技术资料

Brain decellularized extracellular matrix (ECM) can be an attractive scaffold capable of mimicking the native ecosystem of the central nervous system tissue. We studied the in vitro response of neural cultures exposed to region-specific brain decellularized ECM scaffolds from three distinct neuroanatomical sections: cortex,cerebellum and remaining areas. First,each brain region was evaluated with the isotropic fractionator method to understand the cellular composition of the different cerebral areas. Second,the cerebral regions were subjected to the decellularization process and their respective characterization using molecular,histological,and ultrastructural techniques. Third,the levels of neurotrophic factors in the decellularized brain scaffold were analyzed. Fourth,we studied the region-specific brain decellularized ECM as a mimetic platform for the maturation of PC12 cells,as a unidirectional model of differentiation. Finally,in vitro studies were carried out to evaluate the cell recovery capacity of brain decellularized ECM under stroke-mimetic conditions. Our results show that region-specific brain decellularized ECM can serve as a biomimetic scaffold capable of promoting the growth of neural lineage cells and,in addition,it possesses a combination of structural and biochemical signals (e.g.,neurotrophic factors) that are capable of inducing cell phenotypic changes and promote viability and cell recovery in a stroke/ischemia model in vitro. The online version contains supplementary material available at 10.1038/s41598-025-95656-w. View Publication

Genetically encoded calcium ion (Ca 2+ ) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca 2+ dynamics and neuronal activities with single-cell resolution. Here we report the design and development of two far-red fluorescent GECIs,FR-GECO1a and FR-GECO1c,based on the monomeric far-red fluorescent proteins mKelly1 and mKelly2. FR-GECOs have excitation and emission maxima at ~596 nm and ~644 nm,respectively,display large responses to Ca 2+ in vitro (Δ F / F 0 = 6 for FR-GECO1a,18 for FR-GECO1c),are bright under both one-photon and two-photon illumination,and have high affinities (apparent K d = 29 nM for FR-GECO1a,83 nM for FR-GECO1c) for Ca 2+ . FR-GECOs offer sensitive and fast detection of single action potentials in neurons,and enable in vivo all-optical manipulation and measurement of cellular activities in combination with optogenetic actuators. Subject terms: Fluorescent proteins,Optogenetics,Zebrafish,Molecular neuroscience,Calcium signalling View Publication

The lymph node (LN) performs essential roles in immunosurveillance throughout the body. Developing in vitro models of this key tissue is of great importance to enhancing physiological relevance in immunoengineering. The LN consists of stromal populations and immune cells,which are highly organized and bathed in constant interstitial fluid flow (IFF). The stroma,notably the fibroblastic reticular cells (FRCs) and the lymphatic endothelial cells (LECs),play crucial roles in guiding T cell migration and are known to be sensitive to fluid flow. During inflammation,interstitial fluid flow rates drastically increase in the LN. It is unknown how these altered flow rates impact crosstalk and cell behavior in the LN,and most existing in vitro models focus on the interactions between T cells,B cells,and dendritic cells rather than with the stroma. To address this gap,we developed a human engineered model of the LN stroma consisting of FRC-laden hydrogel above a monolayer of LECs in a tissue culture insert with gravity-driven interstitial flow. We found that FRCs had enhanced coverage and proliferation in response to high flow rates,while LECs experienced decreased barrier integrity. We added CD4+ and CD8+ T cells and found that their egress was significantly decreased in the presence of interstitial flow,regardless of magnitude. Interestingly,3.0 μ m/s flow,but not 0.8 μ m/s flow,correlated with enhanced inflammatory cytokine secretion in the LN stroma. Overall,we demonstrate that interstitial flow is an essential consideration in the lymph node for modulating LN stroma morphology,T cell migration,and inflammation. View Publication

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