参考文献

Macrophages are the major source of WNT ligands. However,the regulation of WNT expression in macrophages has not been studied. In the present study,we have discovered that activation of canonical β-Catenin signaling suppresses WNT expression in macrophages. EVs from these pre-conditioned macrophages promoted intestinal stem cell regeneration and mitigated intestinal injury. ChIP-seq analysis and validation studies using recombinant DNA construct expressing Luciferase reporter under WNT promoter (e.g. WNT5a and WNT9b) were conducted to demonstrate the involvement of β-Catenin in the transcriptional regulation of WNT expression. The regulatory role of β-Catenin in WNT expression in macrophages was examined by treating these cells with a Tankyrase inhibitor. In addition,the gene expressing β-Catenin was deleted in macrophages using Csf1r.iCre; Ctnnb1 fl/fl mice model. Both pharmacological and genetically modulated macrophages were examined for WNT expression and activity by qPCR and TCF/LEF luciferase assay respectively. Additionally,Csf1r.iCre; Ctnnb1 fl/fl mice were exposed to irradiation to compare the radiosensitivity with their wildtype littermate. Extracellular vesicles (EVs) were isolated from pre-conditioned WNT-enriched macrophages and infused in irradiated C57BL/6 and Lgr5/eGFP-IRES-Cre-ERT2 ; R26-ACTB-tdTomato-EGFP mice to determine the regenerative response of intestinal stem cell (ISC) and epithelial repair. Regenerative effects of EVs were also examined in mice model DSS induced colitis. ChIP-seq analysis and subsequent validation study suggested physical association of β-Catenin with WNT promoters to suppress WNT expression. Macrophage specific deletion of gene expressing β-Catenin or pharmacological inhibition of Tankyrase improves the WNT expression in macrophages several folds compared to control. Transfusion of these preconditioned macrophages or EVs from these cells delivers optimum level of morphogenic WNT to injured epithelium,activates ISC regeneration and mitigated radiation induced intestinal injury. Intestinal epithelium in Csf1r.iCre; Ctnnb1 fl/fl mice also showed radioresistance compared to wild type littermate. Moreover,EVs derived from WNT enriched macrophages can mitigate intestinal injury in mice model of DSS induced acute colitis. The study provides substantial evidence that macrophage-targeted modulation of canonical WNT signaling induces WNT expression in macrophages. Treatment with preconditioned macrophage derived WNT-enriched EVs can be a promising therapeutic approach against intestinal injury. The online version contains supplementary material available at 10.1186/s12964-025-02065-7. View Publication

Lactate dehydrogenase A (LDHA) can regulate tumorigenesis and cancer progression. Nevertheless,whether the regulation of LDHA is involved in the development of gemcitabine resistance in PDAC has not yet been fully elucidated. Increasing studies have shown that cancer acquired drug resistance led to treatment failure is highly attributed to the cancer stem cell (CSC) properties. Therefore,we aim to demonstrate the functions and regulatory mechanisms of LDHA on cancer stem cell (CSC) properties and gemcitabine resistance in PDAC. We investigate the metabolite profiles by liquid chromatography-mass spectrometry between gemcitabine–resistant PDAC and parental PDAC cells. Additionally,gain-of-function and loss-of-function experiments were conducted to examine the roles of LDHA on CSC properties and gemcitabine resistance in the gemcitabine–resistant PDAC and parental PDAC cells. To investigate regulators involved in LDHA-mediated gemcitabine resistance and CSC of pancreatic cancer cells,we further used a combination of the miRNA microarray results and software predictions and confirmed that miR-4259 is a direct target of LDHA by luciferase assay. Furthermore,we constructed serial miR-4259 promoter reporters and searched for response elements using the TESS 2.0/TFSEARCH software to find the transcription factor binding site in the promoter region of miR-4259. We observed that elevated LDHA expression significantly correlates with recurrent pancreatic cancer patients following gemcitabine treatment and with CSC properties. We further identify that FOXO3a-induced miR-4259 directly targets the 3’untranslated region of LDHA and reduced LDHA expression,leading to decreased gemcitabine resistance and a reduction in the CSC phenotypes of pancreatic cancer. Our results demonstrated that LDHA plays a critical role in cancer stemness and gemcitabine resistance of pancreatic cancer,and indicate that targeting the FOXO3a/miR-4259/LDHA pathway might serve as a new treatment for pancreatic cancer patients with a poor response to gemcitabine chemotherapy. The online version contains supplementary material available at 10.1186/s40170-025-00377-3. View Publication

Cerebral organoids (COs) are valuable tools for studying the intricate interplay between glial cells and neurons in brain development and disease,including HIV-associated neuroinflammation. We developed a novel approach to generate microglia containing COs (CO-iMs) by co-culturing hematopoietic progenitors and inducing pluripotent stem cells. This approach allowed for the differentiation of microglia within the organoids concomitantly with the neuronal progenitors. Compared with conventional COs,CO-iMs were more efficient at generating CD45 + /CD11b + /Iba-1 + microglia and presented a physiologically relevant proportion of microglia (~ 7%). CO-iMs presented substantially increased expression of microglial homeostatic and sensome markers as well as markers for the complement cascade. CO-iMs are susceptible to HIV infection,resulting in a significant increase in several pro-inflammatory cytokines/chemokines,which are abrogated by the addition of antiretrovirals. Thus,CO-iM is a robust model for deciphering neuropathogenesis,neuroinflammation,and viral infections of brain cells in a 3D culture system. The online version contains supplementary material available at 10.1186/s12974-025-03353-2. View Publication

Colorectal cancer (CRC) is a prevalent malignancy with significant morbidity and mortality worldwide. A deeper understanding of the interaction of cancer cells with other cells in the tumor microenvironment is crucial to devise effective therapeutic strategies. MUC2,a major component of the protective mucus layer in the gastrointestinal tract,has been implicated in CRC progression and immune response regulation. In this study,we sought to elucidate the relationship between MUC2 expression and immune infiltration within CRC using in vitro models involving two well-established cell lines,HT-29 and LS-174T. By employing CRISPR-mediated MUC2 knockout,we investigated the influence of MUC2 on tumor immune infiltration and its interplay with T cells and NK cells enriched peripheral blood mononuclear cells (PBMCs) in 3D spheroid cultures. While MUC2 was more abundant in LS-174T cell line compared to HT-29,its knockout resulted in increased immune infiltration solely in the HT-29 cell line,but not in the LS-174T cell line. We revealed that the removal of MUC2 protein was compensated in LS-174T by the expression of other gel-forming mucin proteins (MUC6,MUC5B) commonly expressed in the gastrointestinal epithelium,while this was not observed in HT-29 cell line. Our study is the first to demonstrate that MUC2 functions as a physical barrier to immune infiltration in colorectal cancer (CRC) in vitro . In HT-29 cells,MUC2 knockout increased immune infiltration,while in LS-174T cells,compensatory expression of other mucins (MUC6,MUC5B) maintained the barrier. These findings reveal the complexity of mucin biology in CRC and suggest that targeting mucin pathways could be a novel therapeutic approach. View Publication

The manufacturing of allogeneic cell therapeutics based on human-induced pluripotent stem cells (hiPSCs) holds considerable potential to revolutionize the accessibility and affordability of modern healthcare. However,achieving the cell yields necessary to ensure robust production hinges on identifying suitable and scalable single-use (SU) bioreactor systems. While specific stirred SU bioreactor types have demonstrated proficiency in supporting hiPSC expansion at L -scale,others,notably instrumented SU multiplate and fixed-bed bioreactors,remain relatively unexplored. By characterizing these bioreactors using both computational fluid dynamics and experimental bioengineering methods,operating ranges were identified for the Xpansion ® 10 and Ascent™ 1 m 2 bioreactors in which satisfactory hiPSC expansion under serum-free conditions was achieved. These operating ranges were shown not only to effectively limit cell exposure to wall shear stress but also facilitated sufficient oxygen transfer and mixing. Through their application,almost 5 × 10 9 viable cells could be produced within 5 days,achieving expansion factors of up to 35 without discernable impact on cell viability,identity,or differentiation potential. Key Points • Bioengineering characterizations allowed the identification of operating ranges that supported satisfactory hiPSC expansion • Both the Xpansion ® 10 multiplate and Ascent™ 1 m 2 fixed-bed reactor accommodated the production of almost 5 × 10 9 viable cells within 5 days • Exposing the hiPSCs to a median wall shear stress of up to 8.2 × 10 −5 N cm −2 did not impair quality The online version contains supplementary material available at 10.1007/s00253-024-13373-2. View Publication

To fully utilize the potential of human induced pluripotent stem cells (hiPSCs) for allogeneic stem cell–based therapies,efficient and scalable expansion procedures must be developed. For other adherent human cell types,the combination of microcarriers (MCs) and stirred tank bioreactors has been shown to meet these demands. In this study,a hiPSC quasi-perfusion expansion procedure based on MCs was developed at 100-mL scale in spinner flasks. Process development began by assessing various medium exchange strategies and MC coatings,indicating that the hiPSCs tolerated the gradual exchange of medium well when cultivated on Synthemax II–coated MCs. This procedure was therefore scaled-up to the 1.3-L Eppendorf BioBLU 1c stirred tank bioreactor by applying the lower limit of Zwietering’s suspension criterion ( N s 1 u ),thereby demonstrating proof-of-concept when used in combination with hiPSCs for the first time. To better understand the bioreactor and its bioengineering characteristics,computational fluid dynamics and bioengineering investigations were performed prior to hiPSC cultivation. In this manner,improved process understanding allowed an expansion factor of ≈ 26 to be achieved,yielding more than 3 × 10 9 cells within 5 days. Further quality analyses confirmed that the hiPSCs maintained their viability,identity,and differentiation potential throughout cultivation. • N s 1 u can be used as a scale-up criterion for hiPSC cultivations in MC-operated stirred bioreactors • Uniform distribution and attachment of cells to the MCs are crucial for efficient expansion • Perfusion is advantageous and supports the cultivation of hiPSCs The online version contains supplementary material available at 10.1007/s00253-024-13372-3. View Publication

Light chain (AL) amyloidosis is a serious systemic disease caused by the deposition of free misfolded immunoglobulin light chains (LCs) in the form of amyloid fibrils within tissues. Cardiac involvement determines prognosis and mortality. An important cytotoxic impact of amyloidogenic prefibrillar LC oligomers on cardiomyocytes is by now established in isolated rodent cardiomyocytes,simple animal models,or cardiomyocyte-like cell lines. However,the response of human cardiomyocytes to this pathogenic condition is currently unknown. In this work,we have set up a human cellular disease model of AL cardiac amyloidosis (AL-CA) in the form of cardiac spheroids,to study the cytotoxic effects of amyloidogenic LCs with regard to contractile function and calcium handling. To mimic the disease in a reconstituted system,soluble amyloidogenic LCs purified from urine of AL-CA patients were added to a mixture of induced pluripotent stem cell-issued human cardiomyocytes (hiPSC-CM) and human primary cardiac fibroblasts,which resulted in formation of spheroids within 7 days. This procedure ensured a uniform pericellular LC distribution within spheroids. LC-treated hiPSC-CM cultures and LC-containing spheroids presented structural and functional defects including: (1) decreased levels and subcellular disorganization of sarcomeric protein alpha-actinin; (2) abnormal accumulation of calcium handling SERCA2a protein; (3) impaired contractility of spheroids and altered calcium transients. Three independent patient-derived LCs had similar effects,albeit to varying degrees,highlighting the patient-specific properties of this type of amyloids. Taken together,these results indicate that the present cardiac spheroid disease model could be appropriate to the study of cardiac cytotoxicity caused by different amyloidogenic LCs in AL-CA patients,contributing to a better understanding and therapeutic handling of the disease. The online version contains supplementary material available at 10.1038/s41598-024-82442-3. View Publication

Functional cellular heterogeneity in tumours often underlies incomplete response to therapy and relapse. Previously,we demonstrated that the growth of the paediatric brain malignancy,sonic hedgehog subgroup medulloblastoma,is rooted in a dysregulated developmental hierarchy,the apex of which is defined by characteristically quiescent SOX2 + stem-like cells. Integrating gene expression and chromatin accessibility patterns in distinct cellular compartments,we identify the transcription factor Olig2 as regulating the stem cell fate transition from quiescence to activation,driving the generation of downstream neoplastic progenitors. Inactivation of Olig2 blocks stem cell activation and tumour output. Targeting this rare OLIG2-driven proliferative programme with a small molecule inhibitor,CT-179,dramatically attenuates early tumour formation and tumour regrowth post-therapy,and significantly increases median survival in vivo. We demonstrate that targeting transition from quiescence to proliferation at the level of the tumorigenic cell could be a pivotal medulloblastoma treatment strategy. Subject terms: Cancer stem cells,Mechanisms of disease,Cancer therapy View Publication

Chimeric antigen receptor (CAR)-engineered T cell therapy holds promise for treating myeloid malignancies,but challenges remain in bone marrow (BM) infiltration and targeting BM-resident malignant cells. Current autologous CAR-T therapies also face manufacturing and patient selection issues,underscoring the need for off-the-shelf products. In this study,we characterize primary patient samples and identify a unique therapeutic opportunity for CAR-engineered invariant natural killer T (CAR-NKT) cells. Using stem cell gene engineering and a clinically guided culture method,we generate allogeneic CD33-directed CAR-NKT cells with high yield,purity,and robustness. In preclinical mouse models,CAR-NKT cells exhibit strong BM homing and effectively target BM-resident malignant blast cells,including CD33-low/negative leukemia stem and progenitor cells. Furthermore,CAR-NKT cells synergize with hypomethylating agents,enhancing tumor-killing efficacy. These cells also show minimal off-tumor toxicity,reduced graft-versus-host disease and cytokine release syndrome risks,and resistance to allorejection,highlighting their substantial therapeutic potential for treating myeloid malignancies. Subject terms: Cancer therapy,Immunotherapy,Leukaemia View Publication

Dishevelled-associated activator of morphogenesis1 (DAAM1) is a member of the evolutionarily conserved Formin family and plays a significant role in the malignant progression of various human cancers. This study aims to explore the clinical and biological significance of DAAM1 in pancreatic cancer. Multiple public datasets and an in-house cohort were utilized to assess the clinical relevance of DAAM1 in pancreatic cancer. The LinkedOmics platform was employed to perform enrichment analysis of DAAM1-associated molecular pathways in pancreatic cancer. Subsequently,a series of in vitro and in vivo experiments were conducted to evaluate the biological roles of DAAM1 in pancreatic cancer cells and its effects on intratumoral T cells. DAAM1 was found to be upregulated in pancreatic cancer tissues,with higher expression levels observed in tumor cells. Additionally,high expression of DAAM1 was associated with poor prognosis. DAAM1 acted as an oncogene in pancreatic cancer,and its inhibition suppressed tumor cell proliferation,migration,and invasion,while promoted apoptosis. Furthermore,DAAM1 was involved in the JAK1/STAT1 signaling pathway and regulated PD-L1 expression in pancreatic cancer cells. The inhibition of DAAM1 also significantly reduced the exhaustion levels of CD8+ T cells. In conclusion,DAAM1 functions as an oncogene and is immunologically implicated in pancreatic cancer,these findings suggest that DAAM1 may serve as a promising therapeutic target for the clinical management of pancreatic cancer. The online version contains supplementary material available at 10.1186/s12935-024-03631-8. View Publication

CDK4/6 inhibitors have significantly improved the survival of patients with HR-positive/HER2-negative breast cancer,becoming a first-line treatment option. However,the development of resistance to these inhibitors is inevitable. To address this challenge,novel strategies are required to overcome resistance,necessitating a deeper understanding of its mechanisms. Recent research has identified several dysregulated genes in CDK4/6 inhibitors-resistant breast cancer,but the underlying mechanism is complex due to tumor heterogeneity and warrants further investigation. RNA sequencing and KEGG pathway analysis was carried out to identify the mainly dysregulated genes in CDK4/6 inhibitors-resistant breast cancer cells. The effects of CXCR4 knockdown and overexpression via siRNAs and plasmids transfection were examined by mammosphere formation,RT-qPCR,flow cytometry,MTT and colony formation assays. The regulation mechanisms were analyzed by RT-qPCR,western blotting and immunofluorescence experiments. Mouse xenografts were used to analyze the role of CXCR4 in regulation palbociclib sensitivity in vivo. Additionally,we collected the clinical samples and performed immunohistochemistry to analyze the clinical significance of CXCR4. In our study,we focused on cancer stem cells,a critical contributor to cancer metastasis and therapy resistance,and detected an upregulation of stemness in our established palbociclib-resistant ER-positive breast cancer cells. Additionally,our research pinpointed CXCR4 as a pivotal gene responsible for maintaining cancer stemness and promoting palbociclib resistance. Mechanistically,CXCR4 activates the WNT5A/β-catenin signaling pathway by enhancing the expression of WNT5A and β-catenin,facilitating the nuclear translocation of β-catenin protein. Targeting CXCR4 using siRNAs or small molecular inhibitors effectively reduces cancer stemness and reverses palbociclib resistance both in vitro and in vivo. Clinical sample analysis further underscores the overactivation of the CXCR4/WNT5A/β-catenin axis in palbociclib-resistant breast cancer,suggesting CXCR4 as a potential biomarker for predicting resistance to CDK4/6 inhibitors. Collectively,our study demonstrates that CXCR4 overexpression plays a vital role in maintaining breast cancer stemness and promoting resistance to CDK4/6 inhibitors through the activation of the WNT5A/β-catenin pathway. Targeting CXCR4 may offer a promising therapeutic approach for advanced CDK4/6 inhibitor-resistant ER-positive breast cancer. The online version contains supplementary material available at 10.1186/s13058-025-01965-3. View Publication

Hematopoietic stem and progenitor cells (HSPCs) are critical for the treatment of blood diseases in clinic. However,the limited source of HSPCs severely hinders their clinical application. In the embryo,hematopoietic stem cells (HSCs) arise from hemogenic endothelial (HE) cells lining the major arteries in vivo. In this work,by engineering vascular niche endothelial cells (VN-ECs),we generated functional HSPCs in vitro from ECs at various sites,including the aorta-gonad-mesonephros (AGM) region and the placenta. Firstly,we converted mouse embryonic HE cells from the AGM region (aHE) into induced HSPCs (iHSPCs),which have the abilities for multilineage differentiation and self-renewal. Mechanistically,we found that VN-ECs can promote the generation of iHSPCs via secretion of CX3CL1 and IL1A. Next,through VN-EC co-culture,we showed that placental HE (pHE) cells,a type of extra-embryonic HE cells,were successfully converted into iHSPCs (pHE-iHSPCs),which have multilineage differentiation capacity,but exhibit limited self-renewal ability. Furthermore,comparative transcriptome analysis of aHE-iHSPCs and pHE-iHSPCs showed that aHE-iHSPCs highly expressed HSC-specific and self-renewal-related genes. Moreover,experimental validation showed that retinoic acid (RA) treatment promoted the transformation of pHE cells into iHSPCs that have self-renewal ability. Collectively,our results suggested that pHE cells possess the potential to transform into self-renewing iHSPCs through RA treatment,which will facilitate the clinical application of placental endothelial cells in hematopoietic cell generation. Subject terms: Haematopoietic stem cells,Haematopoietic stem cells View Publication