技术资料

The heart is a metabolic “omnivore” and adjusts its energy source depending on the circulating metabolites. Human cardiac organoids,a three-dimensional in vitro model of the heart wall,are a useful tool to study cardiac physiology and pathology. However,cardiac tissue naturally experiences shear stress and nutrient fluctuations via blood flow in vivo,whilst in vitro models are conventionally cultivated in a static medium. This necessitates the regular refreshing of culture media,which creates acute cellular disturbances and large metabolic fluxes. To culture human cardiac organoids in a more physiological manner,we have developed a perfused bioreactor for cultures in a 96-well plate format. The designed bioreactor is easy to fabricate using a common culture plate and a 3D printer. Its open system allows for the use of traditional molecular biology techniques,prevents flow blockage issues,and provides easy access for sampling and cell assays. We hypothesized that a perfused culture would create more stable environment improving cardiac function and maturation. We found that lactate is rapidly produced by human cardiac organoids,resulting in large fluctuations in this metabolite under static culture. Despite this,neither medium perfusion in bioreactor culture nor lactate supplementation improved cardiac function or maturation. In fact,RNA sequencing revealed little change across the transcriptome. This demonstrates that cardiac organoids are robust in response to fluctuating environmental conditions under normal physiological conditions. Together,we provide a framework for establishing an easily accessible perfusion system that can be adapted to a range of miniaturized cell culture systems. View Publication

Several genetic risk factors for Alzheimer’s disease implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells 1 . However,the relationship between lipid metabolism in glia and Alzheimer’s disease pathology remains poorly understood. Through single-nucleus RNA sequencing of brain tissue in Alzheimer’s disease,we have identified a microglial state defined by the expression of the lipid droplet-associated enzyme ACSL1 with ACSL1-positive microglia being most abundant in patients with Alzheimer’s disease having the APOE4/4 genotype. In human induced pluripotent stem cell-derived microglia,fibrillar Aβ induces ACSL1 expression,triglyceride synthesis and lipid droplet accumulation in an APOE-dependent manner. Additionally,conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for Alzheimer’s disease with microglial lipid droplet accumulation and neurotoxic microglia-derived factors,potentially providing therapeutic strategies for Alzheimer’s disease. Subject terms: Alzheimer's disease,Microglia,Neuroimmunology View Publication

Signal regulatory protein-α (SIRPα,SHPS-1,CD172a) expressed on myeloid cells transmits inhibitory signals when it engages its counter-receptor CD47 on an adjacent cell. Elevated CD47 expression on some cancer cells thereby serves as an innate immune checkpoint that limits phagocytic clearance of tumor cells by macrophages and antigen presentation to T cells. Antibodies and recombinant SIRPα constructs that block the CD47-SIRPα interaction on macrophages exhibit anti-tumor activities in mouse models and are in ongoing clinical trials for treating several human cancers. Based on prior evidence that engaging SIRPα can also alter CD47 signaling in some nonmalignant cells,we compared direct effects of recombinant SIRPα-Fc and a humanized CD47 antibody that inhibits CD47-SIRPα interaction (CC-90002) on CD47 signaling in cancer stem cells derived from the MDA-MB- 231 triple-negative breast carcinoma cell line. Treatment with SIRPα-Fc significantly increased the formation of mammospheres by breast cancer stem cells as compared to CC-90002 treatment or controls. Furthermore,SIRPα-Fc treatment upregulated mRNA and protein expression of ALDH1 and altered the expression of genes involved in epithelial/mesenchymal transition pathways that are associated with a poor prognosis and enhanced metastatic activity. This indicates that SIRPα-Fc has CD47-mediated agonist activities in breast cancer stem cells affecting proliferation and metastasis pathways that differ from those of CC-90002. This SIRPα-induced CD47 signaling in breast carcinoma cells may limit the efficacy of SIRPα decoy therapeutics intended to stimulate innate antitumor immune responses. View Publication

Dou et al. demonstrate that Parkinson’s disease-associated hyperactive LRRK2 decreases the trafficking of synaptic vesicle proteins within neurons by disrupting the regulation of the synaptic vesicle precursor protein RAB3A. Impaired delivery of synaptic proteins to presynaptic sites could contribute to the progression of debilitating non-motor PD symptoms. View Publication

Despite extensive research on microbiome alterations in ulcerative colitis (UC),the role of the constituent stable microbiota remains unclear. This study,employing 16S rRNA-gene sequencing,uncovers a persistent microbial imbalance in both active and quiescent UC patients compared to healthy controls. Using co-occurrence and differential abundance analysis,the study highlights microbial constituents,featuring Phocaeicola,Collinsella,Roseburia,Holdemanella,and Bacteroides,that are not affected during the course of UC. Co-cultivation experiments,utilizing commensal Escherichia coli and Phocaeicola vulgatus,were conducted with intestinal epithelial organoids derived from active UC patients and controls. These experiments reveal a tendency for a differential response in tight junction formation and maintenance in colonic epithelial cells,without inducing pathogen recognition and stress responses,offering further insights into the roles of these microorganisms in UC pathogenesis. These experiments also uncover high variation in patients’ response to the same bacteria,which indicate the need for more comprehensive,stratified analyses with an expanded sample size. This study reveals that a substantial part of the gut microbiota remains stable throughout progression of UC. Functional experiments suggest that members of core microbiota – Escherichia coli and Phocaeicola vulgatus – potentially differentially regulate the expression of tight junction gene in the colonic epithelium of UC patients and healthy individuals. The online version contains supplementary material available at 10.1186/s13099-024-00612-0. View Publication

Genetic deletion of Dusp1 eliminates CSF3R-induced leukemia. Inhibition of Dusp1 induces the expression of Bim and p53 in oncogenic context,resulting in selective demise of leukemic cells. View Publication

Cancer remains one of the leading causes of mortality worldwide,leading to increased interest in utilizing immunotherapy strategies for better cancer treatments. In the past decade,CD103 + T cells have been associated with better clinical prognosis in patients with cancer. However,the specific immune mechanisms contributing toward CD103-mediated protective immunity remain unclear. Here,we show an unexpected and transient CD61 expression,which is paired with CD103 at the synaptic microclusters of T cells. CD61 colocalization with the T cell antigen receptor further modulates downstream T cell antigen receptor signaling,improving antitumor cytotoxicity and promoting physiological control of tumor growth. Clinically,the presence of CD61 + tumor-infiltrating T lymphocytes is associated with improved clinical outcomes,mediated through enhanced effector functions and phenotype with limited evidence of cellular exhaustion. In conclusion,this study identified an unconventional and transient CD61 expression and pairing with CD103 on human immune cells,which potentiates a new target for immune-based cellular therapies. Subject terms: T cells,Tumour immunology,Lymphocyte activation View Publication

Induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet,since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling,it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R. Serial modifications to an existing iMGL protocol were made,including but not limited to changes in growth factor combination to drive microglial differentiation,until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines,the quality of the new iMGL protocol was validated through cell yield assessment,measurement of microglia marker expression,transcriptomic comparison to primary microglia,and evaluation of inflammatory and phagocytic activities. Similarly,molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol,decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally,ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 ( P2RY12 ) expression,a heightened capacity to internalize myelin,as well as heightened inflammatory response to Pam 3 CSK 4 . Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency,as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL,and in CSF1R WT/KO and CSF1R WT/E633K iMGL compared to their respective isogenic controls. We optimized a pre-existing iMGL protocol,generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol,we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant,with preliminary characterization pointing toward functional alterations in migratory,phagocytic and inflammatory activities. The online version contains supplementary material available at 10.1186/s13024-024-00723-x. View Publication

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF),which is not expressed in guided cortical organoids,we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP,LIFR,and HOPX,closely matching human fetal oRG. Finally,incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely. View Publication

Alcohol consumption increases the risk of breast cancer and promotes cancer progression. Alcohol exposure could affect both processes of the mammary carcinogenesis,namely,the cell transformation and onset of tumorigenesis as well as cancer aggressiveness including metastasis and drug resistance/recurrence. However,the cellular and molecular mechanisms underlying alcohol tumor promotion remain unclear. There are four members of the mammalian p38 mitogen-activated protein kinase (MAPK) family,namely,p38α,p38β,p38γ and p38δ. We have previously demonstrated alcohol exposure selectively activated p38γ MAPK in breast cancer cells in vitro and in vivo . Pirfenidone (PFD),an antifibrotic compound approved for the treatment of idiopathic pulmonary fibrosis,is also a pharmacological inhibitor of p38γ MAPK. This study aimed to determine whether PFD is useful to inhibit alcohol-induced promotion of breast cancer. Female adolescent (5 weeks) MMTV-Wnt1 mice were exposed to alcohol with a liquid diet containing 6.7% ethanol. Some mice received intraperitoneal (IP) injection of PFD (100 mg/kg) every other day. After that,the effects of alcohol and PFD on mammary tumorigenesis and metastasis were examined. Alcohol promoted the progression of mammary tumors in adolescent MMTV-Wnt1 mice. Treatment of PFD blocked tumor growth and alcohol-promoted metastasis. It also significantly inhibited alcohol-induced tumorsphere formation and cancer stem cell (CSC) population. PFD inhibited mammary tumor growth and alcohol-promoted metastasis. Since PFD is an FDA-approved drug,the current findings may be helpful to re-purpose its application in treating aggressive breast cancer and alcohol-promoted mammary tumor progression. View Publication

FMS-related tyrosine kinase 3 ligand (FLT3L),encoded by FLT3LG,is a hematopoietic factor essential for the development of natural killer (NK),B cells,and dendritic cells (DCs) in mice. We describe three humans homozygous for a loss-of-function FLT3LG variant,with a history of various recurrent infections,including severe cutaneous warts. The patients’ bone marrow was hypoplastic,with low levels of hematopoietic progenitors,particularly myeloid and B-cell precursors. Counts of B cells,monocytes,and DCs were low in the patients’ blood,whereas the other blood subsets,including NK cells,were affected only moderately,if at all. The patients had normal counts of Langerhans cells and dermal macrophages in the skin but lacked dermal DCs. Thus,FLT3L is required for B-cell and DC development in mice and humans. However,unlike its murine counterpart,human FLT3L is required for the development of monocytes but not NK cells. View Publication

Glucose regulated protein 78 (GRP78) is a chaperone protein that is a central mediator of the unfolded protein response,a key cellular stress response pathway. GRP78 has been shown to be critically required for infection and replication of a number of flaviviruses,and to interact with both non-structural (NS) and structural flavivirus proteins. However,the nature of the specific interaction between GRP78 and viral proteins remains largely unknown. This study aimed to characterize the binding domain and critical amino acid residues that mediate the interaction of GRP78 to ZIKV E and NS1 proteins. Recombinant EGFP fused GRP78 and individual subdomains (the nucleotide binding domain (NBD) and the substrate binding domain (SBD)) were used as a bait protein and co-expressed with full length or truncated ZIKV E and NS1 proteins in HEK293T/17 cells. Protein–protein interactions were determined by a co-immunoprecipitation assay. From the results,both the NBD and the SBD of GRP78 were crucial for an effective interaction. Single amino acid substitutions in the SBD showed that R492E and T518A mutants significantly reduced the binding affinity of GRP78 to ZIKV E and NS1 proteins. Notably,the interaction of GRP78 with ZIKV E was stably maintained against various single amino acid substitutions on ZIKV E domain III and with all truncated ZIKV E and NS1 proteins. Collectively,the results suggest that the principal binding between GRP78 and viral proteins is mainly a classic canonical chaperone protein-client interaction. The blocking of GRP78 chaperone function effectively inhibited ZIKV infection and replication in neuronal progenitor cells. Our findings reveal that GRP78 is a potential host target for anti-ZIKV therapeutics. View Publication