The repair of critical-sized cranial defects resulting from trauma, congenital malformations, or surgery remains a significant clinical challenge. Polyetheretherketone (PEEK) is a commonly used material for cranial defect repair in clinical practice. However, its application is severely limited by drawbacks such as poor structural adaptability, bioinertness, and insufficient osseointegration, which significantly compromise clinical outcomes. In this study, we biomimicked the structure and composition of natural bone to fabricate a biomimetic PEEK/hydroxyapatite (HA) scaffold via a 3D printing strategy. This scaffold exhibits a stochastic lattice structure and mechanical properties similar to natural bone, capable of sustained release of calcium and phosphate ions. An in vitro co-culture study with bone marrow mesenchymal stem cells (BMSCs) confirmed that the biomimetic scaffold promotes cell proliferation and enhances the expression of osteogenesis-related proteins and genes. In a rat model with a critical-sized cranial defect (8 mm diameter), the biomimetic PEEK/HA scaffold demonstrated excellent capability in inducing bone regeneration and promoting osseointegration. This research provides a viable strategy for the repair and functional reconstruction of clinical critical-sized cranial defects.

For several years, scientists have focused on studying mesenchymal stem cells because of their ability for self-regeneration, their potential for differentiation into multiple lineages (e.g., osteogenic, chondrogenic, and adipogenic cells), and their low immunogenicity and remarkable capacity to modulate the immune system. The importance of these cells ranges from preserving tissue health and repairing injured tissues in their nearby areas, to their use in scientific investigation for the treatment of neurodegenerative, autoimmune, and cardiovascular diseases. Despite the availability of various tissue sources, such as bone marrow and adipose tissue, their collection and handling may not always be easily achievable. However, dental tissues, such as the pulp and periodontal ligament, are relatively accessible supplies that do not require complex or stressful interventions for the patient. Here, we provide a detailed description of each step involved in the isolation and characterization of mesenchymal stem cells from the pulp and periodontal ligament using monoclonal antibodies ensuring a high level of effectiveness. In addition, we also describe a technique to generate the cellular presenescence state. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Isolation of human periodontal ligament and dental pulp stem cells Support Protocol 1: Characterization of human periodontal ligament and dental pulp stem cells Support Protocol 2: Induction of presenescence of human periodontal ligament and dental pulp stem cells.

Generation of CAR macrophages from induced pluripotent stem cells(iPSCs) hold great potential for immunotherapy, particularly against T-cell malignancies which are challenging in CAR-T therapy. However, the tumoricidal activity of human iPSCs derived CAR-macrophages (iCAR-Ms) remains less extensively analyzed. Here, we generated human iCAR-Ms targeting CD5 for T-cell malignancy therapy. iCAR-Ms show up-regulation of immunity related functions as well as tumoricidal activity against different T malignant cells expressing CD5. However, the tumoricidal activity of iCAR-Ms is highly related to CD5 density on tumor cells and depends on high dose treatment in vivo. We further reveal that the tumor cells resisting iCAR-M killing show reversible CD5 loss mediated by iCAR-M trogocytosis. In contrast, the retrieved iCAR-Ms from tumor cell co-culture retained tumoricidal activity on new tumor cell expressing CD5. Thus, we identify trogocytosis as an important limiting factor on iCAR-Ms therapy, providing a rationale for developing enhanced CAR-M therapies.

The Par complex regulates cell polarity in diverse animal cells, but how it is restricted to a specific membrane domain remains unclear. The tumor suppressor Lethal giant larvae (Lgl) is thought to inhibit Par complex membrane binding, yet in metaphase Drosophila neural stem cells (NSCs), Lgl is cytoplasmic while the Par complex is apically polarized, raising the question of how Lgl controls Par localization when it is not on the membrane. Using live imaging, we found that Lgl and atypical Protein Kinase C (aPKC) exhibit tightly coordinated, opposing membrane dynamics: aPKC displaces Lgl at mitotic entry, while Lgl displaces aPKC at mitotic exit. In Lgl-depleted NSCs, aPKC is not fully cleared from the membrane after mitosis, and this residual aPKC persists into the subsequent division, disrupting Miranda polarization. Apical aPKC recruitment still occurs, indicating that Lgl is not required for Par polarization per se but for ensuring aPKC absence from the basal membrane before mitosis. These findings reveal a temporal mode of mutual antagonism between Lgl and the Par complex that may license proper asymmetric division.

Rapid and reliable identification of stem cells is a critical prerequisite for regenerative medicine and quality control during cell-based therapies. We report herein a lateral flow assay capable of detecting MSCs by dual-marker recognition using CD105 and CD29 surface antibodies. The biosensing platform relies on gold nanoparticle-conjugated CD29 antibodies as signal probes and immobilized CD105 antibodies as capture agents on the test line, allowing selective MSC recognition through sandwich-type immunocomplex formation with direct colorimetric readout. A distinct red band indicated the successful detection of MSCs, while a control line ensured assay validity and reliability. The developed LFA showed a naked eye detection limit as low as 600 cells within 15 min, which agreed with the qualitative data obtained via a smartphone-based Color Grab application. Notably, the device exhibited excellent stability with no cross-reactivity with non-target cells and successfully distinguished MSCs from their differentiated lineages. Besides this, the device retained stability for up to 28 days under room temperature. Further, MSC stemness was independently validated for CD105 and CD29 by flow cytometry (FACS), which requires a minimum of ≥10 000 cells per analysis. In comparison, the developed LFA provided reliable detection at substantially lower cell numbers, highlighting its significance as a low-input, rapid, and point-of-care alternative. This user-friendly platform represents a promising tool for rapid stemness assessment of MSCs towards their quality control and clinical translation in stem cell research and regenerative medicine applications.

Aplastic anemia (AA) is a rare bone marrow failure syndrome characterized by immune-mediated destruction of hematopoietic stem and progenitor cells (HSPCs). The contribution of the bone marrow microenvironment remains incompletely understood. We analyzed 29 bone marrow biopsies from patients with moderate (mAA), severe (sAA), and very severe AA (vsAA), along with 12 healthy controls and seven subcortical pseudohypocellular samples. Immunohistochemistry for nestin and CXCL12 was performed to quantify stromal niches. RNA sequencing was carried out to investigate immune and niche-related gene expression patterns. sAA patients exhibited a significantly increased number of nestin+ niches compared to mAA and controls. CXCL12+ niches showed no significant differences between groups. RNA sequencing revealed upregulation of immune response genes, as well as pathways related to interferon-gamma signaling, JAK-STAT3 activation, and antigen presentation. Downregulated genes and pathways pointed to impaired DNA repair, cell cycle regulation, and epigenetic stability. Our findings support a model in which AA pathogenesis is driven by both immune injury and compensatory, yet dysfunctional, stromal remodeling. These data underline the importance of the bone marrow microenvironment in AA.

Alpha-gal syndrome (AGS), a distinct form of IgE-mediated hypersensitivity to the carbohydrate galactose-α-1,3-galactose (α-Gal), typically occurs after repeated tick bites and leads to allergic reactions after ingestion of mammalian meat. Patients with AGS may experience a broad spectrum of allergic reactions, from pruritus and urticaria to angioedema and anaphylaxis. Although classically associated with food-triggered reactions, emerging reports describe AGS-related anaphylaxis following exposure to group B blood products, including cases without accompanying gastrointestinal symptoms, highlighting an underrecognized clinical presentation.

Cancer stem cells (CSCs) contribute to an immunosuppressive microenvironment through complex mechanisms and tumor-immune interactions. However, the key determinants of CSC characteristics in driving tumor progression, immune suppression, and response to ICIs remain unclear and require systematic investigation. This study developed a quantitative systems pharmacology (QSP) model covering various CSC properties, thereby capturing the temporal dynamics and CSC-immune interactions in triple-negative breast cancer (TNBC). Using the unified longitudinal dataset of tumor growth, CSC frequency, and immune cell dynamics that we obtained from BALB/c mice bearing wild-type or Cd274-knockout 4T1 cells under various inoculation conditions, which provides multi-dimensional insights into CSC-related biology, the QSP model was calibrated and validated. Simulations and sensitivity analysis indicated that TNBC tumors with strong stemness exhibited significantly accelerated tumor growth and reduced infiltration of cytotoxic immune cells such as cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. These were associated with CSCs' enhanced self-renewal capacity, stemness maintenance, secretion of transforming growth factor beta (TGF-β) and vascular endothelial growth factor (VEGF), and PD-1/PD-L1-mediated immunosuppression. ICIs showed minimal efficacy in tumors with enhanced stemness, which was also linked to the aforementioned characteristics. Both the administration sequence and initiation timing of ICIs differentially influenced the therapeutic outcomes. These findings elucidate the roles of CSCs in TNBC progression, tumor immunity, and ICI efficacy while identifying the key underlying CSC characteristics, suggesting the potential value of assessing CSC biomarkers or abundance before ICI treatment and support the development of ICIs and anti-CSC combination therapies.

Microglia have been implicated in the templated spread of tau aggregates in tauopathies through mouse studies. However, it is unclear whether these findings translate to human disease.

The role of actin and its binding proteins has been discovered in cytoskeleton remodeling as well as in Epithelial-Mesenchymal Transition (EMT) of metastatic cells and apoptosis. Even minor changes in the biomolecular structure of actin and its ABPs (by binding of ligands) can lead to drastic changes in the cytoskeleton with far reaching effects per se. Agents targeting actin can, thus, be viewed as potential anti-metastatic agents. The effect of Withania somnifera (L.) Dunal (WS) on the cytoskeleton has remained relatively unexplored. The present study highlights the interaction between 20 WS phytoconstituents and 10 selected cytoskeletal proteins in silico with a view to validate and analyze the perturbation in growth and differentiation of breast cancer cells in vitro. Pharmacokinetic analyses revealed that the majority of WS phytoconstituents exhibited no violations of Lipinski's rule-of-five parameters. Withanolides A, B, D, M and O displayed the greatest binding affinity particularly for coronin1A, vimentin, gelsolin, ezrin and F-actin. MD simulations of 100 ns revealed maximum stable interaction(s) between Coronin-Viscosalactone B (VISCB) and Vimentin-Withanolide E (WITHE). The prepared methanolic extract of WS stem (WSME), characterized using LC-MS, revealed the presence of Withaferin A (WFA). Both WSME and WFA exhibited potent cytotoxicity against breast cancer MDA-MB-231 cells. WSME increased ROS levels, arrested the cell cycle in S and G2-M phases, decreased the expression of mesenchymal markers, namely, vimentin, N-cadherin and increased levels of the epithelial marker E-cadherin in treated MDA-MB-231 cells. These findings suggest that VISCB, WITHE, and WFA have the potential to emerge as potential antimetastatic agents against breast cancer in the future.

The worldwide diabetes epidemic underscores the urgent need for innovative therapeutic approaches. This review provides an in-depth analysis of the significant potential of marine polysaccharides in both the prevention and management of diabetes. Derived from algae, animals, and microorganisms, these polymers offer distinct structural advantages compared to their terrestrial counterparts, such as increased sulfate content and a wider range of molecular weights. We systematically elucidate their multi-targeted hypoglycemic mechanisms, which include the inhibition of digestive enzymes (α-amylase and α-glucosidase), enhancement of insulin sensitivity, promotion of peripheral glucose metabolism, protection of pancreatic β-cell function, and modulation of gut microbiota. A comprehensive analysis of the intricate structure-activity relationships highlights the influence of specific structural characteristics on bioactivity. Additionally, the utilization of marine polysaccharides in novel drug delivery systems and strategies to enhance efficacy is examined. Despite challenges related to standardization and the necessity for more detailed mechanistic understanding, marine polysaccharides, due to their diverse actions, biocompatibility, and sustainability, present as a promising avenue for the development of next-generation, multi-targeted therapies and functional foods. This approach ultimately offers a holistic strategy for diabetes management, leveraging the extensive resources available within the marine environment.

Skeletal muscle stem cells (SkMSCs) are essential for muscle regeneration and represent a promising therapeutic target for muscle disorders. However, effective strategies to precisely regulate SkMSC fate by integrating biochemical and mechanical cues remain limited.

Temozolomide (TMZ) resistance in glioblastoma (GBM) remains a critical barrier to treatment success, driven by O6-methylguanine-DNA methyltransferase (MGMT) overexpression, glioma stem cell (GSC) persistence, and redox adaptation.

Early-life exposure to general anesthetics, particularly propofol, elevates the risk of neurodevelopmental impairment and cognitive sequelae in pediatric populations, representing a pivotal concern in translational neuroanesthesiology. Although preclinical studies have linked propofol to increased developmental neurotoxicity, the underlying molecular mechanisms remain elusive. Our previous work established that nuclear fragile X mental retardation-interacting protein 1 (NUFIP1)-engineered exosomes from human umbilical cord mesenchymal stem cells could mitigate propofol-induced neurotoxicity and neuronal apoptosis in neonatal rats during a critical postnatal window of synaptogenesis (postnatal days 7-14). The present study provides the first mechanistic insights by performing transcriptomic profiling to link this neuroprotection to the endoplasmic reticulum stress (ERS) apoptotic pathway. Importantly, we directly validated key ERS/apoptosis markers and functionally confirmed the pathway's role through pharmacological rescue experiments with Salubrinal. In conclusion, NUFIP1-engineered exosomes regulate propofol-induced nerve injury through the ERS apoptotic pathway, offering novel mechanistic insights with potential implications for addressing pediatric neurodevelopmental impairments.

Obesity imposes dysfunction of the endogenous cellular reparative system, which may manifest as impaired adipose tissue-derived mesenchymal stem/stromal cells (AT-MSCs) function or altered characteristics of circulating endothelial progenitor cells (EPCs). However, whether both systems are abnormal in patients with obesity remains unclear. We hypothesized that human obesity induces impairment of MSCs and EPCs that would be reversed after weight-loss surgery (WLS).

Information on the maintenance of tissue homeostasis is important for developing effective therapeutic methods. However, reports on the cellular composition and tissue stem cells of the larynx are scarce. Therefore, we analyzed mouse laryngeal mucosa using single-cell RNA- sequencing and spatial transcriptomics by photo-isolation chemistry, and we also generated laryngeal organoids as an in vitro model. Consequently, we found a SOX9-positive basal cell subpopulation and a Lgr5-positive cell subpopulation in the mouse vocal fold, and obtained three types of epithelial organoids from laryngeal epithelium. We also confirmed the differences in pseudostratified ciliated columnar epithelium characters between the supraglottis and subglottis of the mouse larynx. These findings provide valuable insights and tools for future research in laryngology and stem cell biology.

FLT3-ITD inhibitors are approved for acute myeloid leukemia (AML) treatment but relapse is common. In this study, the combined inhibition of FLT3-ITD signal and protein translation by QUIZartinib and Omacetaxine Mepesuccinate (QUIZOM) synergistically suppressed the most critical FLT3-ITD survival signals including mitochondrial respiration and proteostasis, which induced apoptosis and pro-inflammatory response. In a Phase 2 trial (NCT03135054) involving 40 chemo-refractory/unfit FLT3-ITD AML patients, QUIZOM achieved a composite complete remission (CRc) of 83%, a median leukemia-free survival (LFS) of 10 months (Range: 0.7-68.2 months) and a median overall survival (OS) of 12.9 months (Range: 1.8-69.2 months). 13/33 (39%) received allogeneic HSCT after a median of 143 days (Range: 53-367 days). Higher CRc rates were observed in patients with NPM1 mutations, DNMT3A mutations, and wild-type WT1. Single-cell RNA-sequencing of QUIZOM cohort revealed positive correlation between pro-inflammatory response in blasts, CD8 + T activation and clinical responsiveness. Further, we identified a leukemic stem cell (LSC) subpopulation with activated JNK/JUN/HSPA1B axis via PLD1-driven phosphatidylcholine metabolism, which promoted proteostasis and drove QUIZOM resistance. PLD1-inhibitor remodeled phospholipid metabolism, induced ferroptosis and restored QUIZOM response in LSC. Our findings provided the therapeutic and resistant mechanisms of QUIZOM and paved the way for targeted interventions in this AML subtype.

While genetic screens have facilitated the dissection of protein function in animal development, advances in systematic point mutagenesis open new opportunities for forward genetics in mammalian cells. Here, we develop a CRISPR/Cas9-mediated base editing screen that allows functional screening of extensive collections of single amino acid substitutions of endogenous proteins. We demonstrate the application on the X-chromosomal Hprt and the autosomal Msh2 gene in diploid male and haploid mouse embryonic stem cells, respectively. Finally, we use this methodology to generate a sequence-function map of the transcriptional co-repressor SPEN in X chromosome inactivation. We demonstrate that the substitution of the SPEN RRM4-residue W522 abrogates X-linked gene repression by Xist RNA and impairs the establishment of H3K27me3 deposition. Our results demonstrate that screening in haploid cells allows efficient identification of mutations that would be recessive in diploid cells, suggesting applications across a wide range of areas.