Measurable residual disease (MRD) is a key prognostic factor for outcomes following allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with acute myeloid leukemia. To enhance predictive performance, we developed a combined MRD assessment method that integrates the proportion of residual leukemic cells of ten-color multicolor flow cytometry (MFC) with peripheral blood WT1 mRNA expression.

Biomineralization refers to the process by which organisms form inorganic minerals, predominantly through the deposition of calcium phosphates. Calcium ions (Ca2+) serve not only as a fundamental component of the mineral phase but also as key signaling messengers that actively regulate the efficiency and progression of this process. The endoplasmic reticulum (ER) and mitochondria are two major organelles responsible for calcium ion storage and regulation within cells. Contact sites between mitochondria and ER, also called mitochondria-ER contacts (MERCs) or mitochondria-associated ER membranes (MAMs), have been identified as vital spots for calcium transfer. Existing research indicates that calcium ion transport from the ER to mitochondria occupies a pivotal position in biomineralization, but the relevance of MERC integrity in biomineralization is yet to be determined. This study revealed increased MERCs and calcium ion transport during mineralization in vivo and in vitro. Additionally, significantly impaired endoplasmic reticulum-mitochondrial interactions were observed in bone marrow mesenchymal stem cells (BMSCs) from ovariectomy-induced osteoporotic mice. Experimental enhancement of MERCs effectively increased mineralized nodule formation and alleviated ovariectomy-induced osteoporosis, whereas disruption of MERC integrity inhibited mineralization. Our findings indicate that endoplasmic reticulum-mitochondrial calcium ion transport plays a crucial role in biomineralization. This discovery provides a stronger theoretical foundation for elucidating the biomineralization process and may also identify novel therapeutic targets for related diseases.

The accuracy of crucial nuclear processes such as transcription, replication, and repair depends on the local composition of chromatin and the regulatory proteins that reside there. Understanding these DNA-protein interactions at the level of specific genomic loci has remained challenging due to technical limitations. Here, we introduce a method termed 'DNA O-MAP', which uses programmable peroxidase-conjugated oligonucleotide probes to biotinylate nearby proteins. We show that DNA O-MAP can be coupled with label-free or sample multiplexed quantitative proteomics, targeted chemical perturbations, and next-generation sequencing to quantify DNA-proximal proteins and DNA-DNA interactions at specific genomic loci in human and murine cells. Furthermore, we establish that DNA O-MAP is applicable to both repetitive and unique genomic loci of varying sizes, from kilobase HOX gene clusters to megabase alpha-satellite repeats, and that DNA O-MAP can measure proximal molecular effectors in a homolog-specific manner.

Normal mitochondrial function in stem cells is essential for effective bone regeneration, with mitochondrial complex IV (cytochrome c oxidase, CcO) playing a crucial role in sustaining electron transport chain activity and ATP synthesis. To address mitochondrial dysfunction associated with bone defects, we developed a dendritic mesoporous silica nanoparticle (DMSN)-based, CcO-mimetic nanozyme, named triphenylphosphonium (TPP)-DMSN-Fe/Cu. The nanozyme incorporated iron and copper single atoms to mimic the catalytic center of CcO and is modified with the mitochondria-targeting agent TPP. In vitro, TPP-DMSN-Fe/Cu nanozymes colocalized with mitochondria and enhanced mitochondrial function, effectively regulating cellular energy metabolism and promoting stem cell osteogenesis. In vivo, TPP-DMSN-Fe/Cu nanozymes resulted in significantly enhanced bone regeneration compared to the control, resulting in a 177% increase in bone volume and a 12% increase in mineral density at critical-sized bone defects in rats after 4 weeks of treatment. Taken together, these findings demonstrate that bioinspired, mitochondria-targeting TPP-DMSN-Fe/Cu nanozymes hold strong promise for accelerating bone regeneration via regulating cellular energy metabolism.

Extracellular vesicles (EVs) are promising vehicles for targeted therapeutic delivery, capable of encapsulating and transporting biomolecules to specific cells and tissues. Given that inflammation is central to many acute and chronic diseases, understanding EV biodistribution under inflammatory conditions is essential for therapeutic optimisation. This study examines how acute systemic inflammation influences EV biodistribution, clearance and plasma half-life, with a focus on the role of macrophages and their polarisation states. Using a lipopolysaccharide (LPS)-induced inflammation model in wild-type mice and bioluminescent and fluorescent labelling of EVs, we observed that inflammation extends the plasma half-life of EVs by over 600-fold within 2 h and 900-fold at 24 h post-administration, leading to significant enrichment in inflamed organs, particularly the liver and spleen. Enhanced accumulation in specific tissues translated to increased targeting of immune- and epithelial cells within those organs, with notable uptake by hepatocytes in the liver. To probe the mechanism, we profiled the EV protein corona, revealing inflammation-driven remodelling with enrichment of acute-phase proteins, complement factors and cytoskeletal regulators-linking corona composition to altered biodistribution. Yet, despite increased uptake and tissue accumulation, functional EV cargo delivery in vivo remained limited. These findings underscore the complex dynamics between EVs and immune cells under inflammatory conditions and provide critical insights for advancing EV-based therapies in chronic inflammatory diseases.

Advancements in tissue engineering have revolutionized therapeutic paradigms for diabetic tissue defects; however, the lack of applicable scaffold containing various bioactive substance aggregates remained a critical bottleneck hindering satisfactory repair effect. In this study, adipose-derived stem cells (ADSCs) were functionally re-engineered using lipoic acid (LA) to fabricate a novel LA-intervened stem cell spheroid (LA-SCS) with enhanced paracrine activity and extracellular matrix (ECM) biosynthetic capacity. Subsequent decellularization mitigated immunogenicity, yielding LA-intervened decellularized stem cell spheroid (LA-dSCS). In vitro assays confirmed its immunomodulatory potency, as evidenced by the activation of signaling cascades associated with macrophage reprogramming, homeostasis, and autophagy. Furthermore, leveraging the intrinsic viscoelastic properties of the LA-dSCS, a convenient preparation method for preparing LA-dSCS derived injectable material was established, wherein LA-dSCS micro-particles assemble into LA-dSCS granular gel. In vivo studies using diabetic rat models demonstrated closure of both wound and cranial defects. Collectively, this study established a biomimetic engineering strategy that integrates cell-free bioactive aggregates with injectable granular gels, offering a novel proof‑of‑concept strategy for the regeneration of complex diabetic tissue defects.

Regenerative repair of segmental bone defect remains a major clinical challenge. The conventional mental implants suffer from mechanical strength mismatch and long-term foreign bodies presence. While, the osteoinductive materials lack insufficient mechanical strength for the repair of load-bearing bone. Inspired by bamboo, whisker-reinforced Ca-P ceramics (HW) have been developed to accelerate segmental bone regeneration. Bamboo-like whiskers can reduce the stress concentration and impede crack propagation via whisker broken, whisker bridging, and crack deflection, achieving 3 times increase in the compressive strength compared to traditional ceramics. In addition, HW significantly promotes the formation of type-H endothelial cells (ECs) via the integrin-mediated HIF-1 signaling pathway, which can secrete coupling factors (e.g. Noggin), to promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). In turn, HW can stimulate BMSCs to secrete coupling factors (e.g. VEGF) to support angiogenesis. In the segmental bone defect model, DFO is introduced to bamboo-like whiskers (HW+DFO) to enhance type-H vessels formation. HW+DFO can effectively promote the repair of segmental bone defects with mechanical recovery reaching 40% of that of normal femur. In conclusion, this study proposes a novel strategy for promoting bone regeneration by regulating type-H vessels, offering a potential solution for addressing segmental bone defects.

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.