Abstract Adult mammalian cardiomyocytes exhibit limited regenerative capacity, which remains a major challenge for functional recovery after myocardial infarction (MI). Our previous study identified a cocktail of five small molecules (5SM) that promotes cardiomyocyte proliferation and heart regeneration; however, the complexity of this five-compound mixture poses significant barriers to clinical translation and mechanistic studies. Here, we identified a simplified dual-compound cocktail (2SM) composed of phenylephrine hydrochloride (PE) and harmine (HM), which also robustly enhanced cardiomyocyte proliferation in vitro and in vivo. Transcriptomic and metabolomic analyses indicated that 2SM treatment activated cardiomyocyte cell-cycle programs and metabolic reprogramming. PE activated a fetal program and contributed to the up-regulation of CCND1 and WEE1 by activating mTOR signaling pathway, thus enabling cardiomyocytes to enter the G1/S phase but not G2/M phase. HM inhibited DYRK1A and activated MMB and FOXM1-MuvB complex, resulting in the upregulation of G2/M phase gene expression, of which the upregulated CDC25 proteins dephosphorylate CDK1, thereby relieving the inhibitory effect of PE. Sequential treatment of PE followed by HM was essential for completion of cytokinesis. In summary, we identified that 2SM, two compounds of 5SM, reprogrammed cardiomyocytes into an embryonic-like state, and precisely coordinated cell cycle progression for promoting cardiomyocyte proliferation.
While computational deconvolution is routinely used to estimate cell-type proportions from tissue mixtures, reconstructing cell-type-specific transcriptomes at single-sample resolution remains a fundamentally underdetermined algorithmic challenge. Consequently, accurate single-sample, gene-level inference is rarely achieved by existing tools. Here, we systematically benchmarked multiple deconvolution approaches across diverse biological contexts using both pseudo-bulk mixtures and real bulk RNA-seq datasets derived from multiple tissues. Evaluating the critical computational limitations in these models, we developed BayesPrism-DWLS, a framework that enables integrated inference of cell-type proportions and cell-type-specific expression at single-sample resolution. Applied to mouse colon bulk RNA-seq and spatial transcriptomics, BayesPrism-DWLS revealed cell-type-specific genes and pathways that were undetectable at the bulk or spot level. Therefore, this framework provides a robust, high-resolution tool for dissecting cell heterogeneity and supports mechanistic studies informed by cell-type-specific transcriptional programs using cost-effective sequencing data.
Skeletal muscle function deteriorates with injury, disease, or aging. While stem cell therapies offer therapeutic potential, concerns remain regarding the safety and efficacy of transplanted cells. Satellite cells (SCs), the primary endogenous stem cells responsible for muscle repair, demonstrate safe transplantation but limited clinical effectiveness. A deeper understanding of the mechanisms regulating SC activation and differentiation is critical for improving muscle regeneration strategies. We examined the role of CSRP3 in SC-mediated muscle repair using injury models and myoblast differentiation assays. We compared its function in pigs and mice, employing genetic approaches, CSRP3 knockdown (CSRP3KD), Csrp3-/- and overexpression (CSRP3OE), to assess its impact on muscle regeneration. Additionally, we investigated the AKT-SERCA2 signaling axis and its downstream effects on calcium homeostasis, mitochondrial function, and myogenic differentiation. CSRP3 was essential for muscle regeneration and SC differentiation in pigs but not in mice. Genetic ablation of CSRP3 (CSRP3KD) impaired porcine SC differentiation and reduced regenerative capacity, while CSRP3 overexpression (CSRP3OE) enhanced these processes. Mechanistically, CSRP3 deficiency disrupted AKT-SERCA2 signaling, leading to elevated intracellular Ca2⁺, mitochondrial dysfunction, and MYOG protein degradation. Pharmacological AKT activation rescued differentiation defects in CSRP3-deficient cells. Notably, Csrp3-/- mice showed normal SC differentiation and AKT-SERCA2 activity, highlighting a species-specific regulatory role for CSRP3. Our findings identify CSRP3 as a critical regulator of porcine muscle regeneration, with minimal impact in mice. These results suggest that strategically engineered porcine SCs, through CSRP3 modulation, could improve the effectiveness of xenotransplantation-based therapies for muscle repair.
The development of functional human vasculature is essential for tissue engineering, disease modeling, and regenerative medicine. Conventional differentiation protocols of vascular lineages often exhibit lineage heterogeneity and limited control over cellular ratios. Here, we describe a protocol for generating vascular organoids (VOs) via orthogonal forward programming of hPSCs. By utilizing doxycycline-inducible activation of the transcription factors ETV2 and NKX3.1, hPSCs are rapidly directed toward endothelial and mural cell lineages, respectively. This strategy enables the assembly of VOs with precisely tunable cellular compositions within six days. When combined with fluorescent reporter lines (PECAM1-mRuby3 and ACTA2-EGFP), vascular networks can be visualized in real time without the need for tissue clearing or immunostaining. We detail procedures for stable cell line engineering, 3D organoid assembly, in vitro angiogenesis assays for drug screening, and in vivo transplantation under the mouse kidney capsule to form perfusable human vasculature. This platform provides a flexible, standardized, and scalable tool for investigating vascular biology, modeling inherited vasculopathies, and enhancing the vascularization of co-transplant tissues.
Breast cancer remains a leading cause of cancer-related morbidity and mortality worldwide, driven by therapeutic resistance, recurrence, and metastasis. Patient-derived organoids (PDOs) have emerged as a robust platform that preserves histopathological, genetic, and functional features of parental tumors, enabling translational research and precision oncology. However, the lack of standardized procedures across laboratories limits reproducibility and clinical applicability. Here, we present a consensus-oriented standard for the establishment, characterization, quality control and application of human breast cancer organoids derived from diverse clinical sources, including surgical tissues, biopsies, pleural effusion, ascites, and circulating tumor cells (CTCs). This document defines key terminologies, outlines standardized workflows, and introduces quantitative validation criteria for successful organoid establishment. In addition, it provides structured guidance for advanced models, including immune–tumor, neuro–tumor, and microbiota−tumor organoid systems, with defined functional validation endpoints. Standardized protocols for drug sensitivity testing are also proposed, including seeding density, exposure duration, and response metrics such as IC50 and AUC. Representative validation strategies, including histological, molecular and functional concordance with parental tumors, are incorporated to enhance reproducibility and translational relevance. This standard aims to harmonize methodologies, improve cross−study comparability, and accelerate the clinical implementation of breast cancer organoid-based precision medicine.
The olfactory epithelium (OE) maintains lifelong neurogenesis and shows strong regenerative capacity through the coordinated functions of horizontal basal cells (HBCs) and globose basal cells (GBCs). These progenitors are regulated by key transcriptional factors such as Sox2, p63, Pax6, Ascl1, Neurog1 and NeuroD1, as well as signaling pathways including Wnt/β-catenin, Notch, YAP and inflammation-related regulators, which together control lineage specification and injury-induced plasticity. A set of genes such as Lgr5, Tmem59, Notch1, and Chil4 play critical roles in OE homeostasis and regeneration, depending on a broader and highly dynamic network. Recent progress in single-cell transcriptomics, spatial transcriptomics and organoid models has revealed previously unrecognized cell states, differentiation routes and intercellular communications. This review summarizes the molecular and cellular mechanisms that support OE regeneration and highlights emerging technologies that advance understanding the process of olfactory epithelium regeneration and guiding future approaches for restoring olfactory function.
Cells can recover from sub-lethal necrosis by repairing plasma-membrane (PM) rupture through the ESCRT-III machinery, but how this process is regulated remains unknown. Here, we identify Toll-interacting protein (Tollip) as a conserved negative regulator for ESCRT-III–mediated PM repair. Quantitative proteomics revealed the enrichment of Tollip at damaged PM. Additionally, microscopy assays in mammalian cells and C. elegans confirmed the recruitment of Tollip to PM injury sites. Tollip deficiency augmented ESCRT-III assembly, improved long-term cell survival after sub-lethal PM damage, and enhanced PM repair, whereas Tollip overexpression suppressed these processes. Tollip translocation occurred independently of Ca2⁺ influx, different from ESCRT-III. Functionally, by limiting PM repair and maintaining sub-lethal PM integrity loss, Tollip ensured optimal chemokine and cytokine production from the plasma-membrane-integrity (PMI) pathway, which is directly triggered by PM ruptures. Thus, Tollip acts as a molecular rheostat that links membrane damage repair and cell recovery to immune signaling.
Human amniotic membrane (hAM) is a widely used biomaterial with longstanding utility in ophthalmology and emerging therapeutic promise across orthopaedics, obstetrics and gynaecology, and dermatology. Clinically available biological tissue repair materials primarily encompass autologous, allogeneic, and xenogeneic tissues. However, autologous materials are limited by availability, while allogeneic and xenogeneic tissues often present challenges related to immunocompatibility. As a commonly used allogeneic biomaterial, the hAM is regarded as a highly promising tissue repair material owing to its favourable immunological profile and exceptional tissue-regenerative properties. HAM is the innermost natural barrier of the placenta and possesses unique structural and biological characteristics that facilitate tissue repair and regeneration. This review summarizes recent advances and clinical applications of hAM in orthopaedics, obstetrics and gynaecology, and dermatology, specifically focusing on its roles in promoting tendon repair, alleviating osteoarthritis, repairing endometrial injury, treating diabetic foot ulcer, and enhancing burn wound healing. With the continued development of regenerative medicine, hAM is expected to play an increasingly important role in diverse tissue repair and regenerative medicine applications.
Skeletal muscle possesses a remarkable capacity for regeneration, driven by the activation and proliferation of Pax7-positive muscle stem cells within a dynamic niche that includes immune cells, fibro-adipogenic progenitors, endothelial cells, pericytes, and neural elements. Cellular senescence, a stress-induced program featuring stable cell-cycle arrest and the senescence-associated secretory phenotype (SASP), has emerged as a critical yet paradoxical regulator of this process. Accumulating evidence indicates that transient senescence, particularly in FAPs, macrophages, and other niche cells during acute muscle injury, plays a beneficial role in supporting muscle regeneration. These senescent cells promote cellular plasticity, enhance myoblast differentiation, facilitate phagocytic clearance of debris, and modulate inflammation and repair via timely SASP factor secretion. However, conflicting findings suggest that senescent cells exert detrimental effects, impairing regeneration by establishing a sustained pro-inflammatory and pro-fibrotic niche, especially when senescence persists in aged or dystrophic muscle. This review synthesizes the complex and contradictory roles of cellular senescence in skeletal muscle regeneration, underscores the distinction between transient pro-regenerative and persistent deleterious senescence, highlights the importance of cell-type-specific contributions, and emphasizes the need for precise characterization of senescent cell dynamics and fate. Resolving these discrepancies will be critical for developing targeted senotherapeutic strategies to enhance muscle regeneration in aging and degenerative diseases.
Maintenance of pluripotency in embryonic stem cells (ESCs) requires coordinated integration of transcriptional, metabolic, and epigenetic programs. Here, we identify a post-transcriptional regulatory axis linking the RNA-binding protein Musashi-1 (MSI1) to iron dependent DNA demethylation via the ferritin like gene Fthl17c. Genetic ablation of MSI1 and its short isoform MSI1-C in mESCs induced spontaneous differentiation and was accompanied by downregulation of Fthl17 family genes. Among these, Fthl17c was directly bound and stabilized by MSI1, and its depletion reduced intracellular ferrous iron (Fe2⁺), impaired ten eleven translocation (TET) enzyme activity, and increased global 5-methylcytosine (5mC) levels. Restoration of Fthl17c expression rescued TET activity, reduced DNA methylation, and reinstated pluripotency associated gene expression, whereas extracellular Fe2⁺ supplementation alone was insufficient. In contrast, vitamin C, which preserves redox active Fe2⁺, effectively restored DNA demethylation, highlighting the requirement for bioavailable iron in TET mediated epigenetic regulation. Biochemical and imaging analyses further revealed that FTHL17C interacts with TET1 in the nucleus, supporting a role in facilitating iron dependent catalysis. Together, these findings define an MSI1-FTHL17C-Fe2⁺-TET axis that integrates post transcriptional control of iron homeostasis with epigenetic remodeling to preserve the pluripotent and plastic state of embryonic stem cells.
Cytokines from the transforming growth factor-β (TGF-β) superfamily are essential regulators of cell growth, survival, and differentiation, playing a pivotal role in mammalian embryonic development, adult tissue homeostasis, and progression of human diseases. Recently, an international symposium on TGF-β Signaling in Development and Diseases was held in Nanchang, China, from October 21 to 23, 2025. This event showcased the latest advances in TGF-β signaling and its pathophysiological functions. Over ten presentations at the symposium offered new insights on Smad-dependent and non-Smad TGF-β signaling, its spatiotemporal regulation, and multifaceted roles of TGF-β family cytokines in various pathophysiological contexts. The symposium also addressed potential strategies and opportunities for targeting the TGF-β pathway in the treatment of human diseases.
To explore the therapeutic effects of human umbilical cord mesenchymal stem cells (HUMSCs) on repairing CCl₄-induced chronic liver injury in rats via intravenous injection and to identify the associated key metabolites. Cell experiments: THLE-2 cells were divided into blank control, CCl₄-treated, and CCl₄ + Exos groups. Cell viability was assessed using the CCK-8 assay, while levels of AST, ALT, and MDA were determined using commercial kits. Targeted metabolomics analysis was employed to identify differentially expressed metabolites. Transmission electron microscopy (TEM) was used to evaluate mitochondrial morphology, and immunofluorescence staining was performed to examine the colocalization of Exos with mitochondria. Animal experiments: 24 healthy SPF SD rats were randomly divided into healthy, CCl₄, and CCl₄ + HUMSCs groups (n = 8 per group). Serum samples were collected for biochemical detection and targeted metabolomics analyses, while liver tissues underwent histopathological examination. Immunofluorescence staining was employed to monitor HUMSCs enrichment. In the CCl₄ + Exos group, cell viability was significantly restored, and the elevated levels of AST, ALT, and MDA were reversed, while mitochondrial ultrastructure was ameliorated with successful Exos-mitochondria colocalization. Targeted metabolomics confirmed the presence of differentially expressed metabolites exhibiting consistent trends in both cellular and animal models. In the animal study, the CCl₄ group showed significant liver dysfunction and hepatic pathology characterized by hepatocyte steatosis and fibrous tissue hyperplasia. In contrast, the CCl₄ + HUMSCs group demonstrated markedly improved liver function and reduced pathological changes. Biochemical analysis revealed significant differences in ALT, AST, ALB, TBIL, TP, UREA, CR, and UA levels between the CCl₄ + HUMSCs and CCl₄ groups. Serum metabolomics analysis showed that compared with the CCl₄ group, 1,7-Dimethylxanthine and Xanthosine were significantly upregulated, while Succinic Acid, (S)-2-Hydroxybutanoic Acid, oxidized glutathione, and 3'-Sialyllactose were significantly downregulated in the CCl₄ + HUMSCs group. HUMSCs treatment significantly reduced hepatic steatosis and fibrosis compared with the CCl₄ group alone. Metabolomic analysis suggests that the underlying mechanisms may involve upregulation of propanoate metabolism and increased taurochenodeoxycholic acid levels, which warrant further investigation.
Inflammatory bowel disease (IBD) remains a significant clinical challenge with limited curative options. Adipose-derived mesenchymal stem cells (ADSCs) hold therapeutic promise, but their anti-inflammatory efficacy is often compromised by cellular senescence. This study investigates the role of lysine crotonylation (Kcr) in ADSCs senescence and explores its therapeutic potential. We analyzed Pan-Kcr levels in senescent ADSCs and evaluated the effects of sodium crotonate (NaCr), a crotonyl-CoA precursor, on senescence, proliferation, and anti-inflammatory function. A murine colitis model was used to assess therapeutic efficacy. Molecular mechanisms focusing on ACSS2-mediated Kcr regulation and H3K9 crotonylation (H3K9cr) at the ACSS2 promoter. Senescent ADSCs exhibited a marked decline in Pan-Kcr levels. NaCr treatment ameliorated senescence, enhanced proliferation, and improved anti-inflammatory capacity. ACSS2, a key regulator of Kcr, was downregulated in senescent ADSCs. Moreover, the anti-senescence effect of NaCr depended on ACSS2-mediated crotonylation. NaCr promoted H3K9cr modification at the ACSS2 promoter, forming a positive feedback loop that elevated Kcr levels. Mechanistically, ACSS2-mediated Kcr suppressed the NF-κB pathway to delay ADSCs senescence. Our findings reveal an epigenetic pathway (ACSS2-Kcr-H3K9cr) regulating ADSCs senescence and propose Kcr modulation as a novel strategy to enhance ADSC-based therapy for IBD.
Lgr5 marks both adult intestinal stem cells and embryonic intestinal stem/progenitor cells. However, the stemness properties and physiological roles of embryonic intestinal Lgr5⁺ cells prior to villification (PVLCs) remain largely unknown. In this study, we show that PVLCs in the embryonic small intestine exhibit region-specific stemness, with progressively enhanced stemness potential from the proximal to distal region. Through inducible cell ablation and gene knockout experiments, we demonstrate that PVLCs regulate small intestinal morphogenesis via Hedgehog signaling in a region-dependent manner, with distal morphogenesis being more dependent on this mechanism. This study reveals the stemness and functional roles of PVLCs in the embryonic small intestine prior to villification, highlighting regionalized cellular heterogeneity as a critical determinant of intestinal morphogenesis.
The Wnt signaling pathway critically regulates the osteogenic differentiation in periodontal ligament stem cells (PDLSCs). However, the functional contributions of this pathway under inflammatory conditions remain unclear. This study investigated the effect and underlying mechanisms of the FRZB–Wnt5a–mitochondrial axis on the osteogenic differentiation capacity of PDLSCs under inflammatory conditions. PDLSCs were isolated from healthy teeth and exposed to lipopolysaccharide (LPS) to mimic an inflammatory microenvironment. The Wnt pathway-related molecules were assessed, and the osteogenic differentiation capacity and mitochondrial function of PDLSCs were evaluated. To elucidate its regulatory role, we employed gene transfection to establish an FRZB (Frizzled-Related Protein) overexpression model. Results showed that inflammation significantly impaired osteogenic differentiation and activated Wnt/β-catenin signaling. Mitochondrial dysfunction was also observed, including reduced membrane potential, increased calcium and reactive oxygen species (ROS) levels, suppressed autophagic flux, and altered mitochondrial morphology. Notably, FRZB overexpression partially restored mitochondrial function and the osteogenic differentiation capacity of PDLSCs. These results demonstrated that FRZB serves as a pivotal regulator of osteogenic differentiation in PDLSCs. We found that inflammation downregulates FRZB expression, thereby activating Wnt/β-catenin signaling, which leads to mitochondrial dysfunction and ultimately impairs osteogenesis. These findings reveal a mechanism by which inflammation suppresses osteogenesis in PDLSCs and highlight FRZB as a promising therapeutic target for periodontitis.
Skeletal muscle aging is characterized by a functional decline in muscle stem cells (MuSCs), yet the key regulatory mechanisms driving this deterioration remain poorly understood. By integrating transcriptomic profiles from aged MuSCs with data from C2C12 cells exposed to spaceflight conditions (which mimic an aging-like phenotype), we identified MORF4-related gene on chromosome 15 (MRG15) as a putative epigenetic regulator involved in age-related myogenic decline. Using a MuSC-specific inducible knockout (iKO) mouse model, we found that loss of MRG15 severely compromises myogenic differentiation and muscle regeneration. Subsequent RNA sequencing of iKO MuSCs, combined with ChIP-seq analysis of histone modifications, revealed that MRG15 modulates the chromatin landscape of myogenic genes through interaction with MyoD, thereby facilitating transcriptional activation and differentiation. Our findings establish MRG15 as a critical epigenetic regulator that cooperates with MyoD to orchestrate chromatin remodeling, thereby promoting transcriptional activation of the myogenic program. Dysregulation of MRG15 may underlie impaired muscle regeneration during aging.
The precise ablation of specific cell lineages is crucial for functional studies in vivo. Conventional methods, like the Cre-dependent iDTR system, are constrained by the off-target effects and variable efficiency of single-recombinase approaches. Here, we present a novel Cdh5-RL-DTRGFP mouse model that requires both Dre and Cre recombinases to activate diphtheria toxin receptor (DTR) and GFP expression specifically in endothelial cells. This dual-recombinase logic ensures tight control over transgene expression. We demonstrate that diphtheria toxin administration in recombined mice leads to efficient endothelial cell ablation, resulting in severe vascular leakage, rapid organ failure, and mortality. The Cdh5-RL-DTRGFP line thus provides a robust and precise platform for the genetic dissection of endothelial cell function in physiological and pathological contexts.
Currently, effective treatments for skeletal muscle injury remain limited. The self-repair of skeletal muscle relies on the activation and differentiation of satellite cells (SCs), which fuse with damaged myofibers to form new fibers and thereby support muscle regeneration. However, in cases of severe injury, it is difficult for muscle tissue to fully restore its original structure and function, and its regenerative capacity is often markedly reduced. Thus, there is an urgent need to develop therapies that enhance muscle repair and restore physiological function. In this study, we investigated extracellular vesicles derived from neonatal mouse skeletal muscle (NMM-EVs), which are enriched in cargo from Pax7⁺ myogenic progenitor cells. We hypothesized that NMM-EVs could enhance SC activation and improve muscle regeneration following injury. Using glycerol-induced tibialis anterior (TA) muscle injury model, we evaluated the effects of intramuscular NMM-EV administration on skeletal muscle regeneration by histological, immunofluorescence, and functional analyses. In vivo, NMM-EVs significantly promoted skeletal muscle regeneration and functional recovery, upregulated Pax7 expression, increased the cross-sectional area and muscle mass of regenerated TA, and reduced fibrosis and fat infiltration. In vitro, NMM-EVs enhanced the proliferation and myogenic differentiation of mouse SCs and increased the expression of myogenic regulatory factors at both the mRNA and protein levels. In conclusion, this study demonstrates that NMM-EVs activate SCs within injured muscle, promote their proliferation and differentiation, and thereby accelerate injury repair and myofiber regeneration while attenuating fibrotic and adipogenic remodeling. These findings provide a scientific basis for the development of neonatal muscle–derived extracellular vesicle–based, cell-free therapeutic strategies for skeletal muscle injury.
Cardiac fibrosis following myocardial infarction (MI) is a critical determinant of progressive cardiac dysfunction, yet the underlying mechanisms driving this pathological process remain incompletely understood. Elucidating these regulatory pathways holds profound implications for improving post-MI prognosis. Our prior work demonstrated that chronic intermittent hypoxia (CIH) exacerbates cardiac fibrosis while modulating the expression of long non-coding RNA (lncRNA) nonnmmut065573 (tentatively designated LncRNA-IH) in cardiac tissues. Herein, we sought to determine the role of LncRNA-IH in post-MI cardiac fibrosis and its underlying mechanisms. Using a C57BL/6 mouse model of MI, we established a mouse model with cardiac-specific overexpression of LncRNA-IH to evaluate post-MI cardiac fibrosis. In vitro, primary cardiac fibroblasts (MCF) and the PA12 cell line were subjected to LncRNA-IH overexpression or siRNA-mediated knockdown, and cell proliferation and migration were assessed. Transcriptomic profiling was performed to characterize LncRNA-IH-induced changes in cardiac gene expression and signaling pathways, aiming to elucidate the molecular mechanisms involved. Results showed that CIH significantly exacerbated post-MI cardiac fibrosis, and LncRNA-IH was predominantly localized to cardiac fibroblasts. Cardiac-specific overexpression of LncRNA-IH in MI mice markedly exacerbated post-MI cardiac dysfunction and fibrosis. In vitro, LncRNA-IH overexpression significantly enhanced the proliferation and migration capacities of primary cardiac fibroblasts and PA12 cells, whereas these effects were abrogated by LncRNA-IH knockdown. Transcriptomic analysis revealed that LncRNA-IH elicited significant alterations in cardiac gene expression profiles, specifically activating the TGF-β1 signaling pathway and upregulating the expression of its downstream target, ZEB1. Collectively, our findings indicate that LncRNA-IH promotes cardiac fibroblast proliferation and migration, thereby exacerbating post-MI cardiac remodeling, at least in part through activation of the TGF-β1 signaling pathway. This study identifies LncRNA-IH as a potential therapeutic target for mitigating post-MI cardiac fibrosis and preserving cardiac function.
