically expressed in malignant GCTs. HDAC inhibitor treatment may counteract the oncogenic activity of SINHCAF and is a promising therapeutic approach for GCTs. © 2022 The Pathological Society of Great Britain and Ireland.

Myeloid lineage cells present the latent form of transforming growth factor-β1 (L-TGF-β1) to the membrane using an anchor protein LRRC33. Integrin α_Vβ₈ activates extracellular L-TGF-β1 to trigger the downstream signaling functions. However, the mechanism designating the specificity of TGF-β1 presentation and activation remains incompletely understood. Here, we report cryo-EM structures of human L-TGF-β1/LRRC33 and integrin α_Vβ₈/L-TGF-β1 complexes. Combined with biochemical and cell-based analyses, we demonstrate that LRRC33 only presents L-TGF-β1 but not the -β2 or -β3 isoforms due to difference of key residues on the growth factor domains. Moreover, we reveal a 2:2 binding mode of integrin α_Vβ₈ and L-TGF-β1, which shows higher avidity and more efficient L-TGF-β1 activation than previously reported 1:2 binding mode. We also uncover that the disulfide-linked loop of the integrin subunit β₈ determines its exquisite affinity to L-TGF-β1. Together, our findings provide important insights into the specificity of TGF-β1 signaling achieved by LRRC33 and integrin α_Vβ₈.

Human amniotic epithelial cells (hAECs), which are a type of placental stem cell, express stem cell marker genes and are capable of differentiating into all three germ layers under appropriate culture conditions. hAECs are known to undergo TGF-β-dependent epithelial-mesenchymal transition (EMT); however, the impact of EMT on the stemness or differentiation of hAECs has not yet been determined. Here, we first confirmed that hAECs undergo EMT immediately after starting primary culture. Comprehensive transcriptome analysis using RNA-seq revealed that inhibition of TGF-β-dependent EMT maintained the expression of stemness-related genes, including NANOG and POU5F1, in hAECs. Moreover, the maintenance of stemness did not affect the nontumorigenic characteristics of hAECs. We showed for the first time that TGF-β-dependent EMT negatively affected the stemness of hAECs, providing novel insight into cellular processes of placental stem cells.