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Neutrophils are essential innate immune cells with unusual anti-microbial properties while dysfunctions of neutrophils lead to severe health problems such as lethal infections. Generation of neutrophils from human induced pluripotent stem cells (hiPSCs) is highly promising to produce off-the-shelf neutrophils for transfusion therapies. However, the anti-microbial potencies of hiPSCs derived neutrophils (iNEUs) remain less documented. Here, we develop a scalable approach to generate iNEUs in a chemical defined condition. iNEUs display typical neutrophil characters in terms of phagocytosis, migration, formation of neutrophil extracellular traps (NETs), etc. Importantly, iNEUs display a strong killing potency against various bacteria such as K.pneumoniae, P.aeruginosa, E.coli and S.aureus. Moreover, transfusions of iNEUs in mice with neutrophil dysfunction largely enhance their survival in lethal infection of different bacteria. Together, our data show that hiPSCs derived neutrophils hold strong anti-microbial potencies to protect severe infections under neutrophil dysfunction conditions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13619-025-00227-z.

SUMMARY Individuals with Williams syndrome (WS), a neurodevelopmental disorder caused by hemizygous loss of 26–28 genes at 7q11.23, characteristically portray a hypersocial phenotype. Copy-number variations and mutations in one of these genes, GTF2I, are associated with altered sociality and are proposed to underlie hypersociality in WS. However, the contribution of GTF2I to human neurodevelopment remains poorly understood. Here, human cellular models of neurodevelopment, including neural progenitors, neurons, and three-dimensional cortical organoids, are differentiated from CRISPR-Cas9-edited GTF2I-knockout (GTF2I-KO) pluripotent stem cells to investigate the role of GTF2I in human neurodevelopment. GTF2I-KO progenitors exhibit increased proliferation and cell-cycle alterations. Cortical organoids and neurons demonstrate increased cell death and synaptic dysregulation, including synaptic structural dysfunction and decreased electrophysiological activity on a multielectrode array. Our findings suggest that changes in synaptic circuit integrity may be a prominent mediator of the link between alterations in GTF2I and variation in the phenotypic expression of human sociality. Graphical Abstract In brief GTF2I is thought to influence the phenotypic expression of human sociality and is implicated in neurodevelopmental disease. Adams et al. use hiPSC-derived cell platforms to investigate the role of GTF2I in human neurodevelopment. Loss of GTF2I promotes increased cell death, reduced synaptic integrity, and decreased electrical activity of cortical organoids.

IntroductionAccumulating evidence indicates that the increased presence of astrocytes is fundamentally linked to the neurological dysfunctions observed in individuals with Down syndrome (DS). REST (RE1-silencing transcription factor), as a chromatin modifier, regulates 15,450 genes in humans. REST is a key regulatory element that governs astrocyte differentiation, development, and the maintenance of their physiological functions. The downregulation of REST may disrupt the homeostatic balance of astrocytes in DS.MethodsThis study aims to elucidate the role of REST in DS-astrocytes through comprehensive transcriptomic analysis and experimental validation.ResultsTranscriptomic analysis identified that REST-targeted differentially expressed genes (DEGs) in DS astrocytes are enriched in pathways associated with inflammatory response. Notably, our findings in astrocytes derived from DS human induced pluripotent stem cells (hiPSCs) show that the loss of nucleus REST leads to an upregulation of inflammatory mediators and markers indicative of the presence of reactive astrocytes. Lithium treatment, which restored nucleus REST in trisomic astrocytes, significantly suppressed the expression of these inflammatory mediators and reactive astrocyte markers.DiscussionThese findings suggest that REST is pivotal in modulating astrocyte functionality and reactivity in DS. The loss of REST in DS-astrocytes prompts the formation of reactive astrocytes, thereby compromising central nervous system homeostasis. Lithium treatment possesses the potential to rescue astrocyte reactivity in DS by restoring nucleus REST expression.

Background?-catenin, acting as the core effector of canonical Wnt signaling pathway, plays a pivotal role in controlling lineage commitment and the formation of definitive endoderm (DE) during early embryonic development. Despite extensive studies using various animal and cell models, the ?-catenin-centered regulatory mechanisms underlying DE formation remain incompletely understood, partly due to the rapid and complex cell fate transitions during early differentiation.ResultsIn this study, we generated new CTNNB1-/- human ES cells (hESCs) using CRISPR-based insertional gene disruption approach and systematically rescued the DE defect in these cells by introducing various truncated or mutant forms of ?-catenin. Our analysis showed that a truncated ?-catenin lacking both N- and C-terminal domains (?N148C) could robustly rescue the DE formation, whereas hyperactive ?-catenin mutants with S33Y mutation or N-terminal deletion (?N90) had limited ability to induce DE lineage. Notably, the ?N148C mutant exhibited significant nuclear translocation that was positively correlated with successful DE rescue. Transcriptomic analysis further uncovered that two weak ?-catenin mutants lacking the C-terminal transactivation domain (CTD) activated primitive streak (PS) genes, whereas the hyperactive ?-catenin mutants activated mesoderm genes.ConclusionOur study uncovered an unconventional regulatory function of ?-catenin through weak transactivation, indicating that the levels of ?-catenin activity determine the lineage bifurcation from mesendoderm into endoderm and mesoderm.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-024-01279-5.

BackgroundMale factor infertility (MFI) is responsible for 50% of infertility cases and in 15% of the cases sperm is absent due to germ cell aplasia. Human induced pluripotent stem cell (hiPSC)-derived spermatogonial stem cells (hSSCs) could serve as an autologous germ cell source for MFI in patients with an insufficient sperm yield for assisted reproductive technology (ART). The endocannabinoid system (ECS) has been implicated to play a role in mouse embryonic stem cells (mESCs) and the human testicular environment. However, the contribution of the ECS in hiPSCs and hiPSC-derived hSSCs is currently unknown. Here, we aimed to assess whether hiPSCs and hiPSC-derived hSSCs are regulated by components of the ECS and whether manipulation of the ECS could increase the yield of hiPSC-derived SSCs and serve as an autologous cell-based source for treatment of MFI.MethodsWe reprogrammed human dermal fibroblasts (hDFs) to hiPSCs, induced differentiation of hSSC from hiPSCs and evaluated the presence of ECS ligands (AEA, 2-AG) by LC/MS, receptors (CB1R, CB2R, TRPV1, GPR55) by qPCR, flow cytometry and immunofluorescent labeling. We then examined the efficacy of endogenous and synthetic selective ligands (ACPA, CB65, CSP, ML184) on proliferation of hiPSCs using real-time cell analysis (RTCA) and assessed the effects of on CB2R agonism on hiPSC pluripotency and differentiation to hSSCs.ResultshiPSCs from hDFs expressed the pluripotency markers OCT4, SOX2, NANOG, SSEA4 and TRA-1-60; and could be differentiated into ID4+, PLZF?+?hSSCs. hiPSCs and hiPSC-derived hSSCs secreted AEA and 2-AG at 10??10 ??10??9 M levels. Broad expression of all ECS receptors was observed in both hiPSCs and hiPSC-derived hSSCs, with a higher CB2R expression in hSSCs in comparison to hiPSCs. CB2R agonist CB65 promoted proliferation and differentiation of hiPSCs to hiPSC-hSSCs in comparison to AEA, 2-AG, ACPA, CSP and ML184. The EC50 of CB65 was determined to be 2.092?×?10??8 M for support of pluripotency and preservation of stemness on hiPSCs from 78 h. CB65 stimulation at EC50 also increased the yield of ID4?+?hSSCs, PLZF?+?SSPCs and SCP3?+?spermatocytes from day 10 to 12.ConclusionsWe demonstrated here for the first time that stimulation of CB2R results in an increased yield of hiPSCs and hiPSC-derived hSSCs. CB65 is a potent CB2R agonist that can be used to increase the yield of hiPSC-derived hSSCs offering an alternative source of autologous male germ cells for patients with MFI. Increasing the male germ/stem cell pool by CB65 supplementation could be part of the ART-associated protocols in MFI patients with complete germ cell aplasia.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40659-025-00596-4.

Patients with breast cancer show altered expression of genes within the pectoralis major skeletal muscle cells of the breast. Through analyses of The Cancer Genome Atlas (TCGA)-breast cancer (BRCA), we identified three previously uncharacterized putative novel tumor suppressor genes expressed in normal muscle cells, whose expression was downregulated in breast tumors. We found that NEDD4 binding protein 2-like 1 (N4BP2L1), pleckstrin homology domain-containing family A member 4 (PLEKHA4), and brain-enriched guanylate kinase-associated protein (BEGAIN) that are normally highly expressed in breast myoepithelial cells and smooth muscle cells were significantly downregulated in breast tumor tissues of a cohort of 50 patients with this cancer. Our data revealed that the low expression of PLEKHA4 in patients with menopause below 50 years correlated with a higher risk of breast cancer. Moreover, we identified N4BP2L1 and BEGAIN as potential biomarkers of HER2-positive breast cancer. Furthermore, low BEGAIN expression in breast cancer patients with blood fat, heart problems, and diabetes correlated with a higher risk of this cancer. In addition, protein and RNA expression analysis of TCGA-BRCA revealed N4BP2L1 as a promising diagnostic protein biomarker in breast cancer. In addition, the in silico data of scRNA-seq showed high expression of these genes in several cell types of normal breast tissue, including breast myoepithelial cells and smooth muscle cells. Thus, our results suggest their possible tumor-suppressive function in breast cancer and muscle development.