The specification of intestinal stem cells (ISCs) during development is critical for maintaining intestinal homeostasis. However, the mechanisms underlying this process remain elusive. Here, by counting and tracing ISC in Drosophila pupal midgut, we show that ISCs are specified within a narrow 12-hour developmental window, with ~150 ISCs emerging from a pool of ~6000 intestinal epithelial cells. Single-cell sequencing revealed the involvement of Notch and Wnt signaling, with genetic experiments demonstrating that ISC specification requires both Notch suppression and Wnt activation. Furthermore, we showed that Wnt signaling is activated in discrete spatial domains, and Notch-mediated lateral inhibition specifies ISCs in these Wnt-active zones, achieving a ratio of ~1/40. Notably, Notch suppression also promoted the specification of Lgr5 ⁺ progenitors in the mouse embryonic intestine. Together, our data show that Wnt activation defines niches permissive for ISC fate, whereas Notch suppression licenses fate commitment, a spatiotemporal coordination conserved from insects to mammals.

Enteric nervous system (ENS) injury, characterized by progressive degeneration of enteric neurons and glial cells, is a common diabetic complication with no effective cure beyond symptomatic management. Enteric neural precursor cells (ENPCs) play a key role in maintaining neurogenesis and gliogenesis within the adult ENS. Here, we demonstrate that bone marrow mesenchymal stem cell–derived microvesicles (BMSC-MVs) alleviate diabetic ENS injury. In both diabetic patients and mouse models, gastrointestinal transit was delayed, ENS structure was impaired, and neurogenesis and gliogenesis from ENPCs were elevated yet remained functionally insufficient. Transcriptomic profiling revealed activation of ER stress and the pro-apoptotic PERK branch of the unfolded protein response in ENPCs. BMSC-MVs homed to the colon, were internalized by ENPCs, and suppressed ER stress, thereby enhancing functional neurogenesis and gliogenesis, restoring ENS structure, and improving gastrointestinal motility. Mechanistically, vinculin on BMSC-MVs bound talin-1 on ENPCs, activating the ERK pathway to suppress diabetic ER stress. These results identify BMSC-MVs as a promising cell-free therapeutic strategy for diabetic ENS injury.

Background: Hypoxia is a key driver of cancer progression. However, its specific prognostic significance in gastric cancer (GC) remains insufficiently characterized. Methods: Single-sample gene set enrichment analysis (ssGSEA), weighted gene co-expression network analysis (WGCNA), univariate Cox regression, and least absolute shrinkage and selection operator (LASSO) regression were employed to identify a hypoxia-related prognostic signature. Subsequently, immune microenvironment profiling and single-cell RNA sequencing analyses were employed to further characterize the biological characteristics of the signature. In addition, quantitative real-time polymerase chain reaction (qPCR) was used to validate the expression levels of key hypoxia-associated genes in human GC tissues. Results: Elevated hypoxia levels were linked to worse survival outcomes in GC patients. Through integrated WGCNA, Cox, and LASSO analyses, a hypoxia-related prognostic signature (HYS) consisting of four genes—SPARC, AXL, NRP1, and VCAN—was established. Patients in the HYS-high group exhibited markedly poorer overall survival than their HYS-low counterparts [p = 0.000126, hazard ratio (HR) = 1.936]. Moreover, the HYS-high group exhibited increased infiltration of resting CD4⁺ memory T cells, monocytes, M2 macrophages, and resting mast cells, as well as elevated expression of immunosuppressive molecules, including PDCD1LG2 and HAVCR2. Single-cell RNA sequencing analysis revealed that the signature genes were predominantly expressed in cancer-associated fibroblasts. Consistently, qPCR analysis in five paired GC and para-carcinoma tissues confirmed higher expression of these genes in tumor samples (p < 0.01). Conclusions: Our findings indicate that hypoxia is a critical determinant of prognosis in GC and is closely associated with an immunosuppressive tumor microenvironment, highlighting its potential value as a prognostic biomarker and therapeutic target.