�����ƽ��

Rett syndrome arises from loss-of-function mutations in the X-linked chromatin regulator MECP2, yet the earliest molecular derailments in development are poorly defined. Using isogenic human embryonic stem cell (hESC) models carrying three patient-derived MECP2 mutations, we followed the transcriptome from pluripotency through neuroectoderm, neural stem/progenitor stages. Developmental stage dominated transcriptional variance, but mutants shared a secondary program enriched for synaptic-membrane and extracellular matrix genes. Single-cell/bulk profiling at the embryonic stem cell (ESC) stage revealed partial naïve-like drift, marked by the up-regulation of the naïve-enriched factor ZFP42/REX1 and related markers in MECP2-mutant lines. Among convergently dysregulated genes, the cortical determinant EMX1 showed an abnormal developmental trajectory, early repression followed by overshoot, and was consistently altered across independent Rett PSC models. Single-nucleus RNA-seq of cerebral organoids uncovered allele-specific yet convergent disturbances in cortical lineage allocation. These data chart a continuous developmental trajectory for MECP2-mutant cells and nominate naïve-like drift and mis-timed EMX1 expression as tractable entry points for dissecting Rett pathogenesis. Graphical abstract Highlights•MECP2 mutations induce an early naïve-like transcriptional drift in hESCs•EMX1 shows a conserved abnormal developmental trajectory across Rett models•Shared transcriptional programs emerge during neural induction in MECP2 mutants•Rett cerebral organoids display mutation-specific shifts in lineage allocation In this article, Flamier and colleagues show that MECP2 mutations perturb human neurodevelopment from the pluripotent stage onward. Using isogenic hESC and organoid models, they identify an early naïve-like transcriptional drift, abnormal EMX1 timing, and convergent defects in cortical lineage allocation, revealing continuous developmental vulnerability in Rett syndrome.

Human organoids are considered physiological models that reflect human physiology; however, their applications in drug screening studies are limited. To develop a fundamental treatment for recurrent Crohn’s disease (CD), in which tumor necrosis factor (TNF) is a key pathogenic factor, we conducted phenotypic drug screening to prevent TNF-induced cell death in human intestinal epithelial organoids. Glycyrrhizin, a natural product of licorice root, dose-dependently blocked TNF-induced cell death in organoids but not in TNF-sensitive L929 cells; L929 cells exhibited necroptosis, whereas organoid-derived cells preferentially showed apoptosis upon TNF treatment, determining the specificity of glycyrrhizin. Glycyrrhizin inhibited downstream caspase-8 signaling, which is essential for TNF-dependent apoptosis, and ameliorated intestinal inflammation in vivo. These results demonstrate that glycyrrhizin may be a novel therapeutic compound for CD and highlight the importance of using organoids for phenotypic drug screening. Graphical abstract Highlights•Human intestinal organoids are sensitive to TNF-induced cytotoxicity•We developed an assay system to screen for compounds that resist TNF in organoids•Glycyrrhizin inhibited TNF-induced apoptosis but not necroptosis in organoids•Glycyrrhizin ameliorated intestinal inflammation in a murine model in vivo Takahashi et al. developed a high-throughput phenotypic screening platform using human intestinal organoids, which exhibited higher sensitivity to TNF than conventional intestinal epithelial cell lines, and identified glycyrrhizin that protected TNF-induced cell death both in organoids and in vivo. Phenotypic screening using organoids, as demonstrated in this study, paves the way for drug development and chemical biology.

Introduction: Extensive thermal injury remains a formidable clinical challenge, primarily due to the profound deficit of autologous donor skin, which necessitates prolonged hospitalization and escalates healthcare expenditures. While human embryonic stem cells (hESCs) offer a theoretically inexhaustible source for regenerative therapy, optimizing their differentiation and engraftment remains critical for clinical translation. Methods: We used a three-stage protocol to induce the differentiation of hESCs into keratinocytes (KCs). To optimize the delivery of hESC-derived keratinocytes (EKCs), human dermal fibroblasts (HFBs) were utilized to provide essential extracellular matrix (ECM) and microenvironmental support. The two cell types could self-assemble into 3D spheroids. After optimizing the size and cell proportion, these spheroids were subsequently transplanted onto full-thickness dorsal wounds in immunodeficient mice to evaluate their regenerative capacity. Results: hESC-derived keratinocytes exhibited the expression of stage-specific epidermal markers, confirming high differentiation efficiency. In vitro, EKCs demonstrate the capacity to form stratified epidermal structures. By self-assembling into spheres with dermal fibroblasts, the EKCs demonstrated successful engraftment and sustained survival in vivo. The transplantation of these 3D spheroids significantly accelerated wound closure and re-epithelialization compared with controls. Conclusions: This study establishes a robust cell therapy approach characterized by a short preparation cycle with high differentiation efficiency and high transplantation survival rate, offering a novel strategy for the treatment of extensive skin defects.

Embryonic stem cells represent a valuable germplasm resource with significant implications for breed conservation, development, and utilization. However, the scarcity of genetic resources in endangered species poses a fundamental constraint on obtaining gametes for embryonic stem cell derivation. Therefore, generating embryonic stem cells from somatic cell nuclear transfer blastocysts offers an optimal alternative for conservation cloning. In this study, we established ApèiJiaza somatic cell nuclear transfer ESCs (APNT-ESCs) from cloned embryos, using ApèiJiaza cattle ear fibroblasts as nuclear donors. APNT-ESCs could be passaged for over 30 generations in vitro, exhibiting high expression of key pluripotency markers, genomic stability, and the ability to form embryoid bodies and differentiate into cell types of all three germ layers. This research established an effective biotechnological framework for the genetic conservation of other endangered species lacking accessible gametes.

Background: MSCs possess strong immunoregulatory properties and play a central role in maintaining immune homeostasis by limiting inflammatory responses. Their function is highly plastic and influenced by environmental cues, including viral signals. How SARS-CoV-2-derived antigens affect MSC immunoregulation remains incompletely understood. This study aimed to investigate the impact of SARS-CoV-2 peptides on MSC-mediated immune modulation of T-cells. Methods: MSCs were stimulated directly with SARS-CoV-2 spike protein S peptides or cocultured with SARS-CoV-2 peptide-activated T-cells. TLR4 surface expression and receptor downstream signaling were assessed to evaluate pathway activation. MSC immunoregulatory function was analyzed by measuring suppression of TNF-α and IFN-γ expression and induction of CD4+FOXP3+ regulatory T-cells. TLR4 inhibition and lipopolysaccharide (LPS) stimulation were used to examine pathway specificity and interaction. Results: SARS-CoV-2 peptides activated TLR4-associated signaling in MSCs, increasing TLR4 expression and NF-κB phosphorylation. Peptide-treated MSCs showed impaired suppression of pro-inflammatory cytokines and reduced induction of regulatory T-cells. TLR4 inhibition prevented these effects. LPS induced similar effects, while combining LPS and peptide stimulation partially restored physiological T-cell cytokine suppression. Conclusions: SARS-CoV-2 peptides modulate MSC immunoregulatory function on T-cells via TLR4-dependent mechanisms.

Resident cardiac macrophages, derived from primitive yolk sac precursors during embryogenesis, have increasingly been recognized for their distinct phenotype and functions in regulating homeostasis of the human heart. However, the profile of their extracellular vesicles (EVs) in cardiac signaling and regulation remains uncharted. Here, we employ differentiation of human pluripotent stem cell-derived primitive macrophages (Mac), harvesting their secreted EVs and performing in-depth characterization of associated microRNAs (miRNAs). Primitive macrophages secreted nanoscale EVs that expressed canonical EV markers, and miRNA sequencing highlighted a diverse and unique profile of miRNAs when compared to EVs sourced from other principal cardiac cell lineages and published data from monocyte-derived cells. In particular, we noted the abundance and enrichment of vascular-modulatory let-7 miRNAs and miR-126-3p. Functional screening of Mac-EVs in a 3D model of in vitro cardiac vasculogenesis confirmed enhanced early endothelial cell organization and branching. Establishing a reference for the human Mac-EV miRNome enables further hypothesis-driven mechanistic tests of Mac-EV miRNAs in mediating cardiac physiology and disease, opening the door to identification of therapeutic targets and modalities for cardiac repair.

The potential of the immune system to decrease cancer progression is widely recognized and has led to the development of innovative anti-cancer immunotherapies. Here, we studied human macrophages derived from genetically engineered iPSCs (iMac) with angiotensin-converting enzyme (ACE) expression regulatable by a doxycycline (dox)-inducible promoter as a novel anti-cancer immunotherapy. Increased ACE expression in iMac (cells now termed ACE-iMac) augments polarization towards an M1 macrophage phenotype characterized by increased production of proinflammatory cytokines, reactive oxygen species, nitric oxide, and an RNA profile indicating an aggressive immune response. ACE-iMac kills tumor cells in vitro significantly better than iMac. In vivo, studies using tumor xenografts for melanoma, breast cancer, and head and neck squamous cell carcinoma (HNSCC) showed a highly significant 3.4- to 7.2-fold reduction in solid tumor size following ACE-expressing ACE-iMac immunotherapy as compared to results with iMac. To further investigate the impact of ACE on human anti-tumor responses, we developed a humanized BLT-NSG mouse model with a fully functional adaptive immune system. Here, ACE-iMac treatment significantly reduced the growth of human melanoma xenografts by enhancing the activation of human T cells and NK cells. In conclusion, enhancing ACE expression in human-derived macrophages (ACE-iMac) greatly amplifies their anti-cancer phenotype, offering a compelling new therapeutic strategy with the potential to improve clinical outcomes for cancer patients.

Multi-kilobase knock-ins (KIs) are a necessary, yet challenging type of genome editing to create and characterize in cell lines and animals. The combination of rAAV donor transduction and electroporation of single-cell mouse embryos with Cas9/gRNA ribonucleoprotein complex enables highly efficient KI, but the insert size is limited by the viral packaging capacity. Here, we report the creation of up to 6.7 kb precise KI achieved in one step by using three rAAVs designed to insert one after the other. To fully characterize the edited genome with large KIs, we developed LOCK-seq (LOng-read sequencing of Captured Kilo-base targets), where relevant genomic regions are enriched via hybridization, achieving over 100-fold greater coverage compared with other long-read methods with enrichment. LOCK-seq simultaneously detects the presence of precise KI alleles, imprecision in the insert and donor concatenation, genotypes of non-KI alleles, and more importantly, uniquely identifies and localizes random integration of the full or partial donor(s). Additionally, the multi-rAAV donor approach is successfully applied to cell lines, including lines intolerant of plasmid DNA, whereas LOCK-seq reliably and efficiently screens for KI clones. Together, the two approaches significantly improve the creation and precision of knock-in models.

Cryptococcus gattii is an emerging fungal pathogen that is acquired through the respiratory tract and causes invasive infections in both immunocompromised and otherwise healthy people. Many of these apparently immunocompetent patients are subsequently found to have autoantibodies against the pleiotropic cytokine GM-CSF. In this study, we investigated the potential role of GM-CSF (or CSF2) in the host response to C. gattii using a murine model of infection. Interestingly, infected Csf2-/- mice were found to have significantly improved survival and decreased lung fungal burden compared to wild type (WT) mice. We determined that during C. gattii infection, GM-CSF promotes the differentiation of monocytes into alveolar and interstitial macrophages. When these macrophages are ablated in CCR2-DTR+ mice, there is a corresponding improvement in survival with decreased lung fungal burden, thus phenocopying Csf2-/- mice. WT bone-marrow derived macrophages challenged with C. gattii and interstitial and alveolar macrophages from infected WT mice are unable to undergo M1 polarization, suggesting that monocyte-derived macrophages (moMacs) are rendered permissive for fungal proliferation. Therefore, GM-CSF and moMacs mediate immune responses that are harmful to the host. We further found that GM-CSF and moMacs preferentially promote the influx of eosinophils over neutrophils into the infected lung which is associated with substantial inflammatory lung pathology. Ablation of neutrophils using Mrp8cretg iDTR+ mice significantly increased C. gattii burden in the lungs, indicating that GM-CSF and moMacs block the entry of these beneficial, fungal-clearing granulocytes during infection. Altogether, our results show that GM-CSF plays a key role in impeding host anti-fungal responses to C. gattii by coordinating monocyte-derived macrophages and granulocyte activity and crosstalk. Author summaryCryptococcus gattii is an environmental fungus that can cause severe lung and brain infections after inhalation through the respiratory tract. C. gattii causes disease in patients with known immune deficits but also in individuals that are apparently healthy. Studies on otherwise healthy people who become infected with C. gattii suggest that they may have a previously unrecognized problem involving granulocyte macrophage-colony stimulating factor (GM-CSF), a cytokine, or messenger protein, that is an important part of the immune system. Here, we investigate the role of GM-CSF in the immune response to C. gattii using a mouse model of infection. We find that C. gattii increases GM-CSF in the lungs, leading to the influx of immune cells, including monocyte-derived macrophages and eosinophils, while inhibiting the entry of neutrophils. The macrophages and eosinophils allow the fungus to proliferate and cause inflammatory damage to the lungs, which is ultimately fatal. The absence of neutrophils also contributes to fungal growth, as these immune cells would otherwise be able to help kill the fungus. Our study provides new insight into how GM-CSF regulates immunity to C. gattii and has important implications as to the mechanisms that govern susceptibility to this infection.

CD4+ T helper (TH)-17 cells play a pivotal role in mucosal immune defense and are implicated in autoimmune diseases and cancer. Although Th17 cell plasticity is well-studied in mice, the factors driving their transition between pro-inflammatory and immunomodulatory states in humans remain less understood. Our study explored the transcriptional and epigenetic landscapes of single-cell cultures of human memory TH17 cells, focusing on clones that produce either immunomodulatory IL-10 or pro-inflammatory IFNγ and IL-22. We found that IL-10+ TH17 cells exhibit a T cell exhaustion-like profile with increased CTLA-4 expression and reduced IL-2 levels, while Ikaros zinc finger (IkZF) transcription factors, Aiolos and Eos, are differentially expressed in IL-10+ and IL-22+ TH17 cells, respectively. While exogenous IL-2 promotes IL-10 production in TH17 cells, lenalidomide induces IL-2 and promotes inflammatory TH17 cells, shifting TH17 cells towards a pro-inflammatory phenotype by reducing IL-10 and increasing IL-22 and IFNγ levels. Conversely, upregulation of Eos enhanced pro-inflammatory cytokine production. These findings highlight the crucial role of IkZF transcription factors in regulating human TH17 cell functions. Moreover, single-cell RNA sequencing of PBMCs from lenalidomide-treated patients confirmed an enrichment of inflammatory signatures, including interferon and IL-2/STAT5 pathways in TH17 cells. The ability to modulate this axis through targeted interventions, such as lenalidomide-induced Aiolos degradation or enforced Eos expression, presents new therapeutic opportunities for managing TH17 cell states in cancer and autoimmune diseases.

SummaryT cells are the central effectors and regulators of the adaptive immune response. This protocol provides a step-by-step approach for isolating and enriching total T cells by negative selection from complex murine tissues, including bone marrow (BM), liver, heart, and kidneys. We describe steps for tissue harvesting, preparation of single-cell suspensions, and immunomagnetic enrichment. We then outline procedures for flow cytometric assessment of cell purity and viability. This protocol enables efficient recovery of high-quality T cells for reliable downstream analyses. Graphical abstract Highlights•Isolation of leukocytes from murine BM, liver, heart and kidneys•Non-enzymatic dissociation of kidney and heart tissue•Protocol for immunomagnetic enrichment of T cells•Flow cytometry analysis of T cell purity and viability Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. T cells are the central effectors and regulators of the adaptive immune response. This protocol provides a step-by-step approach for isolating and enriching total T cells by negative selection from complex murine tissues, including bone marrow (BM), liver, heart, and kidneys. We describe steps for tissue harvesting, preparation of single-cell suspensions, and immunomagnetic enrichment. We then outline procedures for flow cytometric assessment of cell purity and viability. This protocol enables efficient recovery of high-quality T cells for reliable downstream analyses.

Objective and designChronic rhinosinusitis (CRS) is a heterogeneous inflammatory disease of the paranasal sinuses, which is divided into CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). CRSwNP is typically caused by type 2 inflammation, which is characterized by elevated IL-4 and IL-13 levels, impairment of the epithelial barrier, and tissue remodeling. While the involvement of immune cells is well known, it remains unclear to what extent structural cells intrinsically maintain disease-specific functional programs. The aim of this study was to determine whether epithelial cells and fibroblasts derived from CRSwNP and CRSsNP differ in their barrier properties, inflammatory reactivity, and type 2-associated functional characteristics.MethodsAir–liquid interface (ALI) epithelial cultures and primary fibroblast cultures were generated from CRSwNP and CRSsNP tissue. Epithelial barrier integrity was assessed by transepithelial electrical resistance (TEER), and inflammatory responses to TLR stimulation were analyzed by qRT-PCR. Fibroblast migration was evaluated using scratch assays. Cellular responses to IL-4/IL-13 with or without Dupilumab were quantified by qRT-PCR.ResultsCRSwNP-derived epithelial cells exhibited delayed tight junction formation and impaired differentiation compared to CRSsNP cells. Poly(I:C) stimulation induced stronger expression of Th2-associated cytokines in CRSwNP cultures. CRSwNP fibroblasts showed reduced migratory capacity and a heightened induction of Th2 cytokines and extracellular matrix genes following IL-4/IL-13 stimulation relative to CRSsNP fibroblasts.ConclusionEpithelial cells and fibroblasts derived from CRSwNP retain disease-associated type 2 characteristics in vitro, indicating persistent disease-aligned programmed functional alterations of the polyp microenvironment. In contrast, CRSsNP-derived cells lacked comparable enhanced type 2 responsiveness. These findings support CRSwNP as a distinct, self-sustaining inflammatory endotype and underscore the value of patient-derived models for investigating disease mechanisms and targeted therapies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12950-026-00497-7.