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Leigh syndrome French Canadian type (LSFC) is a recessive neurodegenerative disease characterized by tissue-specific deficiency in cytochrome c oxidase (COX),the fourth complex in the oxidative phosphorylation system. LSFC is caused by mutations in the leucine rich pentatricopeptide repeat containing gene ( LRPPRC ). Most LSFC patients in Quebec are homozygous for an A354V substitution that causes a decrease in the expression of the LRPPRC protein. While LRPPRC is ubiquitously expressed and is involved in multiple cellular functions,tissue-specific expression of LRPPRC and COX activity is correlated with clinical features. In this proof-of-principle study,we developed human induced pluripotent stem cell (hiPSC)-based models from fibroblasts taken from a patient with LSFC,homozygous for the LRPPRC *354V allele,and from a control,homozygous for the LRPPRC *A354 allele. Specifically,for both of these fibroblast lines we generated hiPSC,hiPSC-derived cardiomyocytes (hiPSC-CMs) and hepatocyte-like cell (hiPSC-HLCs) lines,as well as the three germ layers. We observed that LRPPRC protein expression is reduced in all cell lines/layers derived from LSFC patient compared to control cells,with a reduction ranging from ∼70% in hiPSC-CMs to undetectable levels in hiPSC-HLC,reflecting tissue heterogeneity observed in patient tissues. We next performed exploratory analyses of these cell lines and observed that COX protein expression was reduced in all cell lines derived from LSFC patient compared to control cells. We also observed that mutant LRPPRC was associated with altered expression of key markers of endoplasmic reticulum stress response in hiPSC-HLCs but not in other cell types that were tested. While this demonstrates feasibility of the approach to experimentally study genotype-based differences that have tissue-specific impacts,this study will need to be extended to a larger number of patients and controls to not only validate the current observations but also to delve more deeply in the pathogenic mechanisms of LSFC. View Publication

Differentiation of human pluripotent stem cells (hPSCs) into subtype-specific neurons holds substantial potential for disease modeling in vitro . For successful differentiation,a detailed understanding of the transcriptional networks regulating cell fate decisions is critical. The heterochronic nature of neurodevelopment,during which distinct cells in the brain and during in vitro differentiation acquire their fates in an unsynchronized manner,hinders pooled transcriptional comparisons. One approach is to “translate” chronologic time into linear developmental and maturational time. Simple binary promotor-driven fluorescent proteins (FPs) to pool similar cells are unable to achieve this goal,due to asynchronous promotor onset in individual cells. We tested five fluorescent timer (FT) molecules expressed from the endogenous paired box 6 (PAX6) promoter in 293T and human hPSCs. Each of these FT systems faithfully reported chronologic time in 293T cells,but none of the FT constructs followed the same fluorescence kinetics in human neural progenitor cells. Subject areas: Natural sciences,Biological sciences,Biochemistry,Molecular biology,Neuroscience,Cellular neuroscience,Cell biology View Publication

Fibroblastic reticular cells (FRCs) are a subpopulation of stromal cells modulating the immune environments in health and disease. We have previously shown that activation of TLR9 signaling in FRC in fat-associated lymphoid clusters (FALC) regulate peritoneal immunity via suppressing immune cell recruitment and peritoneal resident macrophage (PRM) retention. However,FRCs are heterogeneous across tissues and organs. The functions of each FRC subset and the regulation of TLR9 in distinct FRC subsets are unknown. Here,we confirmed that specific deletion of TLR9 in FRC improved bacterial clearance and survival during peritoneal infection. Furthermore,using single-cell RNA sequencing,we found two subsets of FRCs (CD55 hi and CD55 lo ) in the mesenteric FALC. The CD55 hi FRCs were enriched in gene expression related to extracellular matrix formation. The CD55 lo FRCs were enriched in gene expression related to immune response. Interestingly,we found that TLR9 is dominantly expressed in the CD55 lo subset. Activation of TLR9 signaling suppressed proliferation,cytokine production,and retinoid metabolism in the CD55 lo FRC,but not CD55 hi FRC. Notably,we found that adoptive transfer of Tlr9 -/– CD55 lo FRC from mesenteric FALC more effectively improved the survival during peritonitis compared with WT-FRC or Tlr9 -/– CD55 hi FRC. Furthermore,we identified CD55 hi and CD55 lo subsets in human adipose tissue-derived FRC and confirmed the suppressive effect of TLR9 on the proliferation and cytokine production in the CD55 lo subset. Therefore,inhibition of TLR9 in the CD55 lo FRCs from adipose tissue could be a useful strategy to improve the therapeutic efficacy of FRC-based therapy for peritonitis. View Publication

Chronic obstructive pulmonary disease (COPD) is characterised by persistent airway inflammation even after cigarette smoking cessation. Neutrophil extracellular traps (NETs) have been implicated in COPD severity and acute airway inflammation induced by short-term cigarette smoke (CS). However,whether and how NETs contribute to sustained airway inflammation in COPD remain unclear. This study aimed to elucidate the immunoregulatory mechanism of NETs in COPD,employing human neutrophils,airway epithelial cells (AECs),dendritic cells (DCs),and a long-term CS-induced COPD mouse model,alongside cyclic guanosine monophosphate-adenosine monophosphate synthase and toll-like receptor 9 knockout mice ( cGAS -−/−,TLR9 −/− ); Additionally,bronchoalveolar lavage fluid (BALF) of COPD patients was examined. Neutrophils from COPD patients released greater cigarette smoke extract (CSE)-induced NETs (CSE-NETs) due to mitochondrial respiratory chain dysfunction. These CSE-NETs,containing oxidatively-damaged DNA (NETs-DNA),promoted AECs proliferation,nuclear factor kappa B (NF-κB) activation,NF-κB-dependent cytokines and type-I interferons production,and DC maturation,which were ameliorated/reversed by silencing/inhibition of cGAS/TLR9. In the COPD mouse model,blocking NETs-DNA-sensing via cGAS − /− and TLR9 − /− mice,inhibiting NETosis using mitoTEMPO,and degrading NETs-DNA with DNase-I,respectively,reduced NETs infiltrations,airway inflammation,NF-κB activation and NF-κB-dependent cytokines,but not type-I interferons due to IFN-α/β receptor degradation. Elevated NETs components (myeloperoxidase and neutrophil elastase activity) in BALF of COPD smokers correlated with disease severity and NF-κB-dependent cytokine levels,but not type-I interferon levels. In conclusion,NETs-DNA promotes NF-κB-dependent autoimmunity via cGAS/TLR9 in long-term CS exposure-induced COPD. Therefore,targeting NETs-DNA and cGAS/TLR9 emerges as a potential strategy to alleviate persistent airway inflammation in COPD. Subject terms: Inflammation,Respiratory tract diseases View Publication

Successful cell and gene therapy clinical trials have resulted in the US Food and Drug Administration and European Medicines Agency approving their use for treatment of patients with certain types of cancers and monogenetic diseases. These novel therapies,which rely heavily on lentiviral vectors to deliver therapeutic transgenes to patient cells,have driven additional investigations,increasing demand for both pre-clinical and current Good Manufacturing Practices-grade viral vectors. To better support novel studies by improving current production methods,we report the development of a genetically modified HEK293T-based cell line that is null for expression of both Protein Kinase R and Beta-2 microglobulin and grows in suspension using serum-free media,SJ293TS-DPB. Absence of Protein Kinase R increased anti-sense lentiviral vector titers by more than 7-fold,while absence of Beta-2 microglobulin,a key component of major histocompatibility complex class I molecules,has been reported to reduce the immunogenicity of lentiviral particles. Furthermore,we describe an improved methodology for culturing SJ293TS-DPB that facilitates expansion,reduces handling,and increases titers by 2-fold compared with previous methods. SJ293TS-DPB stably produced lentiviral vectors for over 4 months and generated lentiviral vectors that efficiently transduce healthy human donor T cells and CD34 + hematopoietic stem cells. View Publication

Acute myeloid leukaemia (AML) is a disease of the haematopoietic stem cells(HSCs) that is characterised by the uncontrolled proliferation and impaired differentiation of normal haematopoietic stem/progenitor cells. Several pathways that control the proliferation and differentiation of HSCs are impaired in AML. Activation of the Wnt/beta-catenin signalling pathway has been shown in AML and beta-catenin,which is thought to be the key element of this pathway,has been frequently highlighted. The present study was designed to determine beta-catenin expression levels and beta-catenin-related genes in AML. In this study,beta-catenin gene expression levels were determined in 19 AML patients and 3 controls by qRT-PCR. Transcriptome analysis was performed on AML grouped according to beta-catenin expression levels. Differentially expressed genes(DEGs) were investigated in detail using the Database for Annotation Visualisation and Integrated Discovery(DAVID),Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),STRING online tools. The transcriptome profiles of our AML samples showed different molecular signature profiles according to their beta-catenin levels(high-low). A total of 20 genes have been identified as hub genes. Among these,TTK,HJURP,KIF14,BTF3,RPL17 and RSL1D1 were found to be associated with beta-catenin and poor survival in AML. Furthermore,for the first time in our study,the ELOV6 gene,which is the most highly up-regulated gene in human AML samples,was correlated with a poor prognosis via high beta-catenin levels. It is suggested that the identification of beta-catenin-related gene profiles in AML may help to select new therapeutic targets for the treatment of AML. View Publication

Mesenchymal stem cells (MSCs) have long been postulated as an important source cell in regenerative medicine. During subculture expansion,mesenchymal stem cell (MSC) senescence diminishes their multi-differentiation capabilities,leading to a loss of therapeutic potential. Up to date,the extrachromosomal circular DNAs (eccDNAs) have been demonstrated to be involved in senescence but the roles of eccDNAs during MSC. Here we explored eccDNA profiles in human bone marrow MSCs (BM-MSCs). EccDNA and mRNA was purified and sequenced,followed by quantification and functional annotation. Moreover,we mapped our datasets with the downloading enhancer and transcription factor-regulated genes to explore the potential role of eccDNAs. Sequentially,gene annotation analysis revealed that the majority of eccDNA were mapped in the intron regions with limited BM-MSC enhancer overlaps. We discovered that these eccDNA motifs in senescent BMSCs acted as motifs for binding transcription factors (TFs) of senescence-related genes. These findings are highly significant for identifying biomarkers of senescence and therapeutic targets in mesenchymal stem cells (MSCs) for future clinical applications. The potential of eccDNA as a stable therapeutic target for senescence-related disorders warrants further investigation,particularly exploring chemically synthesized eccDNAs as transcription factor regulatory elements to reverse cellular senescence. View Publication

As autologous induced pluripotent stem cell (iPSC) therapy requires a custom-made small-lot cell production line,and the cell production method differs significantly from the existing processes for producing allogeneic iPSC stocks for clinical use. Specifically,mass culture to produce stock is no longer necessary; instead,a series of operations from iPSC production to induction of differentiation of therapeutic cells must be performed continuously. A three-dimensional (3D) culture method using small,closed-cell manufacturing devices is suitable for autologous iPSC therapy. The use of such devices avoids the need to handle many patient-derived specimens in a single clean room; handling of cell cultures in an open system in a cell processing facility increases the risk of infection. In this study,atelocollagen beads were evaluated as a 3D biomaterial to assist 3D culture in the establishment,expansion culture,and induction of differentiation of iPSCs. It was found that iPSCs can be handled in a closed-cell device with the same ease as use of a two-dimensional (2D) culture when laminin-511 is added to the medium. In conclusion,atelocollagen beads enable 3D culture of iPSCs,and the quality of the obtained cells is at the same level as those derived from 2D culture. View Publication

The roles of the transcriptional factor SIX2 have been identified in several tumors. However,its roles in gastric cancer (GC) progression have not yet been revealed. Our objective is to explore the impact and underlying mechanisms of SIX2 on the stemness of GC cells. Lentivirus infection was employed to establish stable expression SIX2 or PFN2 in GC cells. Gain- and loss-of-function experiments were conducted to detect changes of stemness markers,flow cytometry profiles,tumor spheroid formation,and tumor-initiating ability. ChIP,RNA-sequencing,tissue microarray,and bioinformatics analysis were performed to reveal the correlation between SIX2 and PFN2. The mechanisms underlying the SIX2/PFN2 loop-mediated effects were elucidated through tissue microarray analysis,RNA stability assay,IP-MS,Co-Immunoprecipitation,and inhibition of the JNK signaling pathway. The stemness of GC cells was enhanced by SIX2. Mechanistically,SIX2 directly bound to PFN2’s promoter and promoted PFN2 activity. PFN2,in turn,promoted the mRNA stability of SIX2 by recruiting RNA binding protein YBX-1,subsequently activating the downstream MAPK/JNK pathway. This study unveils the roles of SIX2 in governing GC cell stemness,defining a novel SIX2/PFN2 regulatory loop responsible for this regulation. This suggests the potential of targeting the SIX2/PFN2 loop for GC treatment (Graphical Abstracts). The online version contains supplementary material available at 10.1186/s12967-024-05618-5. View Publication

Hematopoietic stem progenitor cells (HSPCs) undergo phenotypical and functional changes during their emergence and development. Although the molecular programs governing the development of human hematopoietic stem cells (HSCs) have been investigated broadly,the relationships between dynamic metabolic alterations and their functions remain poorly characterized. In this study,we comprehensively described the proteomics of HSPCs in the human fetal liver (FL),umbilical cord blood (UCB),and adult bone marrow (aBM). The metabolic state of human HSPCs was assessed via a Seahorse assay,RT‒PCR,and flow cytometry-based metabolic-related analysis. To investigate whether perturbing glutathione metabolism affects reactive oxygen species (ROS) production,the metabolic state,and the expansion of human HSPCs,HSPCs were treated with buthionine sulfoximine (BSO),an inhibitor of glutathione synthetase,and N-acetyl-L-cysteine (NAC). We investigated the metabolomic landscape of human HSPCs from the fetal,perinatal,and adult developmental stages by in-depth quantitative proteomics and predicted a metabolic switch from the oxidative state to the glycolytic state during human HSPC development. Seahorse assays,mitochondrial activity,ROS level,glucose uptake,and protein synthesis rate analysis supported our findings. In addition,immune-related pathways and antigen presentation were upregulated in UCB or aBM HSPCs,indicating their functional maturation upon development. Glutathione-related metabolic perturbations resulted in distinct responses in human HSPCs and progenitors. Furthermore,the molecular and immunophenotypic differences between human HSPCs at different developmental stages were revealed at the protein level for the first time. The metabolic landscape of human HSPCs at three developmental stages (FL,UCB,and aBM),combined with proteomics and functional validations,substantially extends our understanding of HSC metabolic regulation. These findings provide valuable resources for understanding human HSC function and development during fetal and adult life. The online version contains supplementary material available at 10.1186/s13287-024-03930-x. View Publication

Fatty acid synthase (FASN)-catalyzed endogenous lipogenesis is a hallmark of cancer metabolism. However,whether FASN is an intrinsic mechanism of tumor cell defense against T cell immunity remains unexplored. To test this hypothesis,here we combined bioinformatic analysis of the FASN-related immune cell landscape,real-time assessment of cell-based immunotherapy efficacy in CRISPR/Cas9-based FASN gene knockout ( FASN KO ) cell models,and mathematical and mechanistic evaluation of FASN-driven immunoresistance. FASN expression negatively correlates with infiltrating immune cells associated with cancer suppression,cytolytic activity signatures,and HLA-I expression. Cancer cells engineered to carry a loss-of-function mutation in FASN exhibit an enhanced cytolytic response and an accelerated extinction kinetics upon interaction with cytokine-activated T cells. Depletion of FASN results in reduced carrying capacity,accompanied by the suppression of mitochondrial OXPHOS and strong downregulation of electron transport chain complexes. Targeted FASN depletion primes cancer cells for mitochondrial apoptosis as it synergizes with BCL-2/BCL-X L -targeting BH3 mimetics to render cancer cells more susceptible to T-cell-mediated killing. FASN depletion prevents adaptive induction of PD-L1 in response to interferon-gamma and reduces constitutive overexpression of PD-L1 by abolishing PD-L1 post-translational palmitoylation. FASN is a novel tumor cell-intrinsic metabolic checkpoint that restricts T cell immunity and may be exploited to improve the efficacy of T cell-based immunotherapy. Subject terms: Cancer metabolism,Oncogenesis View Publication

Aging is characterized by the accumulation of proteins that display amyloid-like behavior. However,the molecular mechanisms by which these proteins arise remain unclear. Here,we demonstrate that amyloid-like proteins are produced in a variety of human cell types,including stem cells,brain organoids and fully differentiated neurons by mistakes that occur in messenger RNA molecules. Some of these mistakes generate mutant proteins already known to cause disease,while others generate proteins that have not been observed before. Moreover,we show that these mistakes increase when cells are exposed to DNA damage,a major hallmark of human aging. When taken together,these experiments suggest a mechanistic link between the normal aging process and age-related diseases. Subject terms: Protein aggregation,Mechanisms of disease,Transcription View Publication