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.

Cytokinetic abscission genes are linked to cancers and developmental disorders, but the consequences of disrupted abscission in vivo remain under-explored. Previously we showed that in the forebrain of Cep55 knockout (KO) mouse embryos, a subset of neuroepithelial stem cells (NSCs) fail abscission and become binucleate, and some of those undergo p53-mediated apoptosis. Here we use the Cep55 KO to investigate how stochastic abscission failures in a polarized epithelium affect the epithelial architecture. We find that NSCs in Cep55 KO neuroepithelium have preserved epithelial polarity and integrity. However, they have enlarged apical membranes (called apical endfeet), longer primary cilia, and increased biciliation. We then test whether the enlarged apical endfeet arise from filling the space of apoptotic neighbors. Remarkably, blocking apoptosis does not rescue but exacerbates the phenotypes: extra-large apical endfeet have further increased multiciliation, supernumerary centrosomes, and abnormal or multiple nuclei, although epithelial polarity is maintained. These findings elucidate the importance of proper abscission in maintaining polarized epithelial structure, and reveal that p53-mediated apoptosis is a crucial guardian of tissue architecture when cell division defects arise during development and disease.

Nucleotides and coenzymes play critical roles in energy metabolism and cellular signaling and as building blocks of nucleic acids. This work addresses the challenges in the measurement of the phosphorylated metabolites using hydrophilic interaction liquid chromatography coupled with mass spectrometry, which facilitates the separation and detection of polar metabolites. Here, we present optimized HILIC-MS/MS methods for rapid analysis of polar metabolites including nucleotides and their derivatives in complex biological matrices, such as murine adipose, skeletal, and liver tissues, human plasma, and bacteria. The developed methodologies enable separation of key nucleotides and other phosphorylated metabolites within 6 min and cofactors such as NAD+, NADH, NADP+, and NADPH within 4 min. Validation of these methods demonstrated high accuracy, precision, and sensitivity and stresses the substantial impact of matrix effects. The applicability of the methods was also tested on 13C-labeling experiments with mouse pluripotent stem cells. Additionally, sample pretreatment techniques, such as liquid-liquid extraction and solid-phase extraction, were evaluated as a tool to decrease the negative impact of matrix effects in complex samples. This work enhances the analytical capabilities for nucleotide quantification in metabolomics, facilitating the study of metabolic pathways and disease markers.

Stress urinary incontinence (SUI) adversely impacts millions worldwide due to weakened pelvic floor muscles and urethral sphincter dysfunction. To date, there is a lack of effective non-surgical treatment for SUI, and no clear consensus has been reached on the optimal stem cell source under regenerative therapy. Existing studies have shown no precise molecular mechanisms underlying stem cell-mediated external urethral sphincter (EUS) regeneration. Therefore, we investigated the regenerative and reparative potential of our clinical-grade human amniotic fluid stem cells (hAFSCs) for treating SUI.

Hepatitis C virus (HCV) infection is a major cause of hepatocellular carcinoma (HCC). However, how HCV promotes hepatic progenitor cell (HPC) differentiation into hepatic cancer stem cells (HCSCs) remains unclear, particularly the role of β-catenin signaling and epithelial cell adhesion molecule (EpCAM) regulation in this process. We used mouse HPCs (HP14.5 and HP14.5-Core) and human HCC cell lines (Huh7, HepG2) to investigate HCV core function. Cells were transduced with AdCore, AdGFP, or shRNAs targeting EpCAM or β-catenin. Functional properties were evaluated using spheroid formation, ICG uptake, colony formation, migration, invasion, and in vivo tumorigenesis assays. EpCAM promoter activity and Wnt/β-catenin signaling were examined by luciferase reporter assays. Protein expression was measured by RT-PCR and Western blotting, while immunofluorescence and co-immunoprecipitation were used to analyze HCV core-β-catenin interactions. HCV core expression increased spheroid number, reduced ICG uptake, and accelerated tumor growth in xenograft and orthotopic mouse models, consistent with HPC differentiation toward HCSCs. EpCAM, CD133, CD44, and CD90 were strongly upregulated, whereas EpCAM silencing suppressed proliferation, migration, and invasion. Reporter assays revealed that HCV core enhanced EpCAM promoter activity and activated Wnt/β-catenin signaling. Immunofluorescence demonstrated β-catenin nuclear translocation, and co-immunoprecipitation confirmed direct interaction between HCV core and β-catenin. Knockdown of β-catenin reduced EpCAM, c-Myc, and cyclin D1 expression, diminished spheroid formation, restored ICG uptake, and significantly impaired tumorigenesis in vivo. The HCV core-β-catenin-EpCAM axis drives the conversion of HPCs into HCSCs. This pathway is a central mechanism in HCV-related HCC.IMPORTANCEHCV core protein directly interacts with β-catenin, driving its nuclear translocation and activating EpCAM expression. This promotes HPC differentiation into HCSCs, marked by increased spheroid formation, migration, invasion, and tumorigenesis. Silencing EpCAM or β-catenin reverses these effects, confirming that the HCV core-β-catenin-EpCAM axis is a key pathway in HCV-related HCC.

One nucleotide substitution in codon -18 of HLA-B*40:01:02:01 results in a novel null allele, HLA-B*40:636N.

The identification of cell types remains a major challenge. Even after a decade of single-cell RNA sequencing (scRNA-seq), reasonable cell type annotations almost always include manual non-automated steps. The identification of orthologous cell types across species complicates matters even more, but at the same time strengthens the confidence in the assignment. Here, we generate and analyze a dataset consisting of embryoid bodies (EBs) derived from induced pluripotent stem cells (iPSCs) of four primate species: humans, orangutans, cynomolgus, and rhesus macaques. This kind of data includes a continuum of developmental cell types, multiple batch effects (i.e. species and individuals) and uneven cell type compositions and hence poses many challenges. We developed a semi-automated computational pipeline combining classification and marker-based cluster annotation to identify orthologous cell types across primates. This approach enabled the investigation of cross-species conservation of gene expression. Consistent with previous studies, our data confirm that broadly expressed genes are more conserved than cell type-specific genes, raising the question of how conserved, inherently cell type-specific, marker genes are. Our analyses reveal that human marker genes are less effective in macaques and vice versa, highlighting the limited transferability of markers across species. Overall, our study advances the identification of orthologous cell types across species, provides a well-curated cell type reference for future in vitro studies and informs the transferability of marker genes across species.

Alzheimer's disease (AD) exhibits high genetic and clinical heterogeneity that limits therapeutic success. Patient-derived brain organoids and their extracellular vesicles (EVs) provide physiologically relevant models to study disease mechanisms and individualized drug responses.

The developing embryo harbors multiple hematopoietic programs, categorized as either intra-embryonic or extra-embryonic yolk-sac, that differ in their spatio-temporal origins and developmental potential. In the vertebrate embryo, the hematopoietic stem cell (HSC) derives from the definitive intra-embryonic hematopoietic program and is dependent on stage-specific retinoic acid (RA) signaling. We have recently modelled aspects of this developmental process in vitro using human pluripotent stem cells (hPSCs) and identified a KDR+CXCR4+ mesodermal population that generates definitive hematopoietic progeny in a uniquely RA-dependent manner. A subpopulation of this mesoderm expresses ALDH1A2, an enzyme involved in RA synthesis. Here, we sought to characterize the role of ALDH1A2 in the development of the human RA-dependent hematopoietic lineage and to map its mesodermal origin. Using two different engineered reporter hPSC lines, we show that specification of this lineage requires a functional ALDH1A2 enzyme at the mesoderm stage. Through functional analyses of different mesoderm subpopulations, we demonstrate that this RA-dependent lineage derives from ALDH1A2neg mesoderm by non-cell autonomous RA signaling. Collectively, these studies provide new insight into the differentiation trajectory of hPSCs towards the definitive hematopoietic lineage.

Short QT syndrome is a heritable arrhythmia disorder linked to sudden cardiac death. We recently identified that individuals with alternating hemiplegia of childhood (AHC), a rare neurodevelopmental disorder, can exhibit shortened corrected QT intervals and elevated risk for ventricular fibrillation. This is especially true for patients with AHC heterozygous for the recurrent ATP1A3-D801N variant, though the underlying cardiac mechanism remains unclear. We hypothesized that the D801N missense impairs Na+/K+-ATPase function, causing Ca2+ overload, shortened action potential duration (APD), and arrhythmias. Using in silico modeling and patient-derived induced pluripotent stem cell cardiomyocytes (iPSC-CMsD801N), we observed shorter APD, elevated intracellular and sarcoplasmic reticulum Ca2+ levels, and delayed afterdepolarizations (DADs) compared with WT. Additionally, increased Ca²+ influx via the Na+/Ca2+ exchanger (NCX1) during depolarization was observed in iPSC-CMsD801N. Simulations and in vitro experiments suggest that reduced ATPase function accelerated inactivation of L-type Ca2+ channels. Pharmacologic inhibition of NCX1 with ORM-10103 normalized APD and reduced DADs. These findings support a Ca2+-mediated mechanism for arrhythmogenesis in ATP1A3-D801N carriers and identify NCX1 as a potential therapeutic target.

Bronchopulmonary dysplasia (BPD) is a common and serious complication among preterm infants, particularly those born at extremely low gestational ages. It is primarily characterized by impaired alveolar and vascular development. Inflammation is increasingly recognized as a central mechanism in its pathogenesis. Both prenatal factors, such as intrauterine infection, and postnatal insults, including mechanical ventilation, oxygen toxicity, and infection, can trigger and sustain a dysregulated inflammatory response in the immature lung. This response involves the activation of inflammatory cells, such as neutrophils and macrophages, and the release of pro-inflammatory mediators, reactive oxygen species (ROS), and proteases. These factors disrupt critical developmental signaling pathways and contribute to alveolar simplification and abnormal vascular growth, which are the hallmark features of BPD. Current therapeutic strategies aim to limit these inflammatory processes and support lung development. Established interventions like caffeine and corticosteroids have demonstrated varying levels of effectiveness and safety. Emerging therapies-including anti-cytokine agents, inflammasome inhibitors, and stem cell-based approaches-offer promising avenues by specifically targeting the inflammatory cascade. Additionally, supportive strategies such as non-invasive ventilation, careful oxygen titration, and optimal nutrition play essential roles in reducing initial injury and facilitating recovery. Inflammation is a key mediator linking diverse perinatal insults to the disrupted lung development seen in BPD. A deeper understanding of the inflammatory mechanisms and timely, targeted interventions may offer improved outcomes for this vulnerable population.

Neural progenitor cells (NPCs) are promising candidates for cell replacement therapies, yet maintaining stemness while enabling expansion in chemically defined three-dimensional (3D) hydrogels remains a challenge. By tuning crosslink exchange kinetics, crosslinker functionality and stoichiometry, polymer phase separation behavior, and adhesive ligand presentation, a family of hydrogels was prepared to study the effects of stress relaxation timescale and network connectivity on NPC phenotype. Hydrogels with rapid relaxation and low connectivity promote expansion of NPCs as distributed single-cell networks that maintain stemness marker expression and differentiation capacity. NPCs embedded in slowly relaxing hydrogels maintained stemness marker expression through cell clustering but exhibited impaired proliferation and differentiation. Similarly, in the absence of integrin-binding cell adhesive ligands, NPCs also maintained stem cell marker expression but remained as clusters rather than distributed single-cell networks. Cadherin cell-cell contacts enable downstream β-catenin signaling and stemness maintenance, which are enhanced in rapidly relaxing, low connectivity networks. These findings identify a combination of network connectivity, stress relaxation timescale, and integrin-binding adhesive ligands as crucial design parameters for maintaining NPC stemness and differentiation capacity in 3D hydrogel networks.

Tyrosine kinase inhibitors (TKIs) targeting ABL1 catalytic activity have markedly improved Chronic Myeloid Leukemia (CML) outcomes, inducing unprecedented and durable therapeutic responses. However, while TKIs efficiently target committed leukemic progenitors, they fail to eradicate leukemic stem cells (LSCs), which may drive disease relapse. High BCR::ABL1 transcripts at diagnosis confer a proliferative and survival advantage and are associated with a higher risk of CML progression to the acute phase. Specifically Targeting the ABL Myristoyl Pocket (STAMP) compounds, including asciminib (ASC), provide a novel mechanism to inhibit BCR::ABL1 catalytic activity. ASC is FDA-approved for patients who have failed one or more TKIs, and its efficacy has been evaluated as monotherapy, and in combination with different TKIs, in T315I-positive or advanced-phase CML. We investigated the cytotoxic effects of ASC, alone or with imatinib (IM) or nilotinib (NIL), on committed progenitors and LSCs from CML patients expressing high or low BCR::ABL1 at diagnosis. ASC reduced BCR::ABL1-dependent survival and impaired clonogenicity in committed progenitors with low, but not high, BCR::ABL1 transcripts. ASC also disrupted LSC self-renewal, reducing both frequency and number of Long-Term Culture-Initiating Cells (LTC-ICs). When combined with IM or NIL, ASC restored TKI activity against LTC-ICs expressing high BCR::ABL1 transcripts, with the association of ASC and NIL reducing both LTC-IC division rates and LTC-IC-derived CFUs. These findings suggest that ASC, alone or with NIL, may target LSCs and improve outcomes in patients with high BCR::ABL1 expression at diagnosis.