Cranial sutures are dynamic growth sites that balance bone growth with mesenchymal patency to accommodate cranial expansion during development. While intramembranous ossification has traditionally been considered the default mechanism of suture fusion, accumulating evidence demonstrates that endochondral pathways might also play a significant role under both physiological and pathological conditions. In this review, we contrast normal developmental ossification processes with premature fusion in craniosynostosis, integrating histological, molecular, and imaging data. We highlight the context-dependent nature of cranial suture biology, influenced by embryonic origin, local signalling gradients, and genetic perturbations. Recognizing divergent ossification mechanisms reframes our understanding of both normal and premature suture fusion and has clinical implications for mechanism-specific therapeutic strategies. Finally, we outline areas for future investigation, including high-resolution profiling of human sutures across developmental stages, to establish a normative framework for cranial suture biology and inform mechanism-driven regenerative approaches.

Cytotoxic chemotherapy primarily targets rapidly proliferating cancer cells but also depletes normal myeloid cells. The resulting cell loss triggers reactive myelopoiesis, a compensatory process in which hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) regenerate myeloid lineages. We previously showed that the alkylating agent cyclophosphamide (CTX) induces myelopoiesis leading to the expansion of immunosuppressive monocytes in mice. However, the molecular features and clinical relevance of these cells remain poorly understood. Here, we report the emergence of immunosuppressive monocytes in the peripheral blood of lymphoma patients receiving CTX-containing chemotherapy. To gain mechanistic insight into CTX-induced myelopoiesis, we performed single-cell RNA sequencing (scRNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) on BM monocytes from CTX-treated mice. These analyses revealed a heterogeneous monocyte population and demonstrated that CTX skews myelopoiesis toward the generation of neutrophil-like monocytes (NeuMo). Moreover, CTX-induced NeuMo cells, enriched within the CXCR4⁺CX3CR1⁻ monocyte subset, exhibited potent T-cell suppressive activity. Using the NeuMo gene signature, reanalysis of public scRNA-seq datasets identified a transcriptionally similar monocyte subset in chemotherapy-treated cancer patients. Collectively, our findings suggest that the expansion of NeuMo-like cells following chemotherapy represents a conserved immunoregulatory feedback mechanism with potential impact on tumor response to chemoimmunotherapy.

The replenishment of specialized cells depends on the activity of stem cells. Recent advances in stem cell research have shown that the germline stem cells (GSCs) in Drosophila melanogaster can increase their mitotic activity in response to mating. Here, we show that this ability to respond to mating is eliminated if the males are mutant for either of the ABC transporters, White (W), Brown (Bw) or Scarlet (St), which are known for their role in eye pigmentation and amine production. However, reducing the expression of w specifically from the germline cells also caused a failure to increase GSC mitotic activity upon mating, suggesting that w is required intrinsically in the stem cells. The w gene is a common genetic background for genetic experiments and frequently used as a control. Our findings underline the importance of careful experimental design and control choice.

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.

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.

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.

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.

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.

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.

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.

Hematopoietic stem cells (HSCs) survive many types of cellular stress but often lose their regenerative and lymphopoietic capacities as a result. Such functional decline also occurs with age, and dysfunctional HSCs with impaired mitochondria accumulate during aging. However, the molecular link between HSC stress response and age-related functional decline remains poorly understood. Here we show that multiple stress responses converge on the RIPK3-MLKL axis to induce age-related changes in HSCs. The necroptosis effector MLKL is readily activated by inflammation and replication stress and accumulates in HSC mitochondria. Consequently, activated MLKL does not cause cell death but impairs HSC self-renewal and lymphoid differentiation. Such MLKL-mediated functional decline also occurs in HSCs during organismal aging, with activated MLKL primarily mediating age-related mitochondrial damage and reduced glycolytic flux. Collectively, our results establish the RIPK3-MLKL axis as a key mediator of HSC aging and identify a necroptosis-independent role of MLKL in mitochondrial damage.

Protein tyrosine phosphatase receptor type Z1 (PTPRZ1) is one of the most abundantly expressed and enriched genes in astrocytes during development, yet its function in astrocytes is unknown. Using an astrocyte-neuron co-culture system, we found that knockdown of Ptprz1 in astrocytes significantly impaired astrocyte branching morphogenesis. To investigate the function of PTPRZ1 in astrocytes during brain development, we generated a Ptprz1 conditional knockout mouse and deleted Ptprz1 from astrocytes postnatally, after the bulk of astrogenesis is complete. At postnatal day 21, we found subtle changes in astrocyte morphology and a reduction in the density of co-localized pre and post synaptic excitatory synapse markers across multiple layers of the visual cortex in both male and female mice, suggesting important functions for astrocytic PTPRZ1 in both astrocyte morphogenesis and synaptogenesis. PTPRZ1 is expressed in several neural cell types, including radial glial stem cells and oligodendrocyte progenitor cells (OPCs), and regulates critical aspects of neurodevelopment, including neurite outgrowth, neuronal differentiation, myelination, and extracellular matrix (ECM) development. Moreover, altered PTPRZ1 expression is associated with schizophrenia and glioblastoma. Therefore, this mouse model is a valuable resource for investigating cell-type-specific PTPRZ1 function in numerous neurodevelopmental and neuropathological mechanisms.Significance Statement PTPRZ1 is an abundant, astrocyte-enriched protein linked to neurological dysfunction; however, its astrocyte-specific functions are unknown. We generated a Ptprz1 conditional knockout mouse and found that astrocyte-specific deletion of Ptprz1 reduces the density of co-localized excitatory synapse markers in the developing mouse cortex, with mild impact to astrocyte morphology. PTPRZ1 is an emerging therapeutic target for glioblastoma and neurodegeneration. This study provides a new tool to study PTPRZ1 function in neurodevelopment and neuropathology.

Osteogenesis depends on the self-renewal and differentiation of mesenchymal stem cells (MSCs). Emerging research underscores the regulatory functions of RNA methylation on bone homeostasis. Here, we show PCIF1, the N6,2'-O-dimethyladenosine (m6Am) methyltransferase, is essential for maintaining bone mass and promoting osteogenic differentiation of MSCs. Multiple complementary analyses-including GWAS, TWAS, and single-cell transcriptomics-collectively point to PCIF1 as a regulator of human bone mineral traits and early-stage mesenchymal differentiation. Global or MSC-specific Pcif1 deletion elicits osteoporotic pathology in mice, although myeloid cell-specific Pcif1 knockout does not induce femur bone alterations. Mechanistically, Pcif1 knockout decreases m6Am signals of Wnt-related genes (Wnt11, Fzd4, and Fgfr2) and accelerates mRNA degradation. This down-regulates active β-Catenin protein, and thus impairs osteogenic function of MSCs. Additionally, the WNT agonist attenuates the osteoporosis-like phenotype induced by Pcif1 deletion. These findings highlight the crucial role of PCIF1-mediated m6Am modification in regulating osteogenesis and suggest potential therapeutic implications for bone disorders.

Bone maintains a dynamic and stable state through the orchestration of osteoclasts and osteoblasts. Osteoblasts are derived mainly from mesenchymal stem cell (MSC) and are responsible for bone formation. The inhibition of osteoblast proliferation and differentiation is involved in many diseases, including osteoporosis, osteoarthritis, infected bone defects, and inflammatory aseptic loosening of implants. Given the currently limited treatment options, exploring new methods to promote bone formation is an important focus significant for orthopedists. Platelet-rich plasma (PRP), an autologous substance that is rich in various growth factors, is widely used in regenerative medicine. However, the effect of PRP on inflammatory bone destruction remains unclear. We investigated the effects of PRP on the viability of MSC, and the impact of different concentrations of PRP (1% and 3%, respectively) on cell death, proliferation, and differentiation. Furthermore, we tested the therapeutic effect of different concentrations of PRP (1% and 3%) on LPS-induced inflammatory bone destruction in vivo. PRP enhanced the cellular activity of MSC and promoted osteogenesis. A higher concentration of PRP (3%) primarily reduced the death and increased the proliferation of in LPS-treated MSC via the PI3K/AKT pathway, while a lower concentration of PRP (1%) promoted MSC differentiation into osteoblasts through the MAPK pathway. Consistent with in vitro experiments, we validated the protective effect of PRP against LPS-induced bone loss by increasing bone formation in vivo. These results suggest that different concentrations of PRP can ameliorate LPS-induced inflammatory bone loss through distinct mechanisms.

What is the current landscape of randomized controlled trials (RCTs) evaluating stem cell-based therapies for women's reproductive diseases, and how effectively has preclinical research informed their clinical translation?

Nucleic acid-based therapeutics, which involve the manipulation of genetic materials to treat or prevent diseases, have gained considerable attention, leading to the approval of medicines such as COVID-19 vaccines, patisiran (Onpattro), and nusinersen (Spinraza). However, their clinical application is hindered by challenges such as nuclease degradation, poor biodistribution, limited cellular uptake, and inefficient endosomal escape. Extracellular vesicles (EVs), which are natural nanoscale drug delivery systems derived from various eukaryotic and prokaryotic cells, offer a safe, efficient, specifically targeted, and non-pathogenic method for nucleic acid delivery. In this review, we summarize the classical methods and the latest research advances in EV preparation and nucleic acid loading. Additionally, we review the primary administration routes for nucleic acid-loaded EVs, such as intravenous, local, oral, intranasal, and inhalation delivery. By addressing these aspects, this review aims to guide the optimal design and clinical application of nucleic acid-loaded EVs.