Multiple system atrophy (MSA) is a progressive neurologic disease, known as an α-synucleinopathy. There are currently no effective disease-modifying therapies for MSA. While neuroinflammation is a hallmark of MSA, the contribution of adaptive immune mechanisms remains poorly understood. Here, we profiled peripheral and central T cell responses in patients with MSA, in comparison with Parkinson's disease (PD) and healthy control cohorts, using single-cell transcriptomics, flow cytometry, and antigen-specific functional assays. We demonstrated that peripheral T cells from MSA patients are activated and skewed toward cytotoxic and inflammatory phenotypes. Single-cell transcriptomics further revealed clonal expansion of cytotoxic CD8+ T cells expressing GZMB, GNLY, and chemokine and integrin programs associated with brain homing. We also demonstrated that both CD4+ and CD8+ T cells from MSA patients recognize α-synuclein monomers and preformed fibrils in an HLA class I/II-dependent manner, driving proliferation, clonal expansion, and acquisition of cytotoxic features. Consistent with these peripheral responses, CD8+ T cell density was increased in the parietal cortex of postmortem MSA brain tissues, along with cytotoxic (GZMB+, GZMK+) and proinflammatory (IFNγ+) CD8+ T cells. Together, these findings demonstrate that cytotoxic T cells targeting α-synuclein are engaged in MSA, suggesting that their activity may contribute to neuroinflammation and disease progression, and highlighting this immune axis as a candidate therapeutic target for further investigation.

Cervical cancer is the fourth most common cancer in women and most frequently affects the uterine ectocervix. The uterine ectocervix is lined by stratified squamous epithelium, comprising basal cells and differentiating parabasal and suprabasal cells. The regeneration observed after cervical conization suggests the presence of robust stem cell activity. However, our understanding of the identity and regulatory mechanisms of cervical stem cells and their malignant transformation process has been limited due to the restricted access to human cervical tissues and the lack of optimal systems to assess stem cell activity in the uterine ectocervix. Recently established human cervical organoids can help overcome these limitations and offer new opportunities to study cervical physiology and pathology. However, no standardized method exists for isolating and establishing human cervical organoids. Here, we describe a method for establishing human ectocervical organoids. Specifically, we present a protocol to isolate single cells from the human ectocervical epithelium. By incubating cervical tissue in an optimized enzyme solution, we peel off the cervical epithelium to maximize cell yield while minimizing fibroblast contamination. We further outline the conditions for culturing human cervical organoids using an optimized medium formulation. In addition, we detail the procedures for passaging and genetically manipulating cervical organoids. This protocol provides a highly efficient approach for establishing human cervical organoids and utilizing them to study cervical stem cells and diseases.

Primary mitochondrial disorders (PMDs) are inherited metabolic diseases that most often present with neurological symptoms in infancy or adolescence, underscoring the central importance of mitochondrial function to brain health. Historically, the field has emphasized neurodegeneration-consistent with the high energetic demands of postmitotic neurons. However, neurodevelopmental manifestations are now recognized as common early phenotypes, frequently preceding clinical regression in many PMDs. Given the pivotal role of mitochondria in neural stem/progenitor cell maintenance and cell fate decisions, defects in the respiratory chain are poised to disrupt neurogenesis and gliogenesis. Evidence for such developmental vulnerabilities is reviewed here. Likewise, because mitochondrial metabolism and dynamics shift across the oligodendrocyte lineage-from oligodendrocyte precursor cell expansion to differentiation and the energetically intensive phase of myelin synthesis-callosal atrophy in mitochondrial leukoencephalopathies may, at least in part, reflect developmental shortcomings in oligodendrogenesis and myelination. This possibility warrants focused investigation in cellular and in vivo models.

Fluorescence-guided surgery (FGS) offers a potential strategy to improve complete tumor resection in prostate cancer (PCa); however, clinical application has been limited by an insufficient tumor-to-background ratio (TBR) of available probes. Here, we report the development of GGA-sNIR, a near-infrared (NIR) fluorescent probe engineered by conjugating a high-affinity DNA aptamer specific for a prostate stem cell antigen (PSCA) with a monocarboxy indocyanine green (ICG) derivative. The conjugate adopts a stable three-dimensional structure that not only enables precise target recognition but also protects against nuclease degradation, markedly enhancing its in vivo stability. GGA-sNIR exhibits nanomolar binding affinity (Kd = 54.91 nM) and is specifically internalized by PSCA-positive cells. In subcutaneous tumor models, the probe achieved a peak TBR of 26 and showed prolonged tumor retention exceeding 48 h. Crucially, in an orthotopic PCa model, GGA-sNIR allowed real-time, high-contrast visualization of tumor margins during laparoscopic surgery, facilitating precise and complete resection. Further validation using ex vivo human lymph node specimens confirmed its ability to selectively detect PSCA-positive metastases. With its ultrahigh contrast, demonstrated efficacy in intraoperative guidance, and strong translational potential, GGA-sNIR represents a promising molecular tool for advancing precision surgery in prostate cancer.

Organoid technology provides an experimental in vitro platform for studying human physiology in health and disease. Compared with other in vitro systems, such as cancer-derived cell lines or explants, tissue stem cell (TSC)-derived organoids offer key advantages: they can be expanded indefinitely while maintaining genomic stability and are amenable to CRISPR modification. Since these organoids are generated from normal TSCs, they contain the cell types of their tissue of origin. This makes organoids particularly valuable for investigating interactions between bacteria and differentiated, non-tumoral human cells. Intestinal organoids are grown as two-dimensional polarized monolayers on cell culture inserts coated with Basement Membrane Extract (BME) or invasin, an animal-free, cost-effective alternative, and can be differentiated into major intestinal epithelial cell types for infection with pathogenic adherent-invasive E. coli. Key infection readouts include monitoring epithelial gene expression by Reverse Transcription quantitative PCR (RT-qPCR), visualizing bacteria-epithelium interactions by confocal microscopy, and quantifying epithelial barrier breaching. The protocol can be readily adapted to study other organs and bacterial species, providing a versatile platform for investigating host-microbe interactions in organoids.

Acute Myeloid Leukemia (AML) originates from the hematopoietic stem and progenitor cell compartment and is associated with an overall poor clinical outcome. T-cell engaging bispecific antibodies (TCEs), binding to a tumor-associated antigen and to CD3, redirect T-cells to the target-antigen expressing cells in an MHC/TCR-independent fashion. The development of TCEs for clinical application is facing several challenges. Firstly, it is difficult to identify a tumor-selective, non-MHC-presented tumor target, not expressed on the tissue of tumor origin, thus limiting specificity. Secondly, CD3-directed TCEs do not provide a second, T-cell activating signal, such as the stimulation of CD28 or 41BB, thus possibly limiting T-cell efficacy. To address both aspects, we generated a CD33xCD28 IgG4-scFv2 with CD28 agonistic activity and tested it in combination with a previously by us reported CD117xCD3 TCE in AML. Combining these two bispecific antibodies significantly improved T-cell mediated lysis of AML cell lines and primary AML samples by enhancing T-cell activation, proliferation and cytokine release in vitro. Furthermore, the addition of CD33xCD28 IgG4-scFv2 showed faster time to cell attachment, increased lytic events, and improved specificity towards double-target expressing cells. In summary, the data indicate that combining co-stimulation via a second tumor-associated antigen to CD3-TCEs enhances T-cell lytic activity and simultaneously increases specificity against double-target expressing AML cells.

A plant's indeterminate growth requires constant cell proliferation and stem cell maintenance to support its developmental plasticity. The root meristem is an excellent system to study the developmental control of plant cell cycles due to the organ's accessibility and the tight link between cell cycle and developmental regulation. Studies have uncovered diverse pathways that shape root tissue patterning and cell identity, but how these mechanistically connect to cell cycle components remains unclear. Recent work and new approaches are starting to bridge this gap. In this review, we synthesize recent findings on the developmental regulation of cell cycle progression across distinct cell types and developmental zones in the Arabidopsis thaliana root apical meristem, highlighting cells and regions of the root where these processes have been thoroughly studied, and others where we know little. These discoveries reveal a nuanced relationship between cell identity and cell cycle regulation that implies an active role for cell cycle modulation in the patterning and developmental plasticity that are integral to plant growth.

Successful embryonic development is a collaborative process: the embryo relies on the support of extra-embryonic and maternal tissues. However, understanding early human development is challenging due to the ethical and technical limitations. The generation of stem cell-based models that mimics embryonic and/or extra-embryonic tissues provides in vitro platforms to study human embryogenesis. We recently developed a 3D embryonic stem cell-derived model that mimics the post-gastrulation amnion and extra-embryonic tissues. These structures, which we call post-gastrulation amnioids (PGAs), capture early human amnion development up to the formation of a mature fluid-filled amniotic sac-like membrane composed of two cell layers of inner amniotic ectoderm and an outer extra-embryonic mesoderm. PGAs gradually expand in culture and exhibit functional traits of human amnion. PGAs are highly reproducible and can be scaled for high throughput studies. Here, we describe a step-by-step protocol for PGA generation and their morphological characterization by immunofluorescence staining as well as a co-culture assay to begin to understand interactions between extra-embryonic and embryonic cells.

Wharton's jelly-derived human mesenchymal stem cells (WJ-MSCs) have received much attention in recent years due to their non-invasive isolation method and potential for allogeneic transplantation. This study assessed the differentiation of human WJ-MSCs into neuron-like cells using retinoic acid (RA), 3-isobutyl-1-methylxanthine (IBMX), in combination with reduced graphene oxide nanoparticles (rGO), linalyl acetate (LA), and lavender extract nanoemulsion (LEN). Lavender extract was obtained through sequential extraction using n-hexane, methanol, and ethyl acetate, followed by nanoemulsion preparation. The nanoemulsion properties were analyzed by dynamic light scattering (DLS), zeta potential measurement, and transmission electron microscopy (TEM). Optimal doses were determined via MTT assay for cell viability and Acridine Orange/Ethidium Bromide (AO/EB) staining for cell death detection. The expression of neuron-specific genes (NSE, MAP-2, β-tubulin III, and Oligo-2) was analyzed using quantitative Real-time PCR (qPCR). Furthermore, the protein levels of neuronal markers gamma-enolase (NSE), MAP-2, and β-tubulin III were assessed by immunocytochemistry (ICC) on days 7 and 14. Gene and protein expression analyses demonstrated that rGO-LEN and rGO-LA significantly (p-value ≤ 0.05) enhance the neural differentiation of human WJ-MSCs. These treatments induced a significant increase in the expression of neural markers (both at the gene and protein levels) on day 7 and especially on day 14 compared to the control group and other treatments. These findings can provide initial insights to guide and develop future research to achieve new and effective therapeutic approaches in the field of regenerative medicine and neuroregenerative research.

The combination of hypomethylating agents (HMA) and venetoclax (VEN) has transformed acute myeloid leukemia (AML) treatment. Data on donor lymphocyte infusion (DLI) with HMA/VEN for relapse after allogeneic hematopoietic cell transplantation (alloHCT) remain limited. We retrospectively analyzed 78 adults with relapsed myeloid neoplasms after first alloHCT between 2018 and 2025. DLI was given with HMA, HMA/VEN, or other/no treatments. The primary endpoint was event-free survival (EFS), defined as time to death or second alloHCT. Median time to relapse after alloHCT was 12.9 months (range 2.2-192.4). Initial DLI doses ranged from 0.3 to 8.14 × 10⁶ CD3⁺ cells/kg. Median EFS after first DLI was 15.2 months: with 16.2 (DLI/HMA), 14.3 (DLI/HMA/VEN), and 21.1 (DLI/other), respectively. Death occurred in 25.6% and second alloHCT in 32.1% of patients. GvHD of any kind after DLI treatment manifested in 30.8%, both events comparable across groups. Complete remission (CR) after DLI was achieved in 42.3% after a median of 4.2 months, including patients with TP53 mutations (n = 9). Approximately 40% of patients with relapsed myeloid malignancies were successfully salvaged with DLI and combination treatments. Patients with high-risk features such as morphological relapse were overrepresented in the DLI/HMA/VEN cohort but achieved comparable outcomes to the other treatment groups. This finding suggests successful treatment of even morphological relapse by addition of VEN to DLI/HMA regimens while supporting the need for controlled trials.

This study aimed to evaluate the therapeutic potential of trabecular meshwork stem cells (TMSC) transplantation in enhancing the trabecular meshwork (TM) cell proliferation and intraocular pressure (IOP) reduction in a cell loss human organ cultured anterior segment (HOCAS) model of glaucoma.

In vitro skeletal muscle culture models provide important insight into the cellular mechanisms which underpin skeletal muscle physiology and metabolism in health and disease. The establishment of a model that can be cultured in physiological concentrations of glucose is an important factor in its translatability to more complex models and systems. Using the human skeletal muscle cell line, LHCN-M2 myoblasts, we aimed to determine the effects of different concentrations of glucose in culture media on cell viability, proliferation, ATP production and differentiation. LHCN-M2 myoblasts were cultured in NORM (1 g· L- 1) or HIGH (3.8 g· L- 1) glucose growth media, and cell viability, ATP production, and proliferation were measured. Immunofluorescence microscopy was used to determine LHCN-M2 differentiation into multinucleated myotubes with increasing concentrations of human serum (0.5%, 1% and 2% v/v). There were no differences in the viability, proliferation or basal ATP production rates of LHCN-M2 cells grown in NORM compared to HIGH glucose (P > 0.05). Morphological analysis revealed that myotube area was greater when differentiated in 2% compared to 0.5% human serum (P = 0.02), but myotube number and fusion index were unaffected (P > 0.05). These findings demonstrate that LHCN-M2 cells are capable of proliferating and differentiating into multinucleated myotubes under normal glucose concentrations in the culture media. Further work is required to determine the implications of media glucose concentration on the wider metabolic function and phenotype of LHCN-M2 myoblasts cells and myotubes.

The progressive decline in physiological processes with aging is a complex biological process that increases susceptibility to age-related diseases. Cellular senescence is a primary contributor to this process, a state of sustained cell cycle arrest accompanied by a distinctive secretory profile known as the Senescence-Associated Secretory Phenotype (SASP). In recent years, extracellular vesicles (EVs), including exosomes and microvesicles (MVs), have emerged as key mediators of intercellular communication by carrying bioactive molecules, such as proteins, lipids, and nucleic acids. In this review, we describe how EVs are important SASP vectors that transmit senescent signals to nearby cells, driving immunosenescence, persistent inflammation (inflammaging), and other aging characteristics, such as stem cell exhaustion and genomic instability. Furthermore, we highlight the dual function of EVs as both pathogenic drivers of aging-related dysfunctions and promising therapeutic agents. In addition, we emphasize their potential as diagnostic biomarkers for age-related diseases, including cardiovascular disease, osteoporosis, and Alzheimer's disease. Finally, we explore the emerging therapeutic uses of EVs, particularly those derived from mesenchymal stem cells (MSCs), to promote tissue repair, reduce aging phenotypes, and serve as engineered drug-delivery systems. This study highlights the critical role of EVs in aging mechanisms and establishes them as potent diagnostic and therapeutic tools for anti-aging.

The accumulation of senescent cells during aging contributes to the progression of various age-related pathologies. Ineffective immune clearance, increased half-life of senescent cells, and bystander senescence are considered the primary drivers of this age-associated accumulation. Most of these causes stem from the aging of the immune system, which results in a prolonged persistence of damaged/nonfunctional cells within tissues and allows the internal senescence program to progress to a more severe phenotype. Here, we propose the existence of an additional immune-independent mechanism underlying the accumulation of senescent cells during aging. By reanalyzing existing experimental evidence, we show that cells of diverse identities and tissue origins become increasingly susceptible to senescence with age. The latter implies that epigenetic and molecular changes that cells acquire during aging create a permissive background for the activation of the senescence program. In light of our findings, senotherapeutic interventions alone may be insufficient to substantially alter the trajectory of organismal aging. Effective strategies may need to target upstream drivers of cellular dysfunction, including age-associated epigenetic alterations. Epigenetic rejuvenation could, in principle, enhance cellular stress resilience and thereby reduce the rate at which senescent cells emerge and accumulate.

This study aimed to characterize transcriptional and epigenetic remodeling in the cornea induced by acute inflammation.

ObjectiveTo systematically review and quantitatively synthesize in vivo evidence on extracellular vesicle (EV)-based therapies for regenerative endodontic procedures, focusing on mineralization and angiogenesis outcomes.MethodsWe conducted a systematic review and meta-analysis of in vivo preclinical dentoalveolar/regenerative endodontic models comparing EV-based interventions (exosomes/microvesicles/apoptotic bodies), alone or with scaffolds, versus control conditions. Searches were performed in Web of Science Core Collection and Scopus from inception to 31 December 2025, with reference screening. Random-effects meta-analyses pooled standardized mean differences (SMDs) for mineralization and angiogenesis; heterogeneity was assessed using I2. Risk of bias was evaluated using the Systematic Review Centre for Laboratory animal Experimentation tool.ResultsTwenty-one studies met inclusion criteria; seven were included in the mineralization meta-analysis and five in the angiogenesis meta-analysis. EV-based therapies significantly increased mineralization versus controls (SMD = 6.43; 95% confidence interval (CI) [3.13-9.73]; I2 = 91%) and angiogenesis (SMD = 7.89; 95% CI [3.94-11.85]; I2 = 82%). Subgroup analyses suggested stronger effects for EVs derived from dental pulp stem cells, stem cells from human exfoliated deciduous teeth, and stem cells from apical papilla. Risk of bias was predominantly unclear due to limited reporting of randomization and blinding. Considerable heterogeneity, small numbers of pooled studies, and variability in EV isolation, characterization, dosing, and outcome assessment limit generalizability and translation.ConclusionsEV-based therapies enhance mineralization and angiogenesis in preclinical regenerative endodontic models, with dental stem cell-derived EVs showing the greatest apparent potential. However, effect sizes should be interpreted cautiously given very high heterogeneity and methodological/reporting limitations.PROSPERO registration: Centre for Reviews and Dissemination 420251107328.

Periodontitis is among the most prevalent and challenging oral diseases worldwide. Effective intervention requires strategies that not only eliminate pathogens but also modulate the dysregulated oxidative microenvironment. Hydrogen (H2) therapy holds promise in periodontitis immunotherapy due to its exceptional safety profile and unique selective antioxidant properties. Here, we present a tailored biomaterial platform-Magnesium hydride (MgH2)-Poly(lactic-co-glycolic acid) (PLGA)@Oxygen-deficient titanium dioxide (TiO2-x)-Alginate core-shell microspheres (MTMs)-fabricated via coaxial electrostatic microdroplet technology for localized periodontitis therapy. This system provides rapid antibacterial activity together with sustained remodeling of the local tissue microenvironment. The MTMs achieve long-term antioxidant and anti-inflammatory effects through the synergistic sustained release of hydrogen molecules and Mg2+ ions (up to 7 days). Concurrently, the surface-etched TiO2-x particles embedded in the shell layer, exhibiting peroxidase-like catalytic activity, enable rapid antibacterial effects in the presence of low-level exogenous hydrogen peroxide. In vitro, MTMs attenuated oxidative stress in periodontal ligament stem cells and promoted lipopolysaccharide-stimulated macrophages to polarize from pro-inflammatory M1 toward reparative M2 phenotypes. In a rat periodontitis model, short-term treatment (1 week) reduced bacterial load, mitigated inflammation, and inhibited bone resorption, while long-term application (1 month) promoted bone repair and remodeling. These findings demonstrate that MTMs represent a powerful therapeutic platform for periodontitis and hold potential for broader biomedical applications.

We present an integrated microelectrode array (MEA) platform for high-precision electrophysiological characterization of patient-derived 3D cardiac organoids (COs), enabling dynamic recordings from both healthy and Duchenne muscular dystrophy (DMD) "mini-hearts." A deformable MEA, fabricated by photolithographic patterning of support layers and microelectrodes, is conformally assembled onto a customized scoop-slide well. The concave geometry enables passive self-alignment of millimeter-scale COs within a multichannel electrode array, allowing simultaneous extracellular field potential (EFP) recording and video-based motion analysis. Using COs derived from human induced pluripotent stem cells of healthy donors and a DMD patient, we resolved region-specific EFP waveforms, which were validated by calcium imaging. DMD COs exhibited severe arrhythmic firing and aberrant waveform morphologies, in contrast to the stable, isochronal rhythms of healthy controls. Single-cell transcriptomic analysis revealed a pathological DMD signature marked by excessive extracellular matrix accumulation and disorganized cardiac architecture, contributing to conduction heterogeneity. Pharmacological challenge with the hERG blocker E-4031 prolonged field potential duration in normal COs but triggered disorganized arrhythmogenic storms in DMD COs. By overcoming the limitations of rigid interfaces and ensuring structural preservation with stable impedance coupling, the presented platform provides a robust biointerface for disease phenotyping, drug screening, and mechanistic interrogation of 3D cardiac tissues.

Lung organoids are versatile experimental models, but their broader use in studying human disease is limited by the scarcity of starting material and the complexity of current methods. To align organoid technology with common clinical practice, we developed airway and alveolar organoids using cells obtained from patients' bronchoalveolar lavage (BAL) fluid. Building on existing techniques, we showed that BAL is a reliable, accessible source of primary human epithelial cells, yielding airway and alveolar organoids within 10 days. Organoids can then be expanded over many passages for downstream analysis. Our streamlined methods do not require cell sorting or other complex procedures, all cells are derived from a single patient, and media are based on serum-free, chemically-defined formulations. Here, we present detailed protocols for organoid establishment, standardized passaging and phenotyping, and differentiation of both airway and alveolar models. We provide a time course of BAL-derived airway organoid differentiation at air-liquid interface, and we demonstrate proof of principle for differentiation of BAL-derived alveolar organoids in 3D culture. These methods can be readily adapted to generate and characterize organoids from lung tissue, tracheobronchial specimens, or other primary cells from humans or mice, expanding the potential to use lung organoids for disease modeling.

Glutathione S-transferase omega 1 (GSTO1) is overexpressed in a variety of cancers and plays an important role in the promotion of tumor proliferation and metastasis. Nevertheless, the function of GSTO1 in cervical cancer (CC) is unknown. Immunohistochemistry (IHC) was applied to observe GSTO1 protein levels in CC tissues. Cell proliferation, migration, and invasion capabilities were assessed using CCK8, EdU, plate cloning assays, and Transwell experiments in vitro. Flow cytometry was employed to analyze cell cycle progression and reactive oxygen species (ROS) levels. A subcutaneous xenograft model was utilized to observe cell growth in vivo. Cell stemness was assessed via sphere formation assay. Transcriptome sequencing and enrichment analysis were conducted to explore GSTO1-related signaling pathways. The proteins linked to cell cycle regulation, stemness, epithelial-mesenchymal transition (EMT), and the PI3K/AKT/mTOR signaling pathway were identified through western blotting and IHC. In this study, GSTO1 was highly expressed in CC tissues and associated with poor prognosis of patients. Knockdown of GSTO1 suppressed CC cell proliferation in vivo and in vitro and inhibited the cell cycle, stemness, migration, and EMT as in C1-27 treatment. Conversely, down-regulation of GSTO1 promoted ROS production in CC cells. RNA sequencing indicated that GSTO1 mediated the activation of the PI3K/AKT signaling pathway. In addition, silencing GSTO1 decreased the phosphorylation of PI3K, AKT, and mTOR proteins. Interestingly, 740 Y-P (PI3K activator) reversed the inhibitory effects of GSTO1-induced cell proliferation, cycle, stemness, and EMT via the PI3K/AKT/mTOR signaling axis. GSTO1 was important in CC progression through the PI3K/AKT/mTOR pathway and could serve as a promising therapeutic target.