Breast cancer metastasis remains the leading cause of mortality and frequently targets the bone. Breast cancer cells release soluble factors and extracellular vesicles that disrupt bone marrow (BM)/bone homeostasis, promoting osteoclastogenesis and the accumulation of senescent cells. In line with updated cancer hallmarks, senescent mesenchymal stem/ stromal cells (MSCs), osteoblasts, and osteocytes contribute to remodeling of the BM microenvironment, thereby favoring pre-metastatic niche (PMN) formation and subsequent bone metastasis. We previously demonstrated that untreated stage III-B breast cancer patients (BCPs) exhibit increased oxidative stress and elevated reactive oxygen species (ROS) levels, accompanied by senescent and functionally impaired BM-MSCs-key regulators of BM/bone homeostasis. In the present study, we sought to identify the molecular targets affected by oxidative stress that drive MSC senescence in these patients.

Focal post-traumatic cartilage lesions frequently progress to early osteoarthritis, highlighting the limited regenerative capacity of adult articular cartilage. Compared to native tissue, conventional surgical interventions often produce fibrocartilage with inferior biomechanical properties, representing a persistent therapeutic challenge. This review assessed preclinical studies exploring cartilage repair strategies using autologous mesenchymal stem cells (MSCs) in animal models. MSCs therapies demonstrated superior cartilage regeneration, matrix organization, and integration into the surrounding tissue compared to the control groups. The most efficient source was found to be bone marrow - derived mesenchymal stem cells (BM-MSCs) combined with biodegradable scaffolds, suggesting their potential in tissue engineering applications. Despite methodological heterogeneity across studies - including variations in stem cells sources, implant types, and deliver strategies - cumulative evidence strongly supports the regenerative potential of autologous MSCs for cartilage repair. Current research identifies key knowledge gaps, including the absence of standardized experimental protocols and limited insight into the mechanisms of tissue remodeling and maturation. Collectively, these gaps limit direct clinical translation, highlighting the need for further, standardized studies in large animal models with long-term follow-up (>2 years) to assess integration, functional maturation, and the full regenerative potential of the repair tissue.

Dysfunctional hematopoietic stem cells (HSC) drive the initiation of myelodysplastic syndromes (MDS), yet the genome-wide DNA methylation landscape of primitive MDS HSCs and its mechanistic contribution to disease pathogenesis remain poorly defined. Here, we establish single-base resolution DNA methylomes of bone marrow HSCs from MDS patients and healthy donors. We uncover the widespread hypermethylation in CpG islands, alongside hypomethylation in repetitive elements such as Alu. Differentially methylated regions are enriched for genes involved in cancer-related pathways, as well as extrinsic signaling pathways and intrinsic transcriptional networks essential for HSC function. Among these, we identify GFI1 and BMI1 as key targets of DNA methylation dysregulation in MDS. Notably, using either the MDS or a TET2-deficient mouse model, we demonstrate that loss of TET2, a frequently mutated epigenetic regulator in MDS, induces promoter hypermethylation and transcriptional repression of GFI1, contributing to the expansion of the MDS or aged hematopoietic stem and progenitor cell pool. Our study not only charts the base-resolution DNA methylome of human MDS HSCs but also reveals a TET2-GFI1 axis that safeguards HSC homeostasis. These findings provide mechanistic insight into how aberrant DNA methylation drives HSC dysfunction in MDS and offer an epigenomic resource for discovering regulators and therapeutic targets at the stem cell level.

Poor outcomes in proteinuric kidney diseases are challenging to successfully manage therapeutically due to the heterogeneity of underlying disease pathogenesis and associated risk for progression. The role of cytoskeleton-associated proteins, including the scaffolding protein Anillin (ANLN), are of specific interest in kidney disease given the importance of actin dynamics in the kidney's specialized epithelial cell types. In this study, we identify the prevalence of genetic variants in ANLN , the gene encoding ANLN, in a cohort of deeply phenotyped individuals with non-diabetic proteinuric kidney disease. Thirty-one individuals (of 864 genotyped) harbor heterozygously expressed variants in ANLN ; 7 unrelated individuals shared the same variant (I1109V) in the C-terminal pleckstrin homology (PH) domain, a region necessary for interaction with the plasma membrane. Kidney organoids generated from I1109V induced pluripotent stem cells from 1 of these individuals showed increased epithelial cell mitogen-activated protein kinase 8 network activity and apoptosis, which was enhanced by tumor necrosis factor alpha (TNF-α) and phenocopied by actin polymerization inhibition. TNF-α-treated I1109V organoids also exhibited tubular lumen expansion. Knockdown and re-expression of the analogous ANLN variant in Xenopus laevis embryonic epithelia resulted in defects in cell-cell junction dynamics including wavy cell membranes exhibiting increased transverse movements as well as abnormal junctional F-actin remodeling in response to mechanical stress and leaky barrier function. Taken together, these results indicate that enhanced tubular epithelial cell death, perturbed cell-cell contacts and barrier function defects are associated with a novel ANLN variant discovered in individuals with non-diabetic proteinuric kidney disease.

Multilineage-differentiating stress-enduring (Muse) cells, a subpopulation of mesenchymal stem cells (MSCs) marked by stage-specific embryonic antigen 3 (SSEA3), exhibit superior regenerative capacity compared to conventional MSCs, including enhanced tissue homing, pluripotency, and paracrine effects. However, their natural scarcity (1-5% in MSC populations) limits therapeutic scalability. In this study, we developed a five-compound small molecule method to obtain compound-enriched Muse-like MSCs and assessed their potential use in treating severe veterinary chronic diseases, such as hepatitis and chronic kidney disease (CKD).

Communication between gut microbiota and immune cells within the brain is essential for neurotypical development. Specifically, microglia are known to play a key role in regulating and supporting neural progenitor stem cell production during brain development, and are sensitive to changes in the maternal gut microbial composition during perinatal development. Here, we employed a germ-free (GF) porcine paradigm to examine how the absence of the microbiome affects microglial dynamics during a key epoch of brain development. We utilized automated software to evaluate microglial density and morphology across three developmentally significant regions: the ventricular/subventricular zone (VZ/SVZ), the prefrontal subcortical white matter (PFCSWM), and layers II/III of the prefrontal cortex (PFCII-III). We found no significant differences in microglial morphology or density in the VZ/SVZ or PFCSWM. In contrast, the PFCII-III of P16 piglets exhibited an increase in microglia density paired with morphologies indicative of an activated/reactive functional state. Notably, these effects were identified with no overall changes in microglial density in any of the regions assessed. Transcriptomics on RNA isolated from the PFCII-III revealed a significant upregulation of genes related to neuroinflammation, in agreement with a region-specific microglial and immune response in the absence of microbial colonization during postnatal development. Together, these findings build on the limited knowledge available on how microbiota influence brain development in large animal model organisms with high similarities to human brain anatomy and developmental trajectories.

Investigating single-cell dynamics and morphology in tissues and embryos requires highly accurate quantitative analysis of microscopy images. Despite significant advances in the field of bioimage analysis, even the most sophisticated segmentation and tracking algorithms inevitably produce errors (e.g. : over segmentation, missing objects, miss-connected objects). Although error rate may be small, their propagation throughout a time-lapse sequence has catastrophic effects on the accuracy of tracking and extraction of single cell parameters. Extracting single cell temporal information in the context of tissue/embryo requires thus expert curation to identify and correct segmentation errors. In the movies commonly used in developmental biology and stem cell research, both the number of imaged cells and the duration of recording are large, making this manual correction task extremely time-consuming. This has now become a major bottleneck in the fields of development, stem cell biology and bioimage analysis. We present here EpiCure (Epithelial Curation), a versatile tool designed to streamline and accelerate manual curation of segmentation and tracking in 2D movies of large epithelial tissues. EpiCure uses temporal information and morphometric parameters to automatically identify segmentation and tracking errors and provides user-friendly tools to correct them. It focuses on ergonomics and offers several visualization options to help navigating in movies of tissue covering a large number of cells, speeding up the detection of errors and their curation. EpiCure is highly interoperable and supports input from a wide range of segmentation tools. It also includes multiple export filters, enabling seamless integration with downstream analysis pipelines. In this paper, using movies from several animal models, we highlight the importance of curating cell segmentation and tracking for accurate downstream analysis, and demonstrate how EpiCure helps the curation process for extracting accurate single cell dynamics and cellular events detection, making it faster and amenable on large dataset.

Enhancer-regulating epigenetic modifiers play critical roles in normal physiological processes and human pathogenesis. The major enhancer regulator paralogs MLL3 and MLL4 (MLL3/4) belong to the lysine methyltransferase 2 (KMT2) family, which catalyzes the methylation of lysine 4 on histone H3 (H3K4me). MLL3/4 are required for enhancer activation and are essential for mammalian development and stem cell differentiation. Recent studies have linked MLL3/4 with different metabolic pathways in the context of stem cell self-renewal and cancer cell growth; however, the underlying mechanisms remain elusive. Here, we utilize Seahorse extracellular flux analysis, stable isotope tracing, stem cell biology techniques, and transcriptomic analysis to investigate the functional relationship of MLL3/4, cellular respiration, and stem cell differentiation. Our results indicate that the loss of MLL3/4 impairs glycolytic activity and mitochondrial respiration in murine embryonic stem cells by downregulating the rate-limiting glycolytic enzyme Hexokinase 2 (HK2) and impairing the function of the Alpha-ketoglutarate dehydrogenase (OGDH) complex. Furthermore, simultaneously overexpression of HK2 and OGDH rescues defects in both cellular respiration and differentiation caused by MLL3/4 loss. Taken together, our study reveals a novel mechanism by which epigenetic machineries such as MLL3/4 govern the differentiation of pluripotent stem cells and facilitates the understanding of disease pathogenesis driven by enhancer malfunction.

Primary open-angle glaucoma (POAG), a leading cause of irreversible blindness, has a strong genetic basis. The Primary Open-Angle African Ancestry Glaucoma Genetics study previously identified 46 risk loci. To pinpoint causal variants and their corresponding effector genes, we analyzed gene expression, chromatin accessibility, and conformation in two ocular cell-types: trabecular meshwork cells (hTMCs) and retinal ganglion cells derived from induced pluripotent stem cells (hiPSC-RGCs). We identified 24 candidate genes in hTMCs and 56 in hiPSC-RGCs. The ARHGEF12 gene was selected for further validation because it was nominated by local and distal promoter interactions in both cell-types and has reproducible prior evidence of its association with POAG. While its role in hTMCs is established, its function in RGCs is unclear. hiPSC-RGCs generated from a POAG donor homozygous for the risk allele showed reduced ARHGEF12 expression, altered morphology, and disrupted neuronal activity. This framework enables functional evaluation of additional POAG risk variants.

Despite lacking a robust interferon response, pluripotent stem cells remain highly resistant to viral infection, in part through the constitutive expression of immune genes traditionally classified as interferon-stimulated genes. While interferon signaling has been shown to be incompatible with the maintenance of pluripotency, the molecular mechanisms underlying this relationship remain poorly understood. Here, we investigate the transcriptional response of human embryonic stem cells (hESCs) to infection with a potent activator of the interferon response, an influenza A virus mutant lacking the viral NS1 protein. Single-cell RNA sequencing revealed that while most hESCs remain unresponsive to infection, a distinct subpopulation expresses type I and III interferons. Notably, only interferon-expressing cells mounted a robust antiviral response, characterized by strong induction of interferon-stimulated genes. In contrast to the bulk hESC population, interferon responding cells exhibited reduced expression of core pluripotency factors as well as negative regulators of interferon signaling, such as SOCS1 and SPRY4. Depletion of SOCS1 enabled hESCs to respond robustly to interferon stimulation, showing that this negative regulator is a key suppressor of interferon signaling in pluripotent stem cells. We further show that SOCS1 and additional negative regulators of IFN signaling are intrinsically expressed in hESCs and are transcriptionally controlled by pluripotency factors, such as NANOG, SOX2 and OCT4. Together, our findings support a model in which pluripotency factors regulate intrinsic immune gene expression, including negative regulators of interferon signaling, thereby suppressing canonical interferon signaling to preserve pluripotency while maintaining antiviral resistance.

The lymphatic system is essential for maintaining fluid homeostasis, lipid transport and supporting immune function. Despite its central role in health and disease, advancements in understanding human lymphatic vasculature has been constrained, in part because primary human LECs are difficult to access and study in disease-relevant contexts. This study describes an efficient and scalable feeder-free method to differentiate human iPSCs into lymphatic endothelial cells (LECs) that are transcriptionally and phenotypically similar to primary fetal LECs. An iPSC-derived LEC system overcomes a drawback of primary cells by enabling precise genetic perturbations, supporting study of lymphatic diseases of interest in a human context. By grounding our approach in in vivo stages of lymphangiogenisis, we describe a staged protocol that recapitulates the key milestones of lymphatic development. We first adapted a published method to differentiate human iPSCs into venous endothelial cells (VECs) and then initiate transdifferentiation of VECs into LECs. Using immunocytochemistry, qPCR, as well as flow cytometry, we demonstrated expression of lymphatic-specific markers in the differentiated population. We further characterized our induced VECs (iVECs) and LECs (iLECs) through bulk RNA sequencing analysis and compared the populations to pseudobulk VEC and LEC transcriptomic datasets generated from human fetal heart endothelia at 12, 13 and 14 weeks of gestation. Through this work, we expanded the repertoire of approaches for accessing LECs, with the goal of accelerating discoveries in lymphatic biology and therapeutics.

Oral squamous cell carcinomas (OSCC) account for ∼90% of all oral malignancies and have devastating effects on overall health and quality of life. However, little is known about the early initiating events that drive the development of oral leukoplakia-like premalignant lesions (OPLs) and disease progression. Here, we create a mouse model of tobacco-related premalignant OSCC that takes into consideration its primary risk factors, including advancing age and male sex. This model notably recapitulates the age variant patterns of OSCC risk observed in humans, with a higher prevalence of oral premalignant lesions in older mice. In addition, by building a transcriptomic atlas with this system, we reveal genetic signatures associated with oncogenic progression in the tongue and buccal epithelium, and their resident somatic tissue stem cells. We also identify several novel transcriptomic signatures of premalignancy in OSCC, including enhanced immune response and expansion and dysregulation of head and neck tissue stem cells. These findings offer a new framework for investigating physiologically-relevant risk factors and drivers of OSCC and illuminate novel biological pathways underlying its pathology.

Thrombosis remains a major cause of cardiovascular and cerebrovascular diseases, driven in large part by platelet activation and aggregation. Because platelets are continuously produced from hematopoietic stem cells (HSCs), durable reprogramming of HSC output offers a unique opportunity for a one-time antithrombotic intervention. Here, we show that DNA methylation-based epigenome editors delivered transiently as RNA result in stable, heritable gene silencing in primary human HSCs that persists through long-term self-renewal and megakaryocytic differentiation, while remaining reversible through targeted demethylation. Targeting the platelet integrin β3 ( ITGB3 ), this approach achieves robust, sustained repression and yields platelets with impaired aggregation. Extending this framework to additional genetically-nominated platelet targets establishes HSC epigenome editing as a durable and reversible strategy to modulate thrombotic risk and highlights broader opportunities to engineer hematopoiesis.

Tissue repair is increasingly recognized as a systemic process influenced by age-associated changes beyond the injured organ itself. Clonal hematopoiesis of indeterminate potential (CHIP), a common consequence of somatic evolution in hematopoietic stem cells, has been linked to inflammatory disorders, yet whether it directly regulates tissue remodeling remains unclear. Here, we integrate population genomics, preclinical models, and human lung analyses to examine the role of CHIP in fibrotic lung disease. In large cohorts, idiopathic pulmonary fibrosis (IPF) was associated with a distinct CHIP mutational spectrum enriched for non- DNMT3A variants and for larger mutant clones. In mouse models, hematopoietic mutations exacerbated bleomycin-induced fibrosis and reprogrammed macrophages toward inflammatory, profibrotic states, including expansion of a distinct, injury-responsive SPP1 + population conserved in human disease. CHIP-associated macrophages were sufficient to directly promote fibroblast activation and alter epithelial differentiation, linking hematopoietic genotype to parenchymal remodeling. Consistently, a CHIP-derived macrophage transcriptional signature predicted adverse outcomes in independent IPF cohorts. Notably, immune and epithelial alterations were detectable even in the absence of overt injury, indicating that CHIP establishes a primed tissue environment permissive for maladaptive repair. Together, these findings identify clonal hematopoiesis as a systemic regulator of tissue repair and demonstrate that somatic evolution in blood can actively instruct organ remodeling through immune-parenchymal interactions. This framework supports the possibility that disease-associated selective pressures may shape clonal architecture with functional consequences for organ health.

Adult stem cells are thought to drive the regenerative potential of the endometrium and contribute to the pathogenesis of endometriosis, however, their identity and defining features remain to be characterized. Here, we used in vivo and in vitro approaches to demonstrate that cells with high aldehyde dehydrogenase 1 activity (ALDH HI cells) were long lived progenitors in the endometrial epithelium with a higher organoid formation capacity, long-term passaging potential, and stemness gene signatures. Using lineage tracing with an Aldh1a1cre/ERT2; ROSA26tdTomato reporter mouse, Aldh1a1+ cells expanded during postnatal development, estrus cycling, and following post-partum repair. In response to ovariectomy or exogenous estradiol, we found that ALDH1A1 + cells localized to glandular crypts of the endometrium or throughout the luminal epithelium, respectively, indicating that their spatial localization is hormone sensitive. Functionally, we found that selective ablation of ALDH1A1 + cells in Aldh1a1cre/ERT2; ROSA26- DTR flox/flox mice decreased endometrial gland number and FOXA2 expression . These findings were recapitulated in the human endometrium, where endometrial epithelial organoids with high ALDH activity (ALDH HI cells) showed a higher organoid formation capacity than ALDH LO cells and displayed unique transcriptomes with fewer luminal-like ciliated cells. Overall, our studies indicate that ALDH1A1 + cells are hormone-sensitive adult stem cells in the endometrium with regenerative potential that are critical for endometrial development and function.

Directed differentiation of pluripotent stem cells (PSCs) into pancreatic islets is a cornerstone strategy for diabetes cell therapy. This process relies on growth factor-driven activation of core transcriptional regulators, notably PDX1 and NGN3, to restrict the multi-lineage potential of definitive endoderm to pancreatic progenitors and endocrine cell types. Yet differentiation efficiency and lineage fidelity vary markedly across PSC lines. Here, we demonstrate that a dominant constraint is persistent Polycomb Repressive Complex 2 (PRC2)-mediated epigenetic repression at the PDX1 and NGN3 loci, limiting endocrine specification despite inductive signaling. To directly test whether chromatin states at the PDX1 and NGN3 loci gate developmental competence, we deployed a computationally engineered epigenetic effector (EBdCas9) to transiently and sequentially remove H3K27me3 at those loci during defined developmental windows. Targeted epigenetic resolution robustly enhanced endocrine lineage commitment and accelerated β-cell differentiation across genetically diverse PSC lines. In contrast, direct transcriptional activation with VP64dCas9 increased PDX1 and NGN3 expression but did not improve differentiation outcomes. Integrated cell population studies and genome-wide chromatin and transcriptomic analyses reveal that PRC2-targeted remodeling preferentially activates endocrine gene networks while limiting progenitor expansion and lineage-inappropriate programs. These findings establish that gene-targeted manipulation of PRC2-mediated repression at PDX1 and NGN3 can be used to control cell lineage competence. Collectively, our study reframes variability in PSC differentiation as a failure of epigenetic resolution rather than transcriptional insufficiency and introduces locus-specific chromatin remodeling as a generalizable strategy to enforce developmental fidelity.

Neuroinflammation is characterized by the activation of the brain's immune system, mainly involving microglia and astrocytes, in response to injury, infection, or neurodegenerative processes. It leads to neuronal damage, playing a key role in the onset and progression of neurological disorders. Lipopolysaccharide (LPS)-induced models have become pivotal in the study of neuroinflammation and its related complications. Coumarin derivatives-both natural and synthetic derivatives- have shown a promising effect on neuroinflammatory pathways.

Tumor cells adapt to hypoxia by releasing hiTDExs enriched with bioactive molecules that modulate endothelial behavior and promote tumor progression. This study aimed to characterize how hypoxia-induced HNSCC exosomes reshape the endothelial secretome and contribute to metastatic potential.

Dental follicle stem cells (DFSCs) originate from the dental follicle during tooth development and possess multilineage differentiation potential, contributing to periodontal tissue regeneration, bone repair, and immunomodulation. This review highlights the recent advances in the application of DFSCs and biological scaffolds for regenerative medicine, with a focus on oral and craniofacial tissue. DFSCs exhibit key advantages for regenerative therapies, including high accessibility, robust self-renewal capacity, and multipotent differentiation potential, enabling their differentiation into odontogenic (dentin- and enamel-forming), osteogenic, and fibroblastic lineages. We discuss the embryonic origin of DFSCS and their unique ability to maintain stable cellular properties in long-term in vitro culture. Importantly, DFSCs play a pivotal role in tooth morphogenesis, periodontal tissue formation, and craniofacial bone regeneration, making them promising for functional oral tissue restoration. A critical aspect of DFSC-based regeneration is the integration with bioactive scaffolds, which provide structural support, promote cell adhesion, proliferation, and differentiation, and facilitate vascularization. We analyze how scaffold properties, such as biodegradability, porosity, and permeability, influence DFSC behavior and therapeutic outcomes. Finally, we explore future challenges and opportunities in optimizing DFSC-scaffold interaction, emphasizing advancements in biomaterial design and emerging bioengineering technologies. Preliminary evidence suggests that integrating DFSCs with engineered scaffold systems may offer potential benefits for personalized regenerative therapies, though further validation is required before clinical translation. Such approaches could contribute to advancing tooth and craniofacial reconstruction strategies. This review consolidates existing insights and explores potential avenues for future research to support advancements in DFSC-based regenerative medicine.

Virus-specific CD4+ T cells are essential for coordinating adaptive immunity during infection, but their differentiation and maintenance in chronic infection remain unclear. Using human hepatitis C virus (HCV) infection as a model, we assessed the determinants of virus-specific CD4+ T cell immunity in acute, spontaneously cleared, chronic, and therapeutically cured infections. During acute infection, multiple subsets of progenitor CD4+ T cells emerged, including subsets that are also found in chronic infection. In chronic infection, stem-like Bcl-2+ CD4+ T cells and T-bet+ effector CD4+ T cells existed in a progenitor/progeny relationship. Following therapy-mediated HCV cure, these cells retained their chronic signature but formed a stable memory pool that persisted for years and was distinct from HCV-specific CD4+ T cell memory after spontaneous clearance. Collectively, our findings highlight differences in CD4+ T cell fates that depend on infection outcomes and reveal common principles of CD4+ and exhausted CD8+ T cell maintenance during and after chronic infection.