Stem cell-based embryo models provide innovative ways to explore the principles of human development but also pose new challenges for regulation. Recent guidelines have argued for case-by-case evaluation of stem cell-based embryo model research but it is still unclear what exactly such oversight should consider. Here we argue that effective review requires the identification of a set of attributes by which to evaluate research proposals. To define these, the underlying ethical values and morally relevant biological features present in embryo models must be identified to enable the practical implementation of oversight. We propose that developmental stage, tissue/organ integrity, fetal potential and the capacity to form neural circuits should be used together for evaluation purposes. We also highlight the importance of the wider context, including the proposed uses of embryo models, public perception and individual researcher responsibilities, and thus provide a general framework for regulatory consideration of human embryo model research.

Cell-type-specific promoters are used in gene therapy to restrict expression of the therapeutic payload. However, these promoters often have suboptimal strength, selectivity and size. Here, leveraging recent insights into the function of enhancers, we developed synthetic super-enhancers (SSEs) by assembling functionally validated enhancer fragments into multipart arrays. Focusing on the core SOX2-driven and SOX9-driven transcriptional regulatory network in glioblastoma stem cells (GSCs)1, we engineered SSEs with robust activity and high selectivity. Single-cell profiling, biochemical analyses and genome-binding data indicated that SSEs integrate neurodevelopmental and signalling-state transcription factors to trigger the formation of large multimeric complexes of transcription factors. Moreover, GSC-selective expression of a combination of cytotoxic (HSV-TK and ganciclovir) and immunomodulatory (IL-12) payloads, delivered using adeno-associated virus vectors, as a single treatment led to curative outcomes in a mouse model of aggressive glioblastoma. Notably, IL-12 induced an immunological memory that prevented tumour recurrence. The activity and selectivity of the adeno-associated virus and SSE were validated using primary human glioblastoma tissue and normal cortex samples. In summary, SSEs harness the unique core transcriptional programs that define the GSC phenotype and enable precision immune activation. This approach may have broader applications in other contexts when precise control of transgene expression in specific cell states is necessary.

The human maternal-fetal interface is characterized by mosaic intermingling of maternal and fetal cells1. Yet the underlying cellular, molecular and spatial programmes remain incompletely defined. Here we generate a comprehensive atlas of the human maternal-fetal interface across normal pregnancies from early gestation to term by integrating large-scale paired single-nucleus transcriptomic and chromatin accessibility profiling with submicrometre-resolution spatial transcriptomics and CODEX multiplex protein imaging2, substantially boosting the spatiotemporal resolution of prior research3. This framework delineates common and transient cell types, states and spatial niches across the fetal and maternal compartments, reconstructs transcriptional programmes that guide cytotrophoblast and decidual stromal cell differentiation, and resolves recurrent architecture structural units that build this interface. We identify previously unrecognized arterial endothelial state transitions during cytotrophoblast-mediated spiral artery remodelling and develop a machine learning model that predicts cytotrophoblast invasiveness from transcriptomic signatures. We further discover a decidual stromal cell subtype that suppresses cytotrophoblast invasion via endocannabinoid signalling at the human maternal-fetal interface. By integrating the atlas with genome-wide association data, we pinpoint maternal and fetal cells that are most vulnerable to pre-eclampsia, preterm birth or miscarriage. This resource provides a comprehensive spatially resolved single-cell multiomic reference of the human placenta and decidua and offers a framework for decoding their normal and disordered development.

β-Thalassaemia is caused by reduced or absent production of β-haemoglobin1-4. Previously, we performed laboratory-scale electroporation of CD34+ haematopoietic stem and progenitor cells from patients with β-thalassaemia using a transformer base editor5,6. The aim was to target the binding motif of the transcription repressor BCL11A in the HBG1 and HBG2 promoters7 to reactivate fetal haemoglobin (HbF) production. Here we present results of a phase 1 clinical trial (ClinicalTrials.gov identifier: NCT06024876) of five patients who received autologous CD34+ cells modified using a transformer base editor at clinical scale (CS-101). With a median follow-up of 23.0 months after CS-101 infusion, the median times to neutrophil and platelet engraftment were 16 days and 25 days, respectively. Moreover, all patients had stopped red blood cell transfusions, with a median time to the last transfusion of 18 days after CS-101 infusion. The mean total haemoglobin and HbF concentrations were 12.4 ± 1.0 and 11.5 ± 0.9 g dl-1, respectively, at month 3 after infusion. These levels remained at similar or higher levels throughout the follow-up period, which indicated rapid haematopoietic reconstitution. The adverse events of CS-101 were generally consistent with those of busulfan myeloablative conditioning and autologous haematopoietic stem and progenitor cell transplantation. No deaths or cancer occurrences were reported. In summary, CS-101 can lead to rapid and sustained increases in both total haemoglobin and HbF levels, which resulted in early and enduring transfusion independence.

This study evaluated the effects of 17% trisodium EDTA and 15% disodium EDTA (EDTAd), with or without activation, on SCAP response, antimicrobial activity and dentine chemical alterations. SCAP viability (Live/Dead) and metabolic activity (Alamar Blue) were assessed, along with antibiofilm and intratubular disinfection against Enterococcus faecalis and Streptococcus mutans, after irrigation with EDTA or EDTAd delivered by conventional irrigation (CI), ultrasonic activation (UA) or diode laser activation (LA). Dentine alterations were analysed by Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). EDTA preserved higher SCAP metabolic activity, particularly at early time points (p < 0.05), while cytotoxicity did not differ among groups. Antibiofilm and intratubular antimicrobial effects were similar across irrigants and activation methods (p > 0.05). EDTAd caused greater alterations in the amide III/phosphate ratio (p < 0.05), with increased smear-layer removal and surface irregularities. EDTA showed greater biological compatibility and fewer dentine alterations, whereas EDTAd induced more pronounced structural changes without antimicrobial differences.

Oxidative stress limits mesenchymal stem cell (MSC) efficacy in tissue repair by reducing retention and survival at injury sites. Endogenous production of trehalose may enhance MSC resilience and promote skin wound healing.

Fibrosis is a pathological process characterized by persistent fibroblast activation and excessive ECM accumulation. Aortic carboxypeptidase-like protein (ACLP), a secreted ECM protein that binds fibrillar collagen, is up-regulated in fibrotic tissues and promotes fibroblast differentiation through canonical TGFβ receptor signaling. We hypothesized that when presented within the collagen matrix, ACLP would engage integrin-dependent mechanical signaling pathways that contribute to fibrogenic activation. Using 10T1/2 mouse mesenchymal progenitor cells, we identify a previously unrecognized mechanism through which collagen-bound ACLP induces fibrogenic activation via β1 integrin-mediated signaling. Collagen-bound ACLP induced rapid cell spreading, increased β1 integrin activation, and promoted focal adhesion maturation. These adhesion events triggered activation of the GTPases RhoA and Rac1, accompanied by enhanced F-actin assembly and nuclear accumulation of myocardin-related transcription factor A, a key regulator of fibrogenic gene expression. Transcriptomic profiling revealed enrichment of focal adhesion, ECM-receptor interaction, and actin cytoskeletal pathways downstream of collagen-bound ACLP, which was conserved in primary adipose-derived stromal cells. Together, these findings establish collagen-bound ACLP as a matrix-derived cue that links ECM composition to integrin-dependent fibrogenic activation.

Artemisia argyi Levl. et Van. var. argyi cv. Qiai is a commercially important medicinal plant specifically cultivated in Qichun County, Hubei Province of Central China. Investigation of the antibacterial functional constituents of the endophytic Fusarium asiaticum QA-6, which is derived from Qiai, resulted in the characterization of nine previously undescribed polyketides fusarielins Q-Y (compounds 1-9) and two known congeners fusarielins M (compound 10) and F (compound 11). Structurally, compounds 1-9 possess a highly functionalized decalin moiety, whereas compounds 1-3 feature shorter polyene side chains compared to other fusarielins. The shortened chain in 2 and 3 likely results from a missed S-adenosyl-l-methionine (SAM)-dependent methylation, with 3 being an oxidation product of 2. Conversely, the truncation in 1 may stem from a missed acetyl-CoA addition. The isolated compounds were assayed for antibacterial activity against aquatic bacterial pathogens, among which, compounds 1, 5, 10, and 11 exhibited activity against specific Gram-negative strains, with compound 1 inhibited Aeromonas hydrophilia (MIC = 2.92 μM) and Vibrio alginolyticus (MIC = 5.84 μM), compound 5 targeted the aquatic pathogen Edwardsiella ictarda (MIC = 2.39 μM), compound 10 acted against E. ictarda, Vibrio harveyi, and V. parahaemolyticus (each with an MIC value of 2.60 μM), and compound 11 inhibited V. alginolyticus (MIC = 4.80 μM). All these antibacterial activities were equivalent to or superior to those of the positive control, chloramphenicol (MICs = 1.55 to 6.19 μM). In the preliminary screening against HeLa cells, none of the isolated compounds exhibited significant cytotoxic activity at the concentration 20 μM.

Cholesterol plays critical roles in prostate cancer (PCa) proliferation, immune evasion, and androgen signaling. PCSK9 regulates cholesterol metabolism. Its role in PCa remains unclear.

X-linked Alport syndrome (AS) is a progressive hereditary nephropathy caused by mutations in the COL4A5 gene, leading to end-stage renal disease in young adults. Current treatments are palliative with limited efficacy, highlighting a critical unmet medical need.

Solar ultraviolet A (UVA) radiation makes up nearly 95% of the ultraviolet spectrum at the Earth's surface, forming a major environmental exposure. High-dose UVA is a well-established driver of phototoxicity and tissue degeneration; however, the impact of low-dose UVA on stem-cell homeostasis remains incompletely defined. Here, we investigated whether UVA elicits a dose-dependent biphasic response in human adipose-derived mesenchymal stem cells (AMSCs) and evaluated PRPF40A as a molecular indicator of stemness status. AMSCs were exposed to single or fractionated UVA regimens (0.05-2 J/cm2), followed by assessments of viability, migration, oxidative stress, apoptosis and senescence, stemness programs, multilineage differentiation, and secretome remodeling. In cultured human AMSCs, UVA induced a biphasic dose-response pattern. An ultra-low dose (0.05 J/cm2) enhanced viability and migratory capacity, reduced basal reactive oxygen species, preserved NANOG/SOX2/OCT4 expression, and promoted chondrogenic potential. In contrast, doses of 0.5 J/cm2 or higher, particularly under cumulative exposure, induced oxidative injury, apoptosis, senescence-like changes, reduced stemness, impaired differentiation, and a pro-inflammatory senescence-associated secretory phenotype. Mechanistically, PRPF40A was inversely associated with stemness and showed dose-dependent co-regulation with TGF-β1 and NFAT-related readouts. Cyclosporine A altered PRPF40A- and TGF-β1-associated responses, supporting a provisional regulatory model linking PRPF40A, TGF-β1, and NFAT signaling under UVA stress. In vivo, HR-1 hairless mice showed minimal changes in skin appearance and stemness markers after low-dose UVA, whereas high-dose UVA caused dermal thinning and downregulation of cutaneous stemness markers, without reproducing a definitive biphasic pattern across the tested dose range. Collectively, our findings suggest that human AMSCs may exhibit a narrow in vitro threshold separating adaptive and injurious UVA responses, and identify PRPF40A as a candidate indicator of UVA-driven stemness destabilization.

The P347L RHODOPSIN-related retinal dystrophy leads an autosomal dominant Retinitis Pimentosa. Here we describe the generation of two isogenic human induced pluripotent stem cell (hiPSC) lines carrying the mutation c.1040C > T, p.Pro347Leu in the RHODOPSIN gene using CRISPR/Cas9 engineering from a control hiPSC clone. The two generated hiPSC lines can be differentiated in all the three germ layers, showed pluripotency makers expression and presented a normal karyotype. These hiPSCs will provide a new cell tool to better understand physiopathological mechanisms of retinitis pigmentosa and for the development of innovative treatment.

Exposure of a vital dental pulp following deep caries removal, accidental restorative procedures or trauma can severely affect the dentin-pulp complex in adult permanent teeth. To preserve a biologically functional healthy pulp, promote dentin repair, and minimize invasive and costly procedures, direct pulp capping is commonly used in vital pulp therapies. Despite its well-accepted therapeutic value, current pulp capping inorganic hydraulic calcium-silicate cements like mineral trioxide aggregate show limited targeted bioactive effects. Therefore, repurposing clinically approved drugs or investigational small bioactive agents targeting intracellular signaling pathways relevant to reparative dentinogenesis may translate into novel therapeutic approaches for dentin repair. Here, we review studies published over the past decade that explore two agents for potential repurposing in dentin repair: tideglusib, a glycogen synthase kinase‑3 inhibitor, and metformin, a widely used anti‑diabetic biguanide DESIGN: PubMed was used as the single, web-based search engine and database. Only studies using dental pulp stem cells in which the drugs were investigated as single agents were included.

Nicotine, the principal addictive component of cigarettes, is linked to cognitive decline and neurodegenerative alterations, likely through oxidative stress and impaired iron regulation in neurons. Yet, underlying molecular pathways remain unclear. This study examined the role of pulmonary neuroendocrine cells (PNECs) in smoke-induced neural changes. Using human pluripotent stem cells, we generated induced PNECs (iPNECs) to overcome culture limitations and performed mechanistic analyses. We found that nicotine exposure stimulates iPNECs to secrete exosomes enriched with serotransferrin, an iron-binding glycoprotein. Neurons internalizing these exosomes displayed elevated levels of transferrin receptor 1 (TFR1), divalent metal transporter 1, and duodenal cytochrome b, associated with ferritin accumulation, oxidative stress, and adenosine triphosphate depletion. Inhibition of TFR1 alleviated these effects. Furthermore, nicotine-triggered exosomes increased α-synuclein expression in neurons in a manner consistent with stress- and vulnerability-associated signatures observed in human lungs and nicotine-exposed mice, highlighting PNEC-derived exosomal signaling that may contribute to neuronal dysfunction.

Tauopathies, such as Alzheimer's disease and frontotemporal dementia, are common neurodegenerative diseases characterized by misfolding, hyperphosphorylation, and aggregation of tau. Molecular mechanisms underlying tauopathies are still poorly understood, which is in part due to a lack of human models autonomously developing major disease hallmarks. The formation of late-stage disease phenotypes may require adult tau isoform expression, which contributes to tau pathogenesis but is challenging to replicate in human stem cell-derived systems, thus impeding research on underlying mechanisms and drug development. Here, we show that induction of adult human brain-like 4R tau isoform expression enables cell-intrinsic formation of late-stage tauopathy hallmarks in induced pluripotent stem cell-derived neurons engineered to contain synergistic tau mutations without exogenous sources of tau pathology. Neurons accumulated seeding-competent and hyperphosphorylated tau in tangle-like structures. Furthermore, exclusive expression of mutant 4R in the absence of the 3R tau isoform disproportionately intensified pathology, resulting in abundant tau misfolding and aggregation. Last, we provide proof of principle that our model can be translationally applied both to test chemical disease modulators and evaluate human tau PET tracers. Collectively, our model corroborates the central role of 4R tau isoform expression for pathogenesis in human neurons and enables investigations to elucidate mechanisms underlying human tauopathy formation. Moreover, it may serve as a platform supporting urgently needed development of disease-modifying drugs.

Steroid-resistant acute graft-versus-host disease (SR-aGVHD) is the leading life-threatening complication after allogeneic hematopoietic stem cell transplantation. Therapeutic development is impeded by scarce knowledge of biological pathways leading to steroid resistance after aGVHD diagnosis. To gain insight into circulating immune cell subsets and their functions at the onset of aGVHD, we performed a single-cell deep phenotyping and transcriptome analysis on peripheral blood mononuclear cells from two cohorts of patients (discovery cohort, n = 53; and validation cohort, n = 32) with aGVHD before steroid treatment or without aGVHD. Frequencies of circulating immune subsets were not associated with steroid resistance. However, pathway analysis and inferred ligand-receptor interactions revealed major functional divergences between steroid-sensitive aGVHD (SS-aGVHD) and SR-aGVHD, suggesting that steroid resistance is an intrinsic property of immune cells before any treatment. SR-aGVHD was mainly associated with tumor necrosis factor-α (TNF-α) activation. Steroid resistance resulted from a specific cross-talk characterized by inflammasome and caspase-1 activation in monocyte subsets; by TNF-α/TNF receptor, interleukin-1β (IL-1β), IL-18, CCL3, and CCL4 signaling between myeloid and T cells; and by lower involvement of interferon-α and interferon-γ signaling pathways. Immune trajectories in CD8+ T cells demonstrated a direct transition from an early naïve state to a highly activated one. By contrast, SS-aGVHD involved gene signatures across multiple intermediate differentiation stages during cell-to-cell transitions into CD8+ T subsets. These findings provide evidence that steroid resistance is driven by intrinsic mechanisms already present at the onset of the alloimmune response, which may enable previously unknown therapeutic strategies.

While central circuits governing sleep are well-studied, the contribution of signaling from peripheral tissues remains a critical yet less understood aspect of sleep regulation. The highly conserved RNA-binding protein Pumilio (Pum) is a post-transcriptional regulator expressed in multiple tissues that influence systemic physiology, but its role in modulating basal sleep from peripheral tissues has not been established. Although Pumilio's function in central neurons has been linked to sleep homeostasis following deprivation, whether it regulates sleep through peripheral mechanisms remains unknown. Here, we use conditional genetic tools in the fruit fly Drosophila melanogaster to demonstrate that genetic manipulation of Pumilio targeting the intestinal stem cells (ISCs) and the endocrine corpus allatum (CA) regulates the transition to sleep. Reducing Pumilio function in either the ISCs or the CA independently and significantly accelerates nighttime sleep onset, while overexpression produces the opposite effect. This behavioral change is accompanied by widespread transcriptional alterations in the head, characterized by a robust upregulation of genes involved in cellular stress responses. Our findings reveal a previously unrecognized gut-endocrine-brain signaling axis and identify peripheral post-transcriptional regulation as a key input to the central control of sleep behavior.

Mitochondria are central to energy metabolism and cellular signaling, and mutations in mitochondrial DNA (mtDNA) can disrupt these processes and contribute to human disease. However, progress in defining how mtDNA variation influences adaptation, pathophysiology, and disease susceptibility has been limited by the lack of suitable animal models. Although recent base-editing approaches enable direct mtDNA modification, their low efficiency restricts the generation of diverse models reflecting human mtDNA variation. Here, we develop a scalable embryonic stem (ES) cell-based platform for efficient production of mtDNA mutant mice. Random mutagenesis using an error-prone mtDNA polymerase generates a broad spectrum of mtDNA mutations, which are transferred into ES cells via a multiplexed cybrid fusion strategy coupled with sensitive mutation detection. Optimized ES cell-embryo aggregation enables robust contribution of mtDNA mutant ES cells to host embryos, producing chimeric mice with germline transmission. Using this platform, we generate a library of 155 donor fibroblast lines carrying distinct homoplasmic single-nucleotide mtDNA mutations that produce diverse mitochondrial phenotypes, including impaired oxidative phosphorylation, increased reactive oxygen species, and altered mitochondrial membrane potential. We further generate 34 female C57BL/6 ES cell lines harboring 18 mtDNA mutations across a range of heteroplasmy levels, yielding multiple chimeric mice and achieving germline transmission for one mutation. These data reveal a strong correlation between mitochondrial function and early embryonic development, suggesting a minimal energetic threshold required for normal development. This scalable resource enables systematic investigation of mtDNA variation in physiology, adaptation, disease mechanisms, and therapeutic development.

Accurate interpretation of genetic lineage tracing data is often confounded by the specificity and sensitivity of promoter activity and recombination rates. Thus, reported values may misrepresent targeted event frequency. Here, we present a protocol to validate a genetic tool for marking "dedifferentiation" in Drosophila testes. We describe steps for long-term live imaging to observe reporter activation and detail procedures to estimate false-positive and false-negative rates by integrating these datasets into a mathematical model. This approach enables rigorous evaluation of system performance. For complete details on the use and execution of this protocol, please refer to Bener et al.1.

Children with Down syndrome (DS) have an elevated risk of developing myeloid leukemia in DS (ML-DS). In addition to mutations in GATA1, which generate the truncated isoform GATA1-short (GATA1s), ML-DS requires additional somatic gene mutations, most frequently in cohesion and polycomb repressive complex 2 (PRC2) genes. Here, we show that PRC2 insufficiency underlies ML-DS pathogenesis. Transplantation of Gata1s fetal liver cells followed by deletion of the cohesion subunit Stag2 and/or the PRC2 component Ezh2 induced megakaryocyte-biased differentiation and expansion of megakaryocytic progenitors, culminating in lethal myelofibrosis. Mechanistically, loss of Stag2 or Ezh2 reinforced Gata1s-driven reduced chromatin accessibility at erythroid transcription factor target loci in pre-megakaryocyte/erythroid progenitors (pre-MegEs), thereby promoting megakaryocytic skewing. Ezh2 loss attenuated the Gata1s-mediated global elevation of H3K27me3 in pre-MegEs, resulting in derepression of a broad set of PRC2 target genes and establishing a functionally PRC2-insufficient state. Similarly, Stag2 loss induced a moderate but significant degree of PRC2-insufficient state in Gata1s progenitors. Furthermore, chromosome 21-encoded miR-125b blocked megakaryocytic differentiation of Gata1s progenitors lacking either Stag2 or Ezh2 alone, but drove full transformation and expansion of CD150+Sca-1+c-Kit+ leukemic stem cell-like populations only upon concurrent loss of both Stag2 and Ezh2, leading to acute megakaryoblastic leukemia in mice. These findings reveal that cohesin and PRC2 insufficiencies converge on PRC2 dysfunction while exerting distinct epigenetic effects, and synergize with trisomy 21 and GATA1s to remodel the epigenetic landscape, driving progression from a preleukemic state to overt leukemia.