技术资料

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. We previously discovered that heme oxygenase 1 (HO1) is crucial for chemoresistance in AML,but the detailed molecular mechanism of that remains unclear. RNA sequencing was conducted to assess transcriptomic changes in three pairs of AML cells after regulating the expression of HO1. The molecular mechanism by which HO1 induces gilteritinib resistance in FLT3-ITD (FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD)) AML was evaluated by quantitative real-time PCR (qRT-PCR),CCK-8,flow cytometry,and western blotting. FLT3-ITD AML mouse models were established to investigate the effects of HO1 expression on gilteritinib resistance in vivo. In these three pairs of AML cells,we discovered that HO1-mediated drug resistance is connected to the interleukin-4-mediated signaling pathway (specifically STAT6) only in MV4-11 cells with the FLT3-ITD mutation. Further findings revealed that HO1 overexpression confers gilteritinib resistance in FLT3-ITD AML cell lines and primary individual specimens. While suppression of HO1 sensitized FLT3-ITD AML cell lines and primary individual specimens to gilteritinib. Mechanistically,western blotting and flow cytometry confirmed that HO1-mediated gilteritinib resistance is related to STAT6 phosphorylation in FLT3-ITD AML cell lines and primary individual specimens. Moreover,tumor-bearing mice were employed to determine that HO1 overexpression conferred gilteritinib resistance in vivo. Collectively,these studies illustrate that HO1 may act as a successful treatment target for gilteritinib-resistant FLT3-ITD AML patients. The online version contains supplementary material available at 10.1186/s12935-025-03757-3. View Publication

Trogocytosis is the process by which a recipient cell siphons small membrane fragments and proteins from a donor cell and can be utilized by cancer cells to avoid immune detection. We observed lymphocyte specific protein expressed by triple negative breast cancer (TNBC) cells via immunofluorescence imaging of patient samples. Image analysis of Cluster of Differentiation 45RA (CD45RA) expression,a naïve T cell specific protein,revealed that all stages of TNBCs express CD45RA. Flow cytometry revealed TNBC cells trogocytose CD45 protein from T cells. We also showed that the acquisition of these lymphoid markers is contact dependent. Confocal and super-resolution imaging further revealed CD45+ spherical structures containing T cell genomic DNA inside TNBC cells after co-culture. Trogocytosis between T cells and TNBC cells altered tumor cell expression of PTPRC,the gene that encodes for CD45. Our results revealed that CD45 is obtained by TNBC cells from T cells via trogocytosis and that TNBC cells express CD45 intracellularly and on the membrane. View Publication

Suppression of anticancer immune function is a key driver of tumorigenesis. Identifying molecular pathways that inhibit anticancer immunity is critical for developing novel immunotherapeutics. One such molecule that has recently been identified is the carbohydrate polysialic acid (polySia),whose expression is dramatically upregulated on both cancer cells and immune cells in breast cancer patient tissues. The role of polySia in the anticancer immune response,however,remains incompletely understood. In this study,we profile polySia expression on both healthy primary immune cells and on infiltrating immune cells in the tumour microenvironment (TME). These studies reveal polySia expression on multiple immune cell subsets in patient breast tumors. We find that stimulation of primary T-cells and macrophages in vitro induces a significant upregulation of polySia expression. We subsequently show that polySia is appended to a range of different carrier proteins within these immune cells. Finally,we find that selective removal of polySia can significantly potentiate killing of breast cancer cells by innate immune cells. These studies implicate polySia as a significant negative regulator of anticancer immunity. View Publication

To produce pluripotent stem cells from peripheral blood mononuclear cells (PBMCs) of a patient with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and culture and differentiate them into vascular organoids,producing a disease model for cerebral small vessel disease (CSVD). (1) PMBCs from patients clinically diagnosed with CADASIL ( NOTCH3 p.R141C) were induced to differentiate into pluripotent stem cells (iPSCs); the quality and differentiation ability of the iPSCs were determined. (2) CADASIL-derived iPSCs and control iPSCs were cultured and differentiated into vascular organoids. The differences in the morphological structure of the two differentiated groups of vascular organoids were observed,and both were identified. (1) No mycoplasma infections were detected in the iPSCs prepared from the PBMCs of patients with CADASIL. The short tandem repeat (STR) identification verified that the iPSCs originated from the patient,and the karyotype was normal. Flow cytometry and immunofluorescence detection revealed that the iPSCs expressed SSEA4,OCT4,and NANOG stem proteins. Tri-germ differentiation testing confirmed that the iPSCs expressed the endoderm markers SOX17 and FOXA2,the mesoderm markers Brachyury and α-SMA,and the ectoderm markers Pax6 and β-III Tubulin. (2) CADASIL-derived iPSCs and control iPSCs were induced to differentiate and produce endothelial networks and vascular networks,ultimately forming vascular organoids. Compared with control vascular organoids,CADASIL vascular organoids exhibited lower growth density,earlier blood vessel sprouting,longer and thinner vascular filaments,and smaller final vascular organoids. The vascular organoids from the two sources expressed the endothelial cell marker CD31,the vascular smooth muscle marker α-SMA,and the pericyte marker PDGFR-β. Reprogramming technology can be used to induce PBMCs to become iPSCs,and a CSVD disease model can be successfully constructed by culturing and differentiating the iPSCs into CADASIL vascular organoids. The NOTCH3 p.R141C mutation suppresses the vascular differentiation process in CADASIL. View Publication

The search for an effective cell replacement therapy for diabetes has driven the development of “perfect” pancreatic islets from human pluripotent stem cells (hPSCs). These hPSC-derived pancreatic islet-like β cells can overcome the limitations for disease modelling,drug development and transplantation therapies in diabetes. Nevertheless,challenges remain in generating fully functional and mature β cells from hPSCs. This review underscores the significant efforts made by researchers to optimize various differentiation protocols aimed at enhancing the efficiency and quality of hPSC-derived pancreatic islets and proposes methods for their improvement. By emulating the natural developmental processes of pancreatic embryogenesis,specific growth factors,signaling molecules and culture conditions are employed to guide hPSCs towards the formation of mature β cells capable of secreting insulin in response to glucose. However,the efficiency of these protocols varies greatly among different human embryonic stem cell (hESC) and induced pluripotent stem cell (hiPSC) lines. This variability poses a particular challenge for generating patient-specific β cells. Despite recent advancements,the ultimate goal remains to develop a highly efficient directed differentiation protocol that is applicable across all genetic backgrounds of hPSCs. Although progress has been made,further research is required to optimize the protocols and characterization methods that could ensure the safety and efficacy of hPSC-derived pancreatic islets before they can be utilized in clinical settings. View Publication

Allogeneic transplantation of CCR5 null hematopoietic stem and progenitor cells (HSPCs) is the only known cure for HIV-1 infection. However,this treatment is limited because of the rarity of CCR5 -null matched donors,the morbidities associated with allogeneic transplantation,and the prevalence of HIV-1 strains resistant to CCR5 knockout (KO) alone. Here,we propose a one-time therapy through autologous transplantation of HSPCs genetically engineered ex vivo to produce both CCR5 KO cells and long-term secretion of potent HIV-1 inhibiting antibodies from B cell progeny. CRISPR-Cas9-engineered HSPCs engraft and reconstitute multiple hematopoietic lineages in vivo and can be engineered to express multiple antibodies simultaneously (in pre-clinical models). Human B cells engineered to express each antibody secrete neutralizing concentrations capable of inhibiting HIV-1 pseudovirus infection in vitro. This work lays the foundation for a potential one-time functional cure for HIV-1 through combining the long-term delivery of therapeutic antibodies against HIV-1 and the known efficacy of CCR5 KO HSPC transplantation. Subject terms: Stem-cell biotechnology,Haematopoietic stem cells,CRISPR-Cas9 genome editing View Publication

Motor axon regeneration following peripheral nerve injury is critical for motor recovery but therapeutic interventions enhancing this are not available. We conduct a phenotypic screen on human motor neurons and identified blebbistatin,a non-muscle myosin II inhibitor,as the most effective neurite outgrowth promotor. Despite its efficacy in vitro,its poor bioavailability limits in vivo application. We,therefore,utilize a blebbistatin analog,NMIIi2,to explore its therapeutic potential for promoting axon regeneration. Local NMIIi2 application directly to injured axons enhances regeneration in human motor neurons. Furthermore,following a sciatic nerve crush injury in male mice,local NMIIi2 administration to the axonal injury site facilitates motor neuron regeneration,muscle reinnervation,and functional recovery. NMIIi2 also promotes axon regeneration in sensory,cortical,and retinal ganglion neurons. These findings highlight the therapeutic potential of topical NMII inhibition for treating axon damage. Subject terms: Regeneration and repair in the nervous system,Movement disorders View Publication

Clinical translation of tissue-engineered advanced therapeutic medicinal products is hindered by a lack of patient-dependent and independent in-process biological quality controls that are reflective of in vivo outcomes. Recent insights into the mechanism of native bone repair highlight a robust path dependence. Organoid-based bottom-up developmental engineering mimics this path-dependence to design personalized living implants scaffold-free,with in-build outcome predictability. Yet,adequate (noninvasive) quality metrics of engineered tissues are lacking. Moreover,insufficient insight into the role of donor variability and biological sex as influencing factors for the mechanism toward bone repair hinders the implementation of such protocols for personalized bone implants. Here,male and female bone-forming organoids were compared to non-bone-forming organoids regarding their extracellular matrix composition,transcriptome,and secreted proteome signatures to directly link in vivo outcomes to quality metrics. As a result,donor variability in bone-forming callus organoids pointed towards two distinct pathways to bone,through either a hypertrophic cartilage or a fibrocartilaginous template. The followed pathway was determined early,as a biological sex-dependent activation of distinct progenitor populations. Independent of donor or biological sex,a cartilage-to-bone transition was driven by a common panel of secreted factors that played a role in extracellular matrix remodeling,mineralization,and attraction of vasculature. Hence,the secreted proteome is a source of noninvasive biomarkers that report on biological potency and could be the missing link toward data-driven decision-making in organoid-based bone tissue engineering. Subject terms: Bone,Bone quality and biomechanics View Publication

Cervical cancer (CC) remains a major health challenge with high mortality rates due to chemoresistance and immune escape. However,the underlying mechanisms remain unclear. We investigated the role of TAB2 in CC using cisplatin‐resistant and parental cell lines. Cell proliferation,migration,sphere formation and T cell‐mediated killing assays were performed. Western blot and qRT‐PCR analysed protein and mRNA expression. NF‐κB pathway involvement was examined using the BAY 11–7082 inhibitor. TAB2 expression was significantly elevated in cisplatin‐resistant CC cells. TAB2 overexpression promoted chemoresistance and immune escape through NF‐κB pathway activation. Conversely,TAB2 knockdown or NF‐κB inhibition sensitised resistant cells to cisplatin and enhanced T cell‐mediated killing. The resistant phenotype could be rescued by restoring PD‐L1 expression. Our findings reveal TAB2 as a critical regulator of both chemoresistance and immune escape in CC through NF‐κB pathway activation. This suggests TAB2 as a potential therapeutic target for overcoming treatment resistance in CC. View Publication

Brain organoids have been proposed as suitable human brain model candidates for a variety of applications. However,the lack of appropriate maturation limits the transferability of such functional tools. Here,we present a method to facilitate neuronal maturation by integrating astrocyte-secreted factors into hPSC-derived 2D and 3D neural culture systems. We demonstrate that protein- and nutrient-enriched astrocyte-conditioned medium (ACM) accelerates neuronal differentiation with enlarged neuronal layer and the overproduction of deep-layer cortical neurons. We captured the elevated changes in the functional activity of neuronal networks within ACM-treated organoids using comprehensive electrophysiological recordings. Furthermore,astrocyte-secreted cues can induce lipid droplet accumulation in neural cultures,offering protective effects in neural differentiation to withstand cellular stress. Together,these data indicate the potential of astrocyte secretions to promote neural maturation. Subject terms: Neurological models,Neuronal development View Publication

The airway epithelium represents a central barrier against pathogens and toxins while playing a crucial role in modulating the immune response within the upper respiratory tract. Understanding these mechanisms is particularly relevant for red foxes ( Vulpes vulpes ),which serve as reservoirs for various zoonotic pathogens like rabies or the fox tapeworm ( Echinococcus multilocularis ). The study aimed to develop,establish,and validate an air-liquid interface (ALI) organoid model of the fox respiratory tract using primary airway epithelial cells isolated from the tracheas and main bronchi of hunted red foxes. The resulting ALI cultures exhibited a structurally differentiated,pseudostratified epithelium,characterised by ciliated cells,mucus secretion,and tight junctions,as confirmed through histological and immunohistochemical analysis. Functional assessments using a paracellular permeability assay and measurement of transepithelial electrical resistance,demonstrated a tight epithelial barrier. The potential of model’s utility for studying innate immune responses to respiratory infections was validated by exposing the cultures to lipopolysaccharide,phorbol-12-myristate-13-acetate and ionomycin,and nematode somatic antigens. Quantitative PCR revealed notable changes in the expression of pro-inflammatory cytokines TNF and IL-33. This in vitro model represents a significant advancement in respiratory research for non-classical species that may act as important wildlife reservoirs for a range of zoonotic pathogens. View Publication

Overall survival of acute myeloid leukemia (AML) remains limited. Inhibitors of the master mitotic kinase PLK1 have emerged as promising therapeutics,demonstrating efficacy in an undefined subset of patients with AML. However,the clinical success of PLK1 inhibitors remains hindered by a lack of predictive biomarkers. The Fanconi anemia (FA) pathway,a tumor-suppressive network comprised of at least 22 genes,is frequently mutated in sporadic AML. In this study,we demonstrate that FA pathway disruption sensitizes AML cells to PLK1 inhibition. Mechanistically,we identify novel interactions between PLK1 and both FANCA and FANCD2 at mitotic centromeres. We demonstrate that PLK1 inhibition impairs recruitment of FANCD2 to mitotic centromeres,induces damage to mitotic chromosomes,and triggers mitotic collapse in FANCA-deficient cells. Our findings indicate that PLK1 inhibition targets mitotic vulnerabilities specific to FA pathway–deficient cells and implicate FA pathway mutations as potential biomarkers for the identification of patients likely to benefit from PLK1 inhibitors. This work demonstrates that FA pathway mutations,which are frequently observed in sporadic AML,induce hypersensitivity to PLK1 inhibition,providing rationale for a novel synthetic lethal therapeutic strategy for this patient population. View Publication