技术资料

AbstractThe fabrication of complex and stable vasculature in engineered cardiac tissues represents a significant hurdle towards building physiologically relevant models of the heart. Here,we implemented a 3D model of cardiac vasculogenesis,incorporating endothelial cells (EC),stromal cells,and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) in a fibrin hydrogel. The presence of CMs disrupted vessel formation in 3D tissues,resulting in the upregulation of endothelial activation markers and altered extracellular vesicle (EV) signaling in engineered tissues as determined by the proteomic analysis of culture supernatant. miRNA sequencing of CM- and EC-secreted EVs highlighted key EV-miRNAs that were postulated to play differing roles in cardiac vasculogenesis,including the let-7 family and miR-126-3p in EC-EVs. In the absence of CMs,the supplementation of CM-EVs to EC monolayers attenuated EC migration and proliferation and resulted in shorter and more discontinuous self-assembling vessels when applied to 3D vascular tissues. In contrast,supplementation of EC-EVs to the tissue culture media of 3D vascularized cardiac tissues mitigated some of the deleterious effects of CMs on vascular self-assembly,enhancing the average length and continuity of vessel tubes that formed in the presence of CMs. Direct transfection validated the effects of the key EC-EV miRNAs let-7b-5p and miR-126-3p in improving the maintenance of continuous vascular networks. EC-EV supplementation to biofabricated cardiac tissues and microfluidic devices resulted in tissue vascularization,illustrating the use of this approach in the engineering of enhanced,perfusable,microfluidic models of the myocardium. View Publication

SummaryTissue regeneration following an injury requires dynamic cell-state transitions that allow for establishing the cell identities required for the restoration of tissue homeostasis and function. Here,we present a biochemical intervention that induces an intermediate cell state mirroring a transition identified during normal differentiation of myoblasts and other multipotent and pluripotent cells to mature cells. When applied in somatic differentiated cells,the intervention,composed of one-carbon metabolites,reduces some dedifferentiation markers without losing the lineage identity,thus inducing limited reprogramming into a more flexible cell state. Moreover,the intervention enabled accelerated repair after muscle injury in young and aged mice. Overall,our study uncovers a conserved biochemical transitional phase that enhances cellular plasticity in vivo and hints at potential and scalable biochemical interventions of use in regenerative medicine and rejuvenation interventions that may be more tractable than genetic ones. Graphical abstract Highlights•Early cell transitions in differentiation include metabolites,supporting identity changes•Cell-transition biochemicals can be leveraged to induce plasticity•1C-metabolite supplementation streamlines cell-identity changes in vitro•1C-metabolite in vivo administration impacts acetylation genes,aiding muscle regeneration Hernandez-Benitez et al. identify a metabolomic wave conserved in the early transition of cells differentiating in vitro,and they leverage this finding to customize an in vivo supplementation that facilitates the transition of cell phenotypes when needed,like in regeneration after an injury. View Publication

Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus strains that cause severe infections. Bicomponent PVL kills phagocytes depending on cell surface receptors,such as complement 5a receptor 1 (C5aR1). How the PVL-receptor interaction enables assembly of the leukocidin complex,targeting of membranes,and insertion of a pore channel remains incompletely understood. Here,we demonstrate that PVL binds the anionic phospholipids,phosphatidic acid,and cardiolipin,under acidic conditions and targets lipid bilayers that mimic lysosomal and mitochondrial membranes,but not the plasma membrane. The PVL–lipid interaction was sufficient to enable leukocidin complex formation as determined by neutron reflectometry and the rupture of model membranes,independent of protein receptors. In phagocytes,PVL and its C5aR1 receptor were internalized depending on sphingomyelin and cholesterol,which were dispensable for the interaction of the toxin with the plasma membrane. Internalized PVL compromised the integrity of lysosomes and mitochondria before plasma membrane rupture. Preventing the acidification of organelles or the genetic loss of PVL impaired the escape of intracellular S. aureus from macrophages. Together,the findings advance our understanding of how an S. aureus toxin kills host cells and provide key insights into how leukocidins target membranes. Staphylococcus aureus secretes toxins,such as Panton-Valentine leukocidin (PVL),to kill immune cells,including macrophages. This study shows that PVL binds phosphatidic acid and cardiolipin in acidic conditions,targeting lysosomal and mitochondrial membranes (but not the plasma membrane) to promote bacterial escape. View Publication

Natural killer (NK) cells are cytotoxic immune cells that can eliminate target cells without prior stimulation. Human induced pluripotent stem cells (iPSCs) provide a robust source of NK cells for safe and effective cell-based immunotherapy against aggressive cancers. In this in vitro study,a feeder-free iPSC differentiation was performed to obtain iPSC-NK cells,and distinct maturational stages of iPSC-NK were characterized. Mature cells of CD56bright CD16bright phenotype showed upregulation of CD56,CD16,and NK cell activation markers NKG2D and NKp46 upon IL-15 exposure,while exposure to aggressive atypical teratoid/rhabdoid tumor (ATRT) cell lines enhanced NKG2D and NKp46 expression. Malignant cell exposure also increased CD107a degranulation markers and stimulated IFN-? secretion in activated NK cells. CD56bright CD16bright iPSC-NK cells showed a ratio-dependent killing of ATRT cells,and the percentage lysis of CHLA-05-ATRT was higher than that of CHLA-02-ATRT. The iPSC-NK cells were also cytotoxic against other brain,kidney,and lung cancer cell lines. Further NK maturation yielded CD56?ve CD16bright cells,which lacked activation markers even after exposure to interleukins or ATRT cells - indicating diminished cytotoxicity. Generation and characterization of different NK phenotypes from iPSCs,coupled with their promising anti-tumor activity against ATRT in vitro,offer valuable insights into potential immunotherapeutic strategies for brain tumors. Graphical abstractImage 1 Highlights•Natural killer (NK) cells were derived from human induced pluripotent stem cells (iPSCs) in the absence of feeder cells.•Various maturational subtypes of iPSC-NK cells were characterized,and the phenotypic and functional properties were studied.•iPSC-NK cells of CD56bright CD16bright phenotype expressed activation markers in response to interleukin stimuli.•iPSC-NK cells were cytotoxic toward human atypical teratoid and rhabdoid tumor (ATRT) cells and other human cancer cells.•The cytotoxicity of iPSC-NK cells against various cancer cells in vitro might be translated into an in vivo immunotherapy. View Publication

SummaryMotivated by the cellular heterogeneity in complex tissues,particularly in brain and induced pluripotent stem cell (iPSC)-derived brain models,we developed a complete workflow to reproducibly characterize cell types in complex tissues. Our approach combines a flow cytometry (FC) antibody panel with our computational pipeline CelltypeR,enabling dataset aligning,unsupervised clustering optimization,cell type annotating,and statistical comparisons. Applied to human iPSC derived midbrain organoids,it successfully identified the major brain cell types. We performed fluorescence-activated cell sorting of CelltypeR-defined astrocytes,radial glia,and neurons,exploring transcriptional states by single-cell RNA sequencing. Among the sorted neurons,we identified subgroups of dopamine neurons: one reminiscent of substantia nigra cells most vulnerable in Parkinson’s disease. Finally,we used our workflow to track cell types across a time course of organoid differentiation. Overall,our adaptable analysis framework provides a generalizable method for reproducibly identifying cell types across FC datasets in complex tissues. Graphical abstract Highlights•CelltypeR is a flow cytometry and computational pipeline for cell type quantification•Identified brain cell types in midbrain organoids and measured changes in proportions•Enriched selected populations using FACS and characterized by single-cell RNA sequencing•Identified substantia nigra–like dopaminergic neurons sensitive in Parkinson’s disease Neuroscience; Cell biology; Omics View Publication

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease. Primary symptoms of PD arise with the loss of dopaminergic (DA) neurons in the Substantia Nigra Pars Compacta,but PD also affects the hippocampus and cortex,usually in its later stage. Approximately 15% of PD cases are familial with a genetic mutation. Two of the most associated genes with autosomal recessive (AR) early-onset familial PD are PINK1 and PRKN. In vitro studies of these genetic mutations are needed to understand the neurophysiological changes in patients’ neurons that may contribute to neurodegeneration. In this work,we generated and differentiated DA and hippocampal neurons from human induced pluripotent stem cells (hiPSCs) derived from two patients with a double mutation in their PINK1 and PRKN (one homozygous and one heterozygous) genes and assessed their neurophysiology compared to two healthy controls. We showed that the synaptic activity of PD neurons generated from patients with the PINK1 and PRKN mutations is impaired in the hippocampus and dopaminergic neurons. Mutant dopaminergic neurons had enhanced excitatory post-synaptic activity. In addition,DA neurons with the homozygous mutation of PINK1 exhibited more pronounced electrophysiological differences compared to the control neurons. Signaling network analysis of RNA sequencing results revealed that Focal adhesion and ECM receptor pathway were the top two upregulated pathways in the mutant PD neurons. Our findings reveal that the phenotypes linked to PINK1 and PRKN mutations differ from those from other PD mutations,suggesting a unique interplay between these two mutations that drives different PD mechanisms. View Publication

Dysregulation of TDP-43 as seen in TDP-43 proteinopathies leads to specific RNA splicing dysfunction. While discovery studies have explored novel TDP-43-driven splicing events in induced pluripotent stem cell (iPSC)-derived neurons and TDP-43 negative neuronal nuclei,transcriptome-wide investigations in frontotemporal lobar degeneration with TDP-43 aggregates (FTLD-TDP) brains remain unexplored. Such studies hold promise for identifying widespread novel and relevant splicing alterations in FTLD-TDP patient brains. We conducted the largest differential splicing analysis (DSA) using bulk short-read RNAseq data from frontal cortex (FCX) tissue of 127 FTLD-TDP (A,B,C,GRN and C9orf72 carriers) and 22 control subjects (Mayo Clinic Brain Bank),using Leafcutter. In addition,long-read bulk cDNA sequencing data were generated from FCX of 9 FTLD-TDP and 7 controls and human TARDBP wildtype and knock-down iPSC-derived neurons. Publicly available RNAseq data (MayoRNAseq,MSBB and ROSMAP studies) from Alzheimer’s disease patients (AD) was also analyzed. Our DSA revealed extensive splicing alterations in FTLD-TDP patients with 1881 differentially spliced events,in 892 unique genes. When evaluating differences between FTLD-TDP subtypes,we found that C9orf72 repeat expansion carriers carried the most splicing alterations after accounting for differences in cell-type proportions. Focusing on cryptic splicing events,we identified STMN2 and ARHGAP32 as genes with the most abundant and differentially expressed cryptic exons between FTLD-TDP patients and controls in the brain,and we uncovered a set of 17 cryptic events consistently observed across studies,highlighting their potential relevance as biomarkers for TDP-43 proteinopathies. We also identified 16 cryptic events shared between FTLD-TDP and AD brains,suggesting potential common splicing dysregulation pathways in neurodegenerative diseases. Overall,this study provides a comprehensive map of splicing alterations in FTLD-TDP brains,revealing subtype-specific differences and identifying promising candidates for biomarker development and potential common pathogenic mechanisms between FTLD-TDP and AD.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00401-025-02901-7. View Publication

The emergence of retinal progenitor cells and differentiation to various retinal cell types represent fundamental processes during retinal development. Herein,we provide a comprehensive single cell characterisation of transcriptional and chromatin accessibility changes that underline retinal progenitor cell specification and differentiation over the course of human retinal development up to midgestation. Our lineage trajectory data demonstrate the presence of early retinal progenitors,which transit to late,and further to transient neurogenic progenitors,that give rise to all the retinal neurons. Combining single cell RNA-Seq with spatial transcriptomics of early eye samples,we demonstrate the transient presence of early retinal progenitors in the ciliary margin zone with decreasing occurrence from 8 post-conception week of human development. In retinal progenitor cells,we identified a significant enrichment for transcriptional enhanced associate domain transcription factor binding motifs,which when inhibited led to loss of cycling progenitors and retinal identity in pluripotent stem cell derived organoids. Formation of the retina during development involves the coordinated action of retinal progenitor cells and their differentiated cell types,which is key for producing a functioning eye. Here the authors provide a detailed atlas of human retinal development,combining scRNA-seq and spatial transcriptomics,and identify key genetic factors that mediate retinal progenitor cell proliferation and differentiation. View Publication

Interferon gamma (IFNγ) is a critical cytokine known for its diverse roles in immune regulation,inflammation,and tumor surveillance. However,while IFNγ levels were elevated in sera of most newly diagnosed acute myeloid leukemia (AML) patients,its complex interplay in AML remains insufficiently understood. We aim to characterize these complex interactions through comprehensive bulk and single-cell approaches in bone marrow of newly diagnosed AML patients. We identify monocytic AML as having a unique microenvironment characterized by IFNγ producing T and NK cells,high IFNγ signaling,and immunosuppressive features. IFNγ signaling score strongly correlates with venetoclax resistance in primary AML patient cells. Additionally,IFNγ treatment of primary AML patient cells increased venetoclax resistance. Lastly,a parsimonious 47-gene IFNγ score demonstrates robust prognostic value. In summary,our findings suggest that inhibiting IFNγ is a potential treatment strategy to overcoming venetoclax resistance and immune evasion in AML patients. IFNγ signaling is important in the pathogenesis and immune response,emphasizing the need for investigation of its role. Here,the authors show that IFNγ plays a key role in shaping immune microenvironment in AML and developing resistance,providing insights for potential therapeutic strategies. View Publication

PurposeIsolating circulating tumour cells (CTCs) from the blood is challenging due to their low abundance and heterogeneity. Limitations of conventional CTC detection methods highlight the need for improved strategies to detect and isolate CTCs. Currently,the Food and Drug Administration (FDA)-approved CellSearch™ and other RUO techniques are not available in India. Therefore,we wanted to develop a flexible CTC detection/isolation technique that addresses the limitation(s) of currently available techniques and is suitable for various downstream applications.MethodsWe developed a novel,efficient,user-friendly CTC isolation strategy combining density gradient centrifugation and immuno-magnetic hematogenous cell depletion with fluorescence-activated cell sorting (FACS)-based positive selection using multiple CTC-specific cell-surface markers. For FACS,a stringent gating strategy was optimised to exclude debris and doublets by side scatter/forward scatter (SSC/FSC) discriminator,remove dead cells by 4′,6-diamidino-2-phenylindole (DAPI) staining,and eliminate non-specific fluorescence using a “dump” channel. APC-labelled anti-CD45mAB was used to gate remaining hematogenous cells,while multiple epithelial markers (EpCAM,EGFR,and Pan-Cytokeratin) and an epithelial–mesenchymal transition (EMT) marker (Vimentin) labelled with fluorescein isothiocyanate (FITC) were used to sort cancer cells. The technique was initially developed by spiking Cal 27 cancer cells into the blood of healthy donors and then validated in 95 biopsy-proven oral squamous cell carcinoma (OSCC) patients. CTCs isolated from patients were reconfirmed by Giemsa staining,immuno-staining,and whole transcriptome amplification (WTA),followed by qRT-PCR. In vitro culture and RNA sequencing (RNA-Seq) were also performed to confirm their suitability for various downstream applications.ResultsThe mean detection efficiency for the Cal 27 tongue cancer cells spiked in the whole blood of healthy donors was 32.82% ± 12.71%. While ~75% of our patients (71/95) had detectable CTCs,the CTC positivity was independent of the TNM staging. The isolated potential cancer cells from OSCC patients were heterogeneous in size. They expressed different CTC-specific markers in various combinations as identified by qRT-PCR after WTA in different patients. Isolated CTCs were also found to be suitable for downstream applications like short-term CTC culture and RNA-Seq.ConclusionWe developed a sensitive,specific,flexible,and affordable CTC detection/isolation technique,which is scalable to larger patient cohorts,provides a snapshot of CTC heterogeneity,isolates live CTCs ready for downstream molecular analysis,and,most importantly,is suitable for developing countries. View Publication

α-Defensin 1-3 (DEFA1A3) are host antimicrobial peptides with potent innate immune functions during infectious diseases. Differential UTI risk has been linked to DEFA1A3 DNA polymorphisms. This study elucidates mechanisms of DEFA1A3 gene dose–dependent protection against UTI pathogenesis. Antimicrobial peptides (AMPs) are host defense effectors with potent neutralizing and immunomodulatory functions against invasive pathogens. The AMPs α-Defensin 1-3/DEFA1A3 participate in innate immune responses and influence patient outcomes in various diseases. DNA copy-number variations in DEFA1A3 have been associated with severity and outcomes in infectious diseases including urinary tract infections (UTIs). Specifically,children with lower DNA copy numbers were more susceptible to UTIs. The mechanism of action by which α-Defensin 1-3/DEFA1A3 copy-number variations lead to UTI susceptibility remains to be explored. In this study,we use a previously characterized transgenic knock-in of the human DEFA1A3 gene mouse to dissect α-Defensin 1-3 gene dose–dependent antimicrobial and immunomodulatory roles during uropathogenic Escherichia coli (UPEC) UTI. We elucidate the relationship between kidney neutrophil– and collecting duct intercalated cell–derived α-Defensin 1-3/DEFA1A3 expression and UTI. We further describe cooperative effects between α-Defensin 1-3 and other AMPs that potentiate the neutralizing activity against UPEC. Cumulatively,we demonstrate that DEFA1A3 directly protects against UPEC meanwhile impacting pro-inflammatory innate immune responses in a gene dosage–dependent manner. View Publication

Although Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) have been approved in multiple diseases,including BRCA1/2 mutant breast cancer,responses are usually transient requiring the deployment of combination therapies for optimal efficacy. Here we thus explore mechanisms underlying sensitivity and resistance to PARPi using two intrinsically PARPi sensitive (T22) and resistant (T127) syngeneic murine breast cancer models in female mice. We demonstrate that tumor associated macrophages (TAM) potentially contribute to the differential sensitivity to PARPi. By single-cell RNA-sequencing,we identify a TAM_C3 cluster,expressing genes implicated in anti-inflammatory activity,that is enriched in PARPi resistant T127 tumors and markedly decreased by PARPi in T22 tumors. Rps19/C5aR1 signaling is selectively elevated in TAM_C3. C5aR1 inhibition or transferring C5aR1hi cells increases and decreases PARPi sensitivity,respectively. High C5aR1 levels in human breast cancers are associated with poor responses to immune checkpoint blockade. Thus,targeting C5aR1 may selectively deplete pro-tumoral macrophages and engender sensitivity to PARPi and potentially other therapies. PARP inhibitors (PARPi) have been approved for the treatment of metastatic triple-negative breast cancer (BC),however resistance and recurrence are often observed. Here,in preclinical models of BRCA1/2 wild type and homologous recombination competent BC,the authors show that C5aR1-positive tumor associated macrophages are associated with PARPi-resistance,suggesting targeting C5aR1 as a therapeutic option. View Publication