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A new perspective suggests that a dynamic bidirectional communication system,often referred to as the microbiome-gut-brain axis,exists among the gut,its microbiome,and the central nervous system (CNS). This system may influence brain health and various brain-related diseases,especially in the realms of neurodevelopmental and neurodegenerative conditions. However,the exact mechanism is not yet understood. Metabolites or extracellular vesicles derived from microbes in the gut have the capacity to traverse the intestinal epithelial barrier or blood–brain barrier,gaining access to the systemic circulation. This phenomenon can initiate the physiological responses that directly or indirectly impact the CNS and its function. However,reliable and controllable tools are required to demonstrate the causal effects of gut microbial-derived substances on neurogenesis and neurodegenerative diseases. The integration of microfluidics enhances scientific research by providing advanced in vitro engineering models. In this study,we investigated the impact of microbe-derived metabolites and exosomes on neurodevelopment and neurodegenerative disorders using human induced pluripotent stem cells (iPSCs)-derived neurons in a gut-brain axis chip. While strain-specific,our findings indicate that both microbial-derived metabolites and exosomes exert the significant effects on neural growth,maturation,and synaptic plasticity. Therefore,our results suggest that metabolites and exosomes derived from microbes hold promise as potential candidates and strategies for addressing neurodevelopmental and neurodegenerative disorders. 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

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

Cancer stem cells (CSCs) are a specific subpopulation of cancer cells with the ability of self‐renewal,infinite proliferation,multidifferentiation and tumorigenicity,and play critical roles in cancer progression and treatment resistance. CSCs are tightly regulated by the tumor microenvironment,such as hypoxia; however,how hypoxia regulates CSCs in non‐small cell lung cancer (NSCLC) remains unclear. The proportion of ALDH hi cells was examined using the Aldefluor assay. Tankyrase inhibitor XAV939 and siRNA were used to inhibit β‐catenin while pcDNA3‐β‐catenin (S33Y) plasmid enhanced the expression of β‐catenin. Western blot was administered for protein detection. The mRNA expression was measured by quantitative real‐time PCR. We found that hypoxia led to an increase in the proportion of ALDH hi cells in lung squamous carcinoma (LUSC) H520 cells,while causing a decrease in the ALDH hi cell proportion in lung adenocarcinoma (LUAD) A549 cells. Similarly,β‐catenin expression was upregulated in H520 cells but downregulated in A549 cells upon exposure to hypoxia. Mechanically,the proportion of ALDH hi cells in both cell lines was decreased by β‐catenin inhibitor or siRNA knockdown,whereas increased after β‐catenin overexpression. Furthermore,hypoxia treatment suppressed E‐cadherin expression in H520 cells and enhanced N‐cadherin and β‐catenin expression,while this effect was completely opposite in A549 cells. The hypoxia‐EMT‐β‐catenin axis functions as an important regulator for the proportion of CSCs in NSCLC and could potentially be explored as therapeutic targets in the future. View Publication

Numerous studies have shown that somite development is a necessary stage of myogenesis chondrogenesis and osteogenesis. Our previous study has established a stable presomitic mesoderm progenitor cell line (UiPSM) in vitro. Naturally,we wanted to explore whether UiPSM cell can develop bone and myogenic differentiation. Selective culture conditions yielded PAX3 and PAX7 positive skeletal muscle precursors from UiPSM cells. The skeletal muscle precursors undergo in vitro maturation resulting in myotube formation. MYOD effectively promoted the maturity of the skeletal myocytes in a short time. We found that UiPSM and MYOD mediated UiPSM cell-derived skeletal myocytes were viable after transplantation into the tibialis anterior muscle of MITRG mice,as assessed by bioluminescence imaging and scRNA-seq. Lack of teratoma formation and evidence of long-term myocytes engraftment suggests considerable potential for future therapeutic applications. Moreover,UiPSM cells can differentiate into osteoblast and chondroblast cells in vitro. UiPSM differentiation has potential as a developmental model for musculoskeletal development research and treatment of musculoskeletal disorders. The online version contains supplementary material available at 10.1186/s13578-024-01274-w. View Publication

Acute myeloid leukemia (AML) is a heterogenous disease characterized by the clonal expansion of myeloid progenitor cells. Despite recent advancements in the treatment of AML,relapse still remains a significant challenge,necessitating the development of innovative therapies to eliminate minimal residual disease. One promising approach to address these unmet clinical needs is natural killer (NK) cell immunotherapy. To implement such treatments effectively,it is vital to comprehend how AML cells escape the NK-cell surveillance. Signal transducer and activator of transcription 3 (STAT3),a component of the Janus kinase (JAK)-STAT signaling pathway,is well-known for its role in driving immune evasion in various cancer types. Nevertheless,the specific function of STAT3 in AML cell escape from NK cells has not been deeply investigated. In this study,we unravel a novel role of STAT3 in sensitizing AML cells to NK-cell surveillance. We demonstrate that STAT3-deficient AML cell lines are inefficiently eliminated by NK cells. Mechanistically,AML cells lacking STAT3 fail to form an immune synapse as efficiently as their wild-type counterparts due to significantly reduced surface expression of intercellular adhesion molecule 1 (ICAM-1). The impaired killing of STAT3-deficient cells can be rescued by ICAM-1 overexpression proving its central role in the observed phenotype. Importantly,analysis of our AML patient cohort revealed a positive correlation between ICAM1 and STAT3 expression suggesting a predominant role of STAT3 in ICAM-1 regulation in this disease. In line,high ICAM1 expression correlates with better survival of AML patients underscoring the translational relevance of our findings. Taken together,our data unveil a novel role of STAT3 in preventing AML cells from escaping NK-cell surveillance and highlight the STAT3/ICAM-1 axis as a potential biomarker for NK-cell therapies in AML. View Publication

Delivering macromolecules across biological barriers such as the blood–brain barrier limits their application in vivo. Previous work has demonstrated that Toxoplasma gondii,a parasite that naturally travels from the human gut to the central nervous system (CNS),can deliver proteins to host cells. Here we engineered T. gondii ’s endogenous secretion systems,the rhoptries and dense granules,to deliver multiple large (>100 kDa) therapeutic proteins into neurons via translational fusions to toxofilin and GRA16. We demonstrate delivery in cultured cells,brain organoids and in vivo,and probe protein activity using imaging,pull-down assays,scRNA-seq and fluorescent reporters. We demonstrate robust delivery after intraperitoneal administration in mice and characterize 3D distribution throughout the brain. As proof of concept,we demonstrate GRA16-mediated brain delivery of the MeCP2 protein,a putative therapeutic target for Rett syndrome. By characterizing the potential and current limitations of the system,we aim to guide future improvements that will be required for broader application. Subject terms: Parasitology,Biologics,Synthetic biology View Publication

Streptococcus pneumoniae is a global priority respiratory pathogen that kills over a million people annually. The pore-forming cytotoxin,pneumolysin (PLY) is a major virulence factor. Here,we found that recombinant PLY as well as wild-type pneumococcal strains,but not the isogenic PLY mutant,upregulated the shedding of extracellular vesicles (EVs) harboring membrane-bound toxin from human THP-1 monocytes. PLY-EVs induced cytotoxicity and hemolysis dose-dependently upon internalization by recipient monocyte-derived dendritic cells. Proteomics analysis revealed that PLY-EVs are selectively enriched in key inflammatory host proteins such as IFI16,NLRC4,PTX3,and MMP9. EVs shed from PLY-challenged or infected cells induced dendritic cell maturation and primed them to infection. In vivo,zebrafish administered with PLY-EVs showed pericardial edema and mortality. Adoptive transfer of bronchoalveolar-lavage-derived EVs from infected mice to healthy recipients induced lung damage and inflammation in a PLY-dependent manner. Our findings identify that host EVs released during infection mediate pneumococcal pathogenesis. Subject areas: Microbiology,Bacteriology,Cell biology View Publication

Although bone marrow-derived cells with high aldehyde dehydrogenase activity (ALDH br ) have shown therapeutic potential against various diseases in animal studies,clinical trials have failed to show concurrent findings. We aimed to clarify the optimal conditions for the efficacy of ALDH br cells by using a murine bleomycin-induced pulmonary fibrosis model. We intravenously transferred male or female donor C57BL/6 mice-derived ALDH br cells into recipient C57BL/6 mice under various conditions,and used mCherry-expressing mice as a donor to trace the transferred ALDH br cells. Pulmonary fibrosis improved significantly when (1) female-derived,not male-derived,and (2) lineage (Lin)-negative,not lineage-positive,ALDH br cells were transferred during the (3) fibrotic,not inflammatory,phase. Consistent with the RNA-sequencing results,female-derived Lin − /ALDH br cells were more resistant to oxidative stress than male-derived cells in vitro,and transferred female-derived Lin − /ALDH br cells were more viable than male-derived cells in the fibrotic lung. The mechanism underlying the antifibrotic effects of Lin − /ALDH br cells was strongly associated with reduction of oxidative stress. Our results indicated that Lin − /ALDH br cell therapy could ameliorate pulmonary fibrosis by reducing oxidative stress and suggested that their efficacy was mediated by sex-related differences. Thus,sex-awareness strategies may be important for clinical application of bone marrow ALDH br cells as a therapeutic tool. The online version contains supplementary material available at 10.1186/s13287-024-03933-8. View Publication

Rett syndrome (RTT) is a neurodevelopmental disorder caused by de novo mutations in the MECP2 gene. Although miRNAs in extracellular vesicles (EVs) have been suggested to play an essential role in several neurological conditions,no prior study has utilized brain organoids to profile EV-derived miRNAs during normal and RTT-affected neuronal development. Here we report the spatiotemporal expression pattern of EV-derived miRNAs in region-specific forebrain organoids generated from female hiPSCs with a MeCP2:R255X mutation and the corresponding isogenic control. EV miRNA and protein expression profiles were characterized at day 0,day 13,day 40,and day 75. Several members of the hsa-miR-302/367 cluster were identified as having a time-dependent expression profile with RTT-specific alterations at the latest developmental stage. Moreover,the miRNA species of the chromosome 14 miRNA cluster (C14MC) exhibited strong upregulation in RTT forebrain organoids irrespective of their spatiotemporal location. Together,our results suggest essential roles of the C14MC and hsa-miR-302/367 clusters in EVs during normal and RTT-associated neurodevelopment,displaying promising prospects as biomarkers for monitoring RTT progression. The online version contains supplementary material available at 10.1007/s00018-024-05409-7. View Publication

Head and neck squamous cell carcinoma (HNSCC) presents a significant challenge worldwide due to its aggressiveness and high recurrence rates post-treatment,often linked to cancer stem cells (CSCs). Melatonin shows promise as a potent tumor suppressor; however,the effects of melatonin on CSCs remain unclear,and the development of models that closely resemble tumor heterogeneity could help to better understand the effects of this molecule. This study developed a tumor scaffold based on patient fibroblast-derived decellularized extracellular matrix that mimics the HNSCC microenvironment. Our study investigates the antitumoral effects of melatonin within this context. We validated its strong antiproliferative effect on HNSCC CSCs and the reduction of tumor invasion and migration markers,even in a strongly chemoprotective environment,as it is required to increase the minimum doses necessary to impact tumor viability compared to the non-scaffolded tumorspheres culture. Moreover,melatonin exhibited no cytotoxic effects on healthy cells co-cultured in the tumor hydrogel. This scaffold-based platform allows an in vitro study closer to HNSCC tumor reality,including CSCs,stromal component,and a biomimetic matrix,providing a new valuable research tool in precision oncology. View Publication

Radiotherapy is widely regarded as the primary therapeutic modality for nasopharyngeal cancer (NPC). Studies have shown that cancer cells with high resistance to radiation,known as radioresistant cancer cells,may cause residual illness,which in turn might contribute to the occurrence of cancer recurrence and metastasis. It has been shown that cancer stem-like cells (CSCs) exhibit resistance to radiation therapy. In the present study,fractionated doses of radiation-induced epithelial-mesenchymal transition (EMT) and ALDH+ CSCs phenotype of NPC tumor spheroids. Furthermore,it has been shown that cells with elevated ALDH activity have increased resistance to the effects of fractionated irradiation. Nuclear factor erythroid-2-related factor 2 (Nrf2) plays a pivotal role in regulating cellular antioxidant systems. A large body of evidence suggests that Nrf2 plays a significant role in the development of radioresistance in cancer. The authors’ research revealed that the application of fractionated irradiation resulted in a decline in Nrf2-dependent reactive oxygen species (ROS) levels,thereby mitigating DNA damage in ALDH+ stem-like NPC cells. In addition,immunofluorescence analysis revealed that subsequent to the process of fractionated irradiation of ALDH+ cells,activated Nrf2 was predominantly localized inside the nucleus. Immunofluorescent analysis also revealed that the presence of the nuclear Nrf2+/NQO1+/ALDH1+ axis might potentially serve as an indicator of poor prognosis and resistance to radiotherapy in patients with NPC. Thus,the authors’ findings strongly suggest that the radioresistance of ALDH-positive NPC CSCs to fractionated irradiation is regulated by nuclear Nrf2 accumulation. Nrf2 exerts its effects through the downstream effector NQO1/ALDH1,which depends on ROS attenuation. View Publication