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Diabetes mellitus is a chronic metabolic disorder characterized by the loss or dysfunction of insulin-producing beta (β) cells. Adipose-derived stem cells (ADSCs) represent a promising source for generating functional insulin-producing β cells due to their accessibility and differentiation potential. Photobiomodulation (PBM), a non-invasive light-based therapy, has emerged as an innovative strategy to enhance stem cell differentiation efficiency. Evidence suggests that green (525 nm) and near-infrared (825 nm) wavelengths, applied individually or in combination, can modulate cellular metabolism, ATP production, and differentiation-related signaling pathways, thereby influencing ADSC commitment toward insulin-producing β-cell-like phenotypes. This in vitro study evaluated the effects of PBM at 525 nm and 825 nm, delivered individually and in combination at energy fluences of 5 J/cm2 and 10 J/cm2, on the differentiation of ADSCs cultured in β-cell induction medium into insulin-producing β-cell-like cells under two-dimensional (2D) culture conditions at 24 h, 5 days, and 10 days. Cellular responses were evaluated using adenosine triphosphate (ATP) luminescence assays, lactate dehydrogenase (LDH) activity assays, Giemsa staining, Live/Dead viability assays, and dithizone (DTZ) staining. ATP levels varied significantly among the experimental groups, reflecting changes in cellular metabolic activity associated with β-cell induction and PBM exposure, and reduced LDH activity, suggesting decreased cytotoxicity. Giemsa staining revealed morphological changes consistent with β-cell differentiation, while Live/Dead assays demonstrated the maintenance of cell viability across all experimental groups. Dithizone staining identified the presence of zinc-rich insulin-producing clusters. These findings highlight the importance of PBM wavelength and fluence optimization in regenerative stem cell applications.

The novel models represented by organoids are becoming the key approach to solve the ethical and efficiency problems in drug development, but the effective and low-cost models are still urgently needed. The breakthrough development of artificial cell (AC) technology and synthetic biology has made it possible. In this study, a novel AC for evaluating the efficacy of various antitumor drugs is fabricated by combining cell membrane bionic technology and in situ synthetic biology. After entering ACs, antitumor drugs targeting nucleic acid affect the gene transcription of an artificially designed ribozyme that can catalyze the cleavage of molecular beacons and generate fluorescence signals in situ, indicating the efficacy of antitumor drugs at the cellular level. Specifically, ACs constructed with cell membranes containing drug-resistant proteins show significant drug inhibition, and the 3D coded ACs established based on this method are capable of classifying cell-specific characteristics more accurately to provide support for targeted drug therapy. This platform for in situ pharmacodynamic analysis not only demonstrates the individualized penetration ability of tumor heterogeneous packaging membranes in response to tumor drugs but also fills the gap between non-living and living experiments as a supplementary strategy.

Mesenchymal stem/stromal cells (MSCs), neural stem cells (NSCs), and induced pluripotent stem cells (iPSCs) play a crucial role in human development and cancer biology. While they are known to facilitate tumorigenesis, these stem cell types also hold promise as therapeutic tools in cancer treatment. They can serve as delivery systems for targeted therapies, with potential applications in regenerative medicine and cancer stem cell-targeted therapies. Through mutations, epigenetic modifications, and influences from the tumor microenvironment, somatic or normal stem cells, as well as progenitor or differentiated cells, can evolve into cancer stem cells (CSCs). CSCs represent a small but critical subset of tumor cells involved in tumor formation, metastasis, and treatment resistance. This review examines the dual role of MSCs, NSCs, and iPSCs in cancer by exploring their contribution to tumor initiation and their potential as therapeutic targets. To provide a focused narrative synthesis, we reviewed peer-reviewed studies addressing MSCs, NSCs, iPSCs, CSC formation, and stem cell-based therapeutic strategies in oncology. Additionally, we explore the mechanisms by which somatic or normal stem cells transition into CSCs and the factors contributing to this transformation, along with emerging CSC-targeting therapies. Finally, the role of stem cells and their exosomes as carriers in cancer therapies is examined, alongside their applications in immunotherapy and regenerative medicine, and the challenges associated with their clinical use in cancer treatment.

Mitochondrial function is essential for biology, particularly in cancer. However, cell-type-specific expression patterns of conserved mitochondrial genes in gastric cancer (GC) remain unclear. We herein raised and tested a novel hypothesis of "mitochondrial conserved gene expression homeostasis imbalance" in GC cohorts with single-cell resolution. This work analyzed an open-accessed scRNA-seq dataset (GSE206785, 24 GC patients, 48 samples) with Seurat and defined 43 clusters grouped into 15 cell subtypes. In parallel, Pseudobulk profiles were generated to simulate bulk RNA-seq. A mitochondrial conserved gene score was computed by Seurat AddModuleScore, GSVA, and AUCell. Mitochondria-related biomarkers were also screened, validated, and incorporated into a mitochondria-dependent prognostic model that was further evaluated. Without considering cell-type-specific expression patterns, Pseudobulk analysis showed no significant differences in mitochondrial conserved gene expression between GC and control samples. In contracst, single-cell analysis found a cell-type-specific imbalance, under which tumor-associated epithelial cells displayed relatively elevated mitochondrial conserved gene expression, while non-epithelial cells showed reduced. Notably, survival analyses identified gene KRT7 and KLRC1 as robust prognostic biomarkers for early GC. Our findings support a mitochondrial conserved gene expression homeostasis imbalance in GC, which is characterized by compartment-specific mtGene expression imbalance. Also, KRT7 and KLRC1 emerge as prognostic markers for therapies aimed at restoring mitochondrial homeostasis in GC.

Lymph node metastasis is a pivotal determinant of poor prognosis of gastric cancer, but the molecular orchestrators of lymphatic dissemination remain poorly characterized. Recent research highlights the pivotal role of small extracellular vesicles (sEVs) with specific cargo during the process. Herein, TLN1 was identified as being selectively enriched within sEVs from highly lymph-metastatic gastric cancer cells and in the serum of gastric cancer patients with lymph node metastasis, as identified through proteomic screening and confirmed by western blotting. sEVs act as key autocrine signals that influence gastric cancer cell behaviors such as proliferation, migration, invasion, and adhesion. TLN1 protein levels in cells correlate with their lymphatic metastatic potential and determine TLN1 content in sEVs. Inhibiting TLN1 reduces these cancer cell malignant behaviors and leads to TLN1-depleted sEVs. TLN1 could be transferred to gastric cancer cells and human lymphatic endothelial cells (HLECs) via sEVs. Without TLN1, sEVs cannot enhance cancer cell malignancy or induce HLEC proliferation, tube formation, adhesion, permeability in vitro, or lymphatic metastasis in vivo. Mechanistically, AKT activation was identified as a mediator of the effects exerted by sEV-TLN1 on both gastric cancer cells and HLECs. In conclusion, TLN1 orchestrates lymphatic metastasis in gastric cancer by dual-modulating tumor cell malignancy and lymphatic vessel remodeling via AKT activation. This discovery offers new perspectives on the mechanisms driving lymphatic metastasis in gastric cancer and proposes a promising target for the detection and therapeutic intervention of lymph node metastasis.

Fibroblast growth factor 2 (FGF2) is a canonical member of the fibroblast growth factor family with essential roles in embryonic development, tissue regeneration, and stem cell biology, where it supports pluripotency and high proliferative capacity. Consequently, FGF2 is widely used in experimental models of regeneration and stem cell maintenance; however, its rapid thermal degradation at physiological temperatures limits its practical utility and therapeutic potential. Despite the availability of stabilized variants, developing FGF2 analogs that combine exceptional thermodynamic resilience with favorable pharmacokinetic properties remains a critical challenge for stem cell research and regenerative medicine. Using a combination of consensus sequence analysis and structure-guided protein engineering, we generated novel FGF2 variants with unprecedented thermodynamic stability and optimized extracellular matrix interactions. To enhance bioavailability, we introduced mutations that reduced affinity for heparan sulfate proteoglycans, thereby limiting sequestration within the extracellular matrix. The most stable variant exhibited a denaturation temperature increase of more than 27 °C relative to wild-type FGF2 while fully retaining mitogenic and migratory activity after 5 days of incubation at 70 °C. This exceptional thermal stability was accompanied by markedly increased resistance to proteolytic degradation. In functional cellular models, including adipocyte differentiation and stem cell culture, an optimized long-acting variants demonstrated superior metabolic efficacy and sustained signaling, enabling a reduction in dosing frequency from daily administration to once every three days. We report the development of highly stable, highly bioactive FGF2 variants that retain full receptor specificity and binding affinity even under extreme conditions. By overcoming the intrinsic instability of the wild-type protein, these engineered FGF2s may enable future stem cell expansion and regenerative therapy applications.

Pulsatile cell contraction dynamics play a crucial role in tissue and cell morphogenesis. Previously, we identified a signal network in adherent mammalian cells, that can transduce mechanical signals via cell contraction pulses, which are generated by fast positive feedback amplification of the signal molecule Rho via GEF-H1, and by slow negative feedback that depends on actomyosin. However, the precise mechanism was still unclear, in particular if it is mediated via actin or Myosin-based components. Here, using numerical simulations, we predicted that network dynamics are strongly reduced both by Myosin II inhibition and by constitutive, Rho-activity independent Myosin II activation. We confirmed these predictions experimentally by direct inhibition of Myosin II activity and by constant, non-dynamic activation via constitutively active ROCK1. Furthermore, constitutive activation of Myosin II leads to an accumulation of Myosin II next to the nuclei which locally inhibited Rho activity dynamics. Finally, light-induced recruitment of ROCK1 to the plasma membrane strongly activated Myosin II, and at the same time depleted Actin and inhibited Rho activity. We conclude that negative feedback in the cell contraction signal network of adherent mammalian cells is implemented by Myosin II, and that actin is not the predominant inhibitory factor in this system. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

Tumor budding (TB) is a pathological hallmark of malignant invasion at the invasive front of colorectal cancer (CRC) and provides a morphologic window into early dissemination. In the International Tumor Budding Consensus Conference (ITBCC) framework, buds are single tumor cells or clusters of up to four cells, graded by hotspot counting to support standardized evaluation. Evidence from histopathology, single-cell profiling, spatially resolved analyses, and functional models links TB to invasion-competent tumor states. TB often tracks partial epithelial-mesenchymal transition, with weakened cell-cell adhesion, E-cadherin loss, altered β-catenin localization, and activation of integrin signaling, cytoskeletal remodeling, and extracellular matrix (ECM) degradation while retaining epithelial features. Spatial/trajectory analyses suggest that budding-rich regions concentrate plastic, stem-like programs biased toward migration and stress tolerance and lie close to intravasation. The TB niche also shows immune and metabolic specialization, with constrained dendritic-cell maturation and antigen presentation, reduced or dysfunctional CD8+ T-cell and NK-cell activity, and enrichment of tumor-associated macrophages and other suppressive myeloid programs. Hypoxia-driven glycolysis, lactate-associated acidification, adenosine signaling, and myeloid lipid-metabolic reprogramming can further stabilize invasive phenotypes and raise the threshold for immune control. Digital pathology and AI-enabled whole-slide analysis can improve scoring consistency and add spatial readouts linking TB patterns to immune contexture and stromal organization. Collectively, TB marks an interface between invasive tumor biology and the local microenvironment with direct relevance for risk stratification and therapeutic tailoring in CRC.

Oral squamous cell carcinoma (OSCC) is a major malignancy in South Asia, driven by betel nut/tobacco use, with 5-year survival near 50-60% and frequent recurrences reflecting its aggressive biology. Although tissue resident memory (TRM) T cells mediate innate immunity, their spatial organization and associated regulatory role of cytokine, interleukin-15 (IL-15) in the OSCC tissues and their microenvironment remain unclear.This study aimed to map CD4&#x207a; and CD8&#x207a; T lymphocytes, and TRM markers (CD103, CD49a, CD69) across tumor and stromal compartments, to assess the regulatory role of IL-15 and CD103 associated epithelial E-cadherin interactions and build a multivariate prognostic model.Method Eighty OSCC cases confirmed by histopathological examination. Formalin-fixed, paraffin-embedded tumor tissues (~&#x2009;1&#xa0;g) from surgical resections underwent immunohistochemistry. Whole-slide digital imaging (&#xd7;40) quantified CD4&#x207a; and CD8&#x207a; T lymphocytes, and TRM markers across tumor and stromal fields. Staining intensity and percent scores were generated as a composite score (0-3). Inter-observer reliability (Kendall's &#x3c4;&#x2009;&#x2265;&#x2009;0.7) was verified. Associations were tested using &#x3c7;&#xb2;/Fisher's exact, while survival was analyzed by Kaplan-Meier and Cox regression (HR, 95% CI).Results Immune and epithelial marker expression varied significantly across tumor and stromal compartments (p&#x2009;<&#x2009;0.001). Advanced tumor stage predicted poor survival (p&#x2009;=&#x2009;0.044). Retained E-cadherin correlated with improved survival (p&#x2009;=&#x2009;0.027), while CD49a expression was associated with adverse outcomes (p&#x2009;=&#x2009;0.006). Multivariate Cox regression identified CD103 (Adj.HR 2.02, 95% CI 1.03-3.96) and E-cadherin (Adj.HR 3.01, 95% CI 1.25-7.23) as independent adverse predictors, whereas stromal CD8 (HR 0.35, 95% CI 0.14-0.99) and CD49a (HR 0.37, 95% CI 0.14-0.98) were protective. High IL-15 expression showed a positive association with CD103+ TRM T cell infiltration, particularly within the stromal compartment, although the association did not reach statistical significance (p&#x2009;=&#x2009;0.076).Conclusion The spatial interplay among IL-15 expression, CD103&#x207a; immune infiltration, and the localization of TRM-associated markers suggests the presence of an integrated immune-epithelial axis associated with OSCC progression and may provide prognostic and therapeutic insights.

Reconstructing and investigating the geometry underlying data is a fundamental task in single-cell analysis, yet no unified framework exists for learning, evaluating, and diagnosing representations that faithfully preserve it. We present TopoMetry, a geometry-aware framework that learns intrinsic coordinate systems directly from the data and refines them into high-fidelity spectral scaffolds. These scaffolds capture both local neighborhoods and global structures, supporting downstream analyses such as clustering and visualization. In benchmarks across diverse single-cell datasets, TopoMetry preserved geometry more reliably than standard workflows and revealed biological signals otherwise obscured, including unexpected transcriptional diversity among T cells and links between RNA-defined subpopulations, and clonal expansion. The full analysis can be executed with a single line of code to generate a comprehensive report, making the framework both powerful and accessible. Beyond individual findings, TopoMetry warrants a shift of focus from static two-dimensional projections to the systematic learning and evaluation of geometry itself, enabling more accurate exploration of cellular diversity.

Accurate monitoring of thermal dynamics at the single-cellular scale is crucial for elucidating metabolism, signaling pathways and disease mechanisms, yet remains hampered by the limited stability and spatiotemporal resolution of current sensors. Here, we report a fiber-optic quantum dots sensor (FOQDS), which is based on a tapered fiber optic tip functionalized with CdSe/CdS/ZnS QDs and encapsulated by a dense Al2O3 layer to ensure exceptional long-term stability. FOQDS features a linear thermal response with high sensitivity (145 pm/&#xb0;C), a temporal resolution of <10 &#x3bc;s, with the potential to achieve single-cellular-scale spatial precision. This performance enables quantitative and real-time monitoring of pharmacologically induced thermal fluctuations within living three-dimensional glioblastoma spheroids. This work provides a stable and minimally invasive platform for monitoring cellular thermogenesis in physiologically relevant microenvironments, with promising prospects in applications of fundamental biology and precision therapeutics.

We have established an iPSC cell line carrying mutations in Plakophillin 2 (PKP2) and the A-kinase anchor protein 9 (AKAP9). Plakophilin 2 is an important protein of desmosomes, facilitating cell attachment and tissue strength. Its absence/mutation is associated with impaired cell adhesion and intracellular processes including calcium cycling and actin rearrangement. AKAP9, together with potassium channels and other enzymes, regulates cardiac repolarization. Mutations in both proteins are associated with arrhythmic cardiomyopathy, making this a valuable resource for the purposes of disease modeling and anti-arrhythmia drug testing.

CAR-T therapy is effective in hematologic cancers but faces challenges in solid tumors due to antigen heterogeneity and an immunosuppressive tumor microenvironment (TME). Systemic CTLA-4 blockade enhances immunity but often causes severe adverse events. To overcome these limitations, we developed a dual-modular nanobody-based CAR-T platform targeting fibroblast activation protein (FAP) on cancer-associated fibroblasts and locally releasing an anti-CTLA-4 nanobody within the tumor stroma. FAP/CTLA-4 dual-module CAR-T cells were generated and assessed in vitro for antigen-specific cytotoxicity, cytokine release, and exhaustion. Antitumor efficacy was evaluated in xenograft models, measuring tumor growth, survival, and T-cell infiltration (Ethics Approval Number: 202001011). One patient with refractory glioblastoma received intrathecal infusion; clinical response, cerebrospinal fluid (CSF) cytokines (Ethics Approval Number 2022-0553-01), and safety were monitored. Tumor and immune microenvironment changes were analyzed via transcriptomic sequencing and multiplex immunofluorescence staining. In vitro, engineered CAR-T cells showed potent cytotoxicity, cytokine production, and reduced exhaustion. In vivo, they induced tumor regression, prolonged survival, and increased T-cell infiltration. In the glioblastoma patient, intrathecal administration resulted in disease stabilization, elevated CSF cytokines, and a favorable safety profile. Transcriptomic sequencing and multiplex immunofluorescence staining indicated TME remodeling toward an immunologically active state. FAP/CTLA-4 DMN CAR-T overcomes the immunosuppressive solid tumor microenvironment through localized immunomodulation, demonstrating promising efficacy in preclinical models and a patient with refractory glioblastoma.

Light chain monoclonal gammopathy of undetermined significance (LC-MGUS) is defined by an abnormal serum free light chain ratio and elevated involved light chain in the absence of a detectable immunoglobulin heavy chain on immunofixation and of end-organ damage attributable to a plasma cell disorder. It is the least characterized of the MGUS subtypes and the one whose accurate diagnosis is most dependent on the precision of the reference intervals used to interpret free light chain measurements. Population-based screening has demonstrated that standard reference intervals substantially overdiagnose LC-MGUS through three independent mechanisms: age-related physiological free light chain elevation, impaired renal clearance in chronic kidney disease, and ancestry-related differences in individuals of African descent. Revised age-stratified and kidney function-adjusted reference intervals reduce LC-MGUS prevalence by 82%, with no observed progressions among reclassified individuals during available follow-up of 4.6&#x2009;years and have been validated across multiple independent international cohorts. For individuals of African ancestry, ancestry-adjusted reference intervals provide a further essential and independent correction. True LC-MGUS progresses along two biologically distinct trajectories: clonal expansion toward light chain multiple myeloma and toxic light chain misfolding toward AL amyloidosis. These pathways differ fundamentally in biology, determinants of progression, and clinical consequences. Risk models designed to predict myeloma progression incompletely capture the risk of amyloidogenic transformation. Because amyloidogenic transformation can occur in the setting of low clonal burden and only modestly abnormal free light chain levels, clinical evaluation must independently and explicitly address both trajectories at every patient encounter.

Type I interferons (IFNs) are central antiviral cytokines in vertebrates, yet the mechanisms underlying their functional diversification in early vertebrates remain unclear. Teleost fish, whose IFN repertoires expanded through whole-genome duplication, provide a powerful model to address this question. Here, we systematically characterize grass carp (Ctenopharyngodon idella) IFNd to elucidate the mechanisms underlying its functional divergence from IFNa. IFNd used the same receptors of IFNa to activate JAK-STAT pathway, albeit with significantly lower antiviral potency than IFNa. Structural analysis revealed significant differences in F helix length and key receptor-binding residues between IFNa and IFNd. Furthermore, single-cell RNA sequencing demonstrated high heterogeneity among immune cell subpopulations responding to IFNa and IFNd, Notably, the signaling pathways enriched by differentially expressed genes in monocytes and macrophages were distinct for the 2 cytokines. Together, our findings link ligand structural conformation and receptor-binding composition to IFN signaling strength and immune cell specificity, providing mechanistic insight into the functional diversification of type I IFNs in lower vertebrates.

Lipid-binding domains, traditionally isolated from natural proteins, are essential tools for probing membrane lipid dynamics and specialized cellular compartments. Despite diverse applications, a general strategy for their engineering remains elusive. Here we present a robust and high-throughput method for monitoring protein-lipid interactions, named the cell surface liposome binding (CLiB) assay. Using the assay, we conducted directed evolution of the PX domain from SnxA, isolating high-affinity variants specific for phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). Combining the CLiB assay with next-generation sequencing enabled parallel analysis of >6,000 clones, comprehensively identifying key residues critical for lipid binding. An engineered variant, PX-SnxAGV, functioned as a lipid biosensor in yeast and mammalian cells, visualizing PI(3,5)P2-enriched membrane subdomains upon hyperosmotic shock and during microautophagy, thereby suggesting localized PI(3,5)P2 synthesis within spatially restricted regions. This study provides a framework for on-demand generation of lipid-binding probes, facilitating the discovery of membrane compartments characterized by unique lipid compositions.

Reelin signaling regulates multiple pathways in neurodegenerative conditions, including neuronal migration, synaptic plasticity, tau phosphorylation, and amyloidogenic processing of amyloid precursor protein (APP). This study aimed to investigate the impact of reelin downregulation on the expression of topoisomerase II&#x3b2; (topo II&#x3b2;), given its crucial role in neuronal differentiation and its established association with neurodegenerative disorders such as Alzheimer's disease (AD). Furthermore, we sought to elucidate the potential relationship between reelin downregulation and proteins implicated in the pathophysiology of AD. Firstly, the optimum concentration of small interfering RNAs (siRNA) targeting reelin was transfected into SH-SY5Y cells using Lipofectamine RNAiMAX reagent. The downregulation of reelin was confirmed at the mRNA level by real-time quantitative polymerase chain reaction (qRT-PCR). Reelin-mediated molecular alterations at both the mRNA and protein levels were analyzed by qRT-PCR and Western blotting. Reelin downregulation led to a decrease in the number of viable cells as determined by the MTT assay. Consistent with the downregulation of reelin gene expression, topo II&#x3b2;, Psen1, and BACE1 expressions were also reduced, whereas tau and APP expressions were upregulated. Although siRNA treatment effectively decreased reelin mRNA levels and the proteolytic fragment of reelin protein, no significant change was observed in total full-length reelin protein levels, suggesting the involvement of post-transcriptional regulatory mechanisms. Moreover, pTAU and APP protein expressions were increased, while Nurr1 protein was decreased in reelin-silenced cells. These findings suggest that downregulation of reelin gene expression may contribute to neurodegeneration through alterations in topo II&#x3b2; and nurr1 expression, in addition to changes in proteins associated with AD pathology.

Despite rapid advances in human stem cell-based embryo model (SCBEM) research, national regulatory frameworks remain limited. On April 1, 2026, Japan implemented revised guidelines extending their scope to encompass human SCBEM research. We examine the revisions, differences from ISSCR guidelines, and practical challenges arising during the international regulatory transition period.

Mitochondrial lipid peroxidation is a major component of oxidative damage and is also thought to contribute to ferroptosis. Lipid peroxidation is generally assessed from the accumulation of oxidized end products, such as 4-hydroxynonenal (HNE). However, these report on damage throughout the cell and are affected by changes in how oxidized phospholipids are turned over. To overcome these constraints, we developed MitoLiPOX, a mitochondria-targeted mass spectrometry probe. Mitochondria targeting and detection sensitivity were achieved by incorporating a lipophilic triphenylphosphonium cation. Responsiveness to lipid peroxidation was brought about by building in a bis-allylic carbon-hydrogen bond mimic that, upon oxidation and processing, generated a single product, MitoLiPOX-OH. LC-MS/MS quantification of MitoLiPOX-OH followed by normalization to the amount of MitoLiPOX present enabled ratiometric quantification of mitochondrial lipid peroxidation. We then used MitoLiPOX to assess mitochondrial lipid peroxidation during ferroptosis in vitro and in zebrafish in vivo.