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Biology subjects

Chen, Y.-W.

Publications and source records attributed to Chen, Y.-W..

At least 19 recordsLinked to original sources

Multi-Angle Lateral-View Breast Tissue Classification in Thermographic Images Using Deep Reinforcement Learning and Statistical Shape Modelling

This study is a multi-stage method for accurately segmenting and classifying breast tissue in thermographic images, with a specific focus on lateral views captured at multiple angles (30{degrees}, 45{degrees}, and 90{degrees}). Our algorithm is a hybrid boundary detection framework that combines scanning, tracking, and a deep reinforcement learning (DRL) algorithm to delineate the external breast tissue boundary precisely. The DRL agent corrects inaccurately detected landmark points and reduces the average localization error to 17.4 pixels. A Statistical Shape Model (SSM) captures shape variability and refines landmark alignment. A geometric arc-based method estimates the posterior breast boundary, which facilitates robust breast tissue isolation across various lateral view angles. After that, we pull texture features using GLCM (Grey Level Co-occurrence Matrix) and feed them through an SVM classifier. The proposed pipeline boosted diagnostic performance - the SVM hit 74.1% accuracy on our segmented images. It is about 15% better than raw originals and 21% above cropped images. The results prove the effectiveness of our multi-angle lateral view analysis combined with reinforcement learning and statistical shape modelling in enhancing breast cancer detection from thermographic imaging.

bioengineering↗

Airway Diameter-Matched Injury Improves Severity and Reproducibility of Experimental Rabbit Tracheal Stenosis

ObjectiveReliable animal models of tracheal stenosis are necessary for the development and translational testing of anti-fibrotic and regenerative therapies, but existing rabbit models frequently demonstrate substantial variability in stenosis severity, which limits their translational utility. The objective of our study was to determine whether airway diameter-matched mechanical injury improves the severity and reproducibility of experimental tracheal stenosis in a rabbit model, and to evaluate whether rabbit body weight is a reliable surrogate for tracheal luminal diameter during model creation. MethodsFourteen male New Zealand White rabbits (weight range, 2.7-3.5 kg) underwent tracheal injury using steel-bristle brushes introduced through a tracheotomy. Animals were assigned to receive either airway diameter-matched injury, in which brush size was selected to closely approximate the directly measured tracheal lumen diameter, or non-matched injury, in which brush size was selected without regard to measured lumen diameter. At postoperative day 21 (POD21), the injured tracheal segment and a native uninjured segment from the same animal were harvested and compared. Stenosis degree was quantified grossly, and lamina propria-to-cartilage (LP:C) ratio was quantified histologically by three blinded reviewers. The relationship between rabbit weight and airway diameter was assessed, and inter-rater reliability was calculated using the intraclass correlation coefficient (ICC). ResultsTwelve of fourteen rabbits reached the POD21 endpoint; two were euthanized early for severe airway compromise meeting humane endpoint criteria, both with approximately 80% stenosis. Injured tracheas demonstrated significantly greater stenosis than native controls (66.0 {+/-} 13.0% vs 16.0 {+/-} 2.7%; p = 0.00012), with a corresponding increase in LP:C ratio (p = 0.031). Airway diameter-matched injury produced significantly greater stenosis than non-matched injury (74.6 {+/-} 6.1% vs 50.6 {+/-} 4.0%; p = 0.001), while LP:C ratio did not differ between injury techniques (p = 1.0). Rabbit weight did not correlate with airway diameter (r = 0.176, p = 0.515; R2 = 0.031). Inter-rater reliability was excellent for both stenosis degree (ICC = 0.989) and LP:C ratio (ICC = 0.992). ConclusionsDirect measurement and matching of injury instrument diameter to native airway diameter substantially improves both the severity and the reproducibility of stenosis in a rabbit tracheal injury model, whereas body weight is an unreliable surrogate for airway size. This optimized, standardized protocol offers a reproducible platform for future translational studies of airway fibrosis and anti-fibrotic or regenerative therapies.

cell biology↗

Engineering a pacemaker-driven human mini-heart guided by spatial multi-omics of sinoatrial node development

The human sinoatrial node (SAN) functions as the primary pacemaker of the heart and coordinates the hierarchical electrical activity that drives cardiac contraction. However, experimental systems capable of reconstructing pacemaker-driven cardiac organization in human tissues remain limited. Here we integrate spatial multi-omics of the human fetal SAN with stem-cell engineering to generate pacemaker organoids ("Sinoids") and assemble them into a pacemaker-driven human mini-heart composed of sinoatrial, atrial and ventricular cardiac modules. High-resolution spatial transcriptomics and single-nucleus multi-omic analyses of human fetal SAN tissues identify regulatory pathways guiding pacemaker lineage specification, which we leverage to engineer human pluripotent stem cell-derived SAN organoids with robust pacemaker identity and electrophysiological activity. When integrated with atrial and ventricular cardioids, Sinoids initiate and coordinate electrical activation across assembled cardiac tissues, establishing directional propagation of electrophysiological signals within structured mini-heart organoids. Combining AI-guided perturbation modeling with functional validation further identifies conserved regulatory pathways controlling pacemaker specification and regionalization, including YAP-TEAD and NRG-ERBB signaling. Together, these results establish a multi-omic-guided strategy for engineering pacemaker tissues and reconstructing cardiac conduction hierarchy in vitro. The pacemaker-driven mini-heart platform provides a modular human cardiac system for studying pacemaker biology, modeling arrhythmia mechanisms and enabling electrophysiological drug discovery.

bioinformatics↗

Social Disconnection in the Brain: Loneliness and Age across Networks using Graph Theory

Loneliness, conceptualized as a multi-dimensional construct of unmet social needs, has been linked to adverse health outcomes across the lifespan, prompting significant interest in its underlying neural processes. Our study aimed to address the limitations of prior neuroimaging studies of loneliness by leveraging the Lifespan Human Connectome Project Aging dataset and applying graph theory to characterize its relationship with age and resting-state brain network organization. Socio-demographic measures confirmed prior work that higher loneliness was associated with younger age, being male, unmarried, and living alone. While loneliness showed no main effects on neural graph measures, a significant interaction between loneliness and age emerged for the local interconnectivity of the Default Model and Frontoparietal networks after adjusting for key socio-demographic factors. Conversely, older age was associated with lower functional connectivity, reduced global efficiency, and less modular brain network organization. Different graph measures showed distinct age-related associations, highlighting the heterogeneous nature of brain aging. The absence of a main effect of loneliness, while unexpected, underscores the complex, subjective nature of loneliness and suggests that its neural correlates may manifest differently across ages.

neuroscience↗

Phenotypes and Cellular Mechanics of Primary Human Aorta- and Pluripotent Stem Cell-derived Vascular Smooth Muscle Cells

AimsMature, contractile vascular smooth muscle cells (vSMC) in the medial layer of muscular blood vessels can de-differentiate into synthetic vSMC, while human pluripotent stem cells (hPSC) differentiate into proliferating, fetal-like vSMC that can be induced to form contractile vSMC. Our aim was to define the mechanical properties of well-characterized in vitro-differentiated vSMC compared to vSMC derived from adult human aorta (AoSMC). Methods and ResultsWe generated paraxial mesoderm (PM)-derived synthetic and contractile vSMC from hPSC lines, and we obtained proliferating AoSMC. Immature, synthetic hPSC-vSMC are proliferative, sensitive to contact inhibition and exhibit phenotype switching. Contractile hPSC-vSMCs are non-proliferative, have elevated levels of contractile proteins, and can undergo phenotype switching in vitro into a proliferative form. Immunostaining of contractile proteins present in vSMCs (CNN1, MYH11, TAGLN, ACTA2) display similar cell-to-cell heterogeneities between AoSMC and hPSC-vSMCs. The fluorescent signals are relatively uniform at sub-confluency, but highly variable at confluency. Human PSC-vSMC maintain doubling rates more readily and are more easily enriched using lactate medium than AoSMC. The single cell mechanics of AoSMC and hPSC-vSMC, assessed using micropost array detectors (mPADs), are highly comparable and have an average force production per micropost in the nN range. When cultivated to form humanized smooth muscle cell microtissues (SMTs), developed forces normalized to cell numbers are similar to that seen using microarray post detectors. Maximum developed forces in these tissues could be measured in response to endothelin-1. ConclusionsThese results demonstrate that synthetic hPSC-vSMCs and proliferating AoSMCs are highly comparable, and mechanically similar, but results with hPSC-vSMC are more reproducible. This study establishes the hPSC-vSMCs as a reproducible system to study how changes in cellular mechanics may contribute to development and disease. Translational PerspectiveThe mechanical properties and responses of lineage-specific human vSMC undergoing phenotype switching during perinatal development and vascular pathologies remain poorly understood. We have employed paraxial-mesoderm derived hPSC-vSMC, which recapitulate phenotypic properties of AoSMC, to define the mechanical properties of synthetic and contractile vSMCs. We quantitatively measured the forces of individual vSMC and of multicellular vSMC constructs. By defining the mechanical changes of normal vSMCs to phenotype switching, this validated, adaptable system advance studies of vSMC mechanics that may contribute to vascular pathologies in patients.

cell biology↗

Leveraging AI for facioscapulohumeral muscular dystrophy prediction and omics biomarker identification

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscle disorder characterized by a complex genetic etiology, variable prognosis, and a lack of effective therapies. Previous studies have identified candidate protein and miRNA biomarkers using various profiling techniques, underscoring their potential for monitoring FSHD, assessing prognosis, and evaluating pharmacodynamic responses. However, the feasibility of applying machine learning (ML) models to predict FSHD using these molecular signatures has not been explored. In this study, we developed ML models to predict FSHD using a multi-omics dataset comprising protein abundance and miRNA expression profiles. Key predictive features were identified using Random Forest and the Support Vector Machine-Recursive Feature Elimination (SVM-RFE) methods. Performance evaluations demonstrated the robustness of the ML classifiers, with logistic regression consistently achieving the robust predictive accuracy in distinguishing FSHD from healthy conditions. Additionally, we assessed the predictive power of the identified features by comparing them with biomarker sets reported in previous studies. Our findings highlight the potential of AI to improve prediction accuracy and facilitate the cost- and time-efficient strategy for identifying FSHD biomarker candidates, even with limited sample sizes in the context of rare diseases.

bioinformatics↗

Generation of Region-specific Airway Basal Stem Cells from Human Pluripotent Stem Cells via Regulation of BMP-NOGGIN Axis

Basal cells (BCs) are the primary stem cell population of adult human airways and a key target for hPSC-based models of airway development, disease and regenerative medicine. Recent studies have revealed substantial regional differences between human proximal and distal airway cell types, including BCs. Here, we show that the NOGGIN-BMP signaling axis governs proximal-distal patterning of hPSC-derived lung progenitors leading to the generation of region-specific induced BCs (iBCs). Continuous BMP inhibition through NOGGIN, coupled with tapered WNT activation, generates proximal iBCs that molecularly and functionally resemble human proximal airway BCs and differentiate in vitro and in vivo into the full repertoire of specialized proximal airway cell types, including ionocytes and pulmonary neuroendocrine cells. In sharp contrast, BMP activation with tapered WNT generates distal airway-like cells and distal BCs with limited ionocyte differentiation potential. Progeny of proximal iBCs derived from G551D CFTR mutant hiPSCs also recapitulate a cystic fibrosis-associated ionocyte phenotype.

cell biology↗

Blocking apoptosis promotes survival and alters developmental dynamics of human retinal ganglion cells in retinal organoids

Retinal ganglion cells (RGCs) are the projection neurons that transmit visual information from the retina to the brain. In many species, a substantial proportion of RGCs are eliminated by programmed cell death during development to regulate their final number, but how cell death impacts human RGC development remains poorly understood. Here, we characterized the timing and cell-type-specificity of cell death in human fetal retinas and retinal organoids. Both retinas and organoids exhibited two waves of apoptosis: an early wave targeting neurogenic retinal progenitor cells and neuronal precursors, and a late wave affecting RGCs and other neurons. Additionally, organoids displayed a distinct wave of necrosis. To investigate how the apoptotic waves affect retinal development, we differentiated human BAX/BAK double mutant organoids deficient in apoptosis. In these mutants, RGC lifespan and survival increased, while RGC neurogenesis and maturation were delayed. Thus, developmental apoptosis controls not only the quantity of RGCs but also their developmental dynamics. Together, our results highlight the roles of apoptosis in human RGC development and the challenges in retinal organoid design. Addressing these limitations will improve the utility of organoids for studying human retinal development and modeling optic neuropathies like glaucoma.

developmental biology↗

Inactivation of the Fusobacterium nucleatum Rnf complex reduces FadA-mediated amyloid formation and tumor development

The Gram-negative anaerobe Fusobacterium nucleatum is an oral oncobacterium that promotes colorectal cancer (CRC) development with the amyloid-forming cell surface adhesin FadA integral to CRC tumorigenesis. We describe here molecular genetic studies uncovering a novel mode of metabolic regulation of FadA-mediated tumor formation by a highly conserved respiratory enzyme known as the Rnf complex. First, we show that genetic disruption of Rnf, via rnfC deletion, significantly reduces the level of fadA transcript, accompanied by a near-complete abolishment of the precursor form of FadA (pFadA), reduced assembly of FadA at the mature cell pole, and severe defects in the osmotic stress-induced formation of FadA amyloids. We show further that the Rnf complex regulates three response regulators (CarR, ArlR, and S1), which modulate the expression of pFadA, without affecting fadA transcript. Consistent with our hypothesis that these response regulators control factors that process FadA, deletion of rnfC, carR, arlR, or s1 each impairs expression of the signal peptidase gene lepB, and FadA production is nearly abolished by CRISPR-induced depletion of lepB. Importantly, while rnfC deletion does not affect the ability of the mutant cells to adhere to CRC cells, rnfC deficiency significantly diminishes the fusobacterial invasion of CRC cells and formation of spheroid tumors in vitro. Evidently, the Rnf complex modulates the expression of the FadA adhesin and tumorigenesis through a gene regulatory network consisting of multiple response regulators, each controlling a signal peptidase that is critical for the post-translational processing of FadA and surface assembly of FadA amyloids. IMPORTANCEThe Rhodobacter nitrogen-fixation (Rnf) complex of Fusobacterium nucleatum plays an important role in the pathophysiology of this oral pathobiont, since genetic disruption of this conserved respiratory enzyme negatively impacts a wide range of metabolic pathways, as well as bacterial virulence in mice. Nonetheless, how Rnf deficiency weakens the virulence potential of F. nucleatum is not well understood. Here, we show that genetic disruption of the Rnf complex reduces surface assembly of adhesin FadA and FadA-mediated amyloid formation, via regulation of signal peptidase LepB by multiple response regulators. As FadA is critical in the carcinogenesis of colorectal cancer (CRC), the ability to invade CRC cells and promote spheroid tumor growth is strongly diminished in an Rnf-deficient mutant. Thus, this work uncovers a molecular linkage between the Rnf complex and LepB-regulated processing of FadA - likely via metabolic signaling - that maintains the virulence potential of this oncobacterium in various cellular niches.

microbiology↗

Proteomic profiling uncovers sexual dimorphism in the muscle response to wheel running exercise in the FLExDUX4 murine model of facioscapulohumeral muscular dystrophy

FLExDUX4 is a murine experimental model of facioscapulohumeral muscular dystrophy (FSHD) characterized by chronic, low levels of leaky expression of the human full-length double homeobox 4 gene (DUX4-fl). FLExDUX4 mice exhibit mild pathologies and functional deficits similar to people affected by FSHD. Proteomic studies in FSHD could offer new insights into disease mechanisms underpinned by post-transcriptional processes. We used mass spectrometry-based proteomics to quantify the abundance of 1322 proteins in triceps brachii muscle, encompassing both male and female mice in control and free voluntary wheel running (VWR) in Wild-type (n=3) and FLExDUX4 (n=3) genotypes. We report the triceps brachii proteome of FLExDUX4 mice recapitulates key skeletal muscle clinical characteristics of human FSHD, including alterations to mitochondria, RNA metabolism, oxidative stress, and apoptosis. RNA-binding proteins exhibit a sex-specific difference in FLExDUX4 mice. Sexual dimorphism of mitochondrial protein adaptation to exercise was uncovered specifically in FLExDUX4 mice, where females increased, but males decreased mitochondrial proteins after a 6-week of VWR. Our results highlight the importance of identifying sex-specific diagnostic biomarkers to enable more reliable monitoring of FSHD therapeutic targets. Our data provides a resource for the FSHD research community to explore the burgeoning aspect of sexual dimorphism in FSHD. In BriefNishimura et al. conducted proteomic analysis of triceps brachii muscle in the FLExDUX4 murine model of FSHD and verified FLExDUX4 mice recapitulate key skeletal muscle clinical characteristics of human FSHD, including disruptions to the mitochondrial proteome and proteins associated with RNA metabolism, oxidative stress, and apoptosis. RNA-binding proteins and the mitochondrial proteome response to exercise exhibited sexual dimorphism in FLExDUX4 mice. Specifically, females exhibited increases, whereas males exhibited decreases in mitochondrial protein abundance after 6-weeks voluntary wheel running. HighlightsO_LIFLExDUX4 muscle proteome mirrors pathophysiology of FSHD patient myoblasts C_LIO_LIMitochondrial proteins are more abundant in FLExDUX4 as compared to WT mice C_LIO_LISeverity of proteome disruption is greater in male than female mice C_LIO_LIRNA-binding proteins exhibit a sex-specific difference in FLExDUX4 mice C_LIO_LISex-specific mitochondrial proteome response to VWR in FLExDUX4 mice C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/639012v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1bf8a40org.highwire.dtl.DTLVardef@dad2borg.highwire.dtl.DTLVardef@1e402e7org.highwire.dtl.DTLVardef@7aad7c_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

Innovative Carnitine-Fed Rats Model Reveals Resveratrol Butyrate Ester's Multimechanistic Role in Reducing TMAO and Cardiovascular Risk

BACKGROUNDTrimethylamine-N-oxide (TMAO), a metabolite produced from dietary carnitine through gut microbiota, is a recognized risk factor for cardiovascular disease (CVD). High-fat diets and carnitine supplementation exacerbate TMAO levels and related risks, making them valuable in developing experimental models for studying CVD. Resveratrol butyrate ester (RBE) and its monomer ED4 have shown potential in reducing TMAO levels and improving cardiovascular outcomes through metabolic and microbial modulation, but their precise mechanisms remain unclear. METHODS AND RESULTSA novel animal model was established using 36 male Sprague-Dawley rats fed a high-fat diet supplemented with carnitine to elevate serum TMAO levels, simulating dietary-induced CVD risks. Rats were divided into six groups: control diet (CN), high-fat diet (HFD), high-fat diet with carnitine (HFDC), HFDC with dimethylbutanol (HFDCB), CN with ED4 (CNM), and HFDC with ED4 (HFDCM). Physiological parameters, serum lipid profiles, SCFA levels, microbiota composition, and gene expression (OCTN2 and FMO3) were analyzed. ED4 supplementation reduced serum TMAO levels by upregulating OCTN2 expression, promoting urinary TMAO excretion, and restoring SCFA levels. ED4 also modulated gut microbiota, reducing TMA-producing bacteria (e.g., Bacteroides), and improved cardiovascular markers, including reduced blood lipid levels and fat accumulation. While dimethylbutanol inhibited FMO3 expression to reduce TMAO, ED4 acted primarily through carnitine utilization and microbiota modulation. Both treatments enhanced urinary TMAO excretion and altered gut microbiota composition. CONCLUSIONSThis study introduced an innovative animal model combining high-fat and carnitine-enriched diets to study TMAO-related cardiovascular risks. ED4 demonstrated multimechanistic effects in reducing TMAO levels and CVD risk factors by modulating gut microbiota, restoring SCFA levels, and enhancing carnitine metabolism. These findings highlight ED4s therapeutic potential in cardiovascular protection and metabolic regulation. However, further research is needed to elucidate the molecular pathways underlying ED4s effects on TMAO metabolism and its broader translational applications.

molecular biology↗

Ethanolamine-induced assembly of microcompartments is required for Fusobacterium nucleatum virulence

Many bacteria metabolize ethanolamine as a nutrient source through cytoplasmic organelles named bacterial microcompartments (BMCs). Here we investigated the molecular assembly, regulation, and function of BMCs in Fusobacterium nucleatum - a Gram-negative oral pathobiont that is associated with adverse pregnancy outcomes. The F. nucleatum genome harbors a conserved ethanolamine utilization (eut) locus with 21 genes that encode several putative BMC shell proteins and a two-component signal transduction system (TCS), in addition to the enzymes for ethanolamine transport and catabolism. We show that the expression of most of these genes as well as BMC formation is highly increased in wild type fusobacteria when cultured in the presence of ethanolamine as a nutrient source. Deletion of the response regulator EutV eliminated this induction of eut mRNAs and BMCs, thus demonstrating that BMC formation is transcriptionally regulated by the TCS EutV-EutW in response to ethanolamine. Mass spectrometry of isolated BMCs unveiled the identity of the constituent proteins EutL, EutM1, EutM2, and EutN. Consistent with the role of these proteins in BMC assembly and metabolism, deletion of eutN, eutL/eutM1/eutM2, or eutL/eutM1/eutM2/eutN not only affected BMC formation, but also ethanolamine utilization, causing cell growth defects with ethanolamine as nutrient. BMCs also assembled in fusobacteria cultured with placental cells or the culture media, a process that is dependent on the BMC shell proteins. Significantly, we show that the eutN mutant is defective in inducing preterm birth in a mouse model. Together, these results establish that BMC-mediated metabolism of ethanolamine is critical for fusobacterial virulence. IMPORTANCEThe oral anaerobe Fusobacterium nucleatum can spread to distal internal organs, such as the colon and placenta, and thereby promote the development of colorectal cancer and induce preterm birth, respectively. Yet, how this opportunistic pathogen adapts to the various metabolically distinct host cellular niches remains poorly understood. We demonstrated here that this microbe assembles specialized metabolic organelles, termed bacterial microcompartments (BMCs), to utilize environmental ethanolamine (EA) as a key environmental nutrient source. The formation of F. nucleatum BMCs, containing BMC shell proteins EutLM1M2N, is controlled by a two-component system, EutV-EutW, responsive to EA. Significantly, this ability of F. nucleatum to form BMCs in response to EA is crucial for its pathogenicity evidenced by the fact that the genetic disruption of BMC formation reduces fusobacterial virulence in a mouse model of preterm birth.

microbiology↗

Combination of mitomycin C and low-dose metronidazole synergistically against Clostridioides difficile infection and recurrence prevention

Clostridioides difficile is an anaerobic, spore-forming, Gram-positive pathogen responsible for various conditions from mild diarrhea to severe toxic megacolon and potentially death. Current treatments for C. difficile infection (CDI) rely on antibiotics like vancomycin and metronidazole (MTZ); however, the high doses required often disrupt gut microbiota, leading to a high recurrence rate. Mitomycin C (MMC), a chemotherapy drug approved by the FDA, is known for inducing phage production in lysogenic bacterial strains, effectively targeting the host bacteria. Given that 70% of C. difficile strains harbor prophages, this study investigates MMCs potential to enhance antibiotic efficacy against CDI. Our in vitro experiments indicate that MMC acts synergistically with MTZ to inhibit the growth of C. difficile strain R20291. Furthermore, this combination decreases biofilm-resident vegetative cell resistance and reduces the MTZ concentration needed to kill C. difficile in stool samples ex vivo. In a CDI mouse relapse model, in vivo results show that MMC combined with a low dose of MTZ significantly improves survival rates and reduces fecal spore counts after antibiotic treatment. Overall, these findings suggest that a low-dose combination of MMC and MTZ offers enhanced therapeutic efficacy for CDI, both in vitro and in vivo, and may provide a promising new approach for treatment.

microbiology↗

Self-Organization of Sinusoidal Vessels in Pluripotent Stem Cell-derived Human Liver Bud Organoids

The induction of tissue-specific vessels in in vitro living tissue systems remains challenging. Here, we directly differentiated human pluripotent stem cells into CD32b+ putative liver sinusoidal progenitors (iLSEP) by dictating developmental pathways. By devising an inverted multilayered air-liquid interface (IMALI) culture, hepatic endoderm, septum mesenchyme, arterial and sinusoidal quadruple progenitors self-organized to generate and sustain hepatocyte-like cells neighbored by divergent endothelial subsets composed of CD32blowCD31high, LYVE1+STAB1+CD32bhighCD31lowTHBD-vWF-, and LYVE1-THBD+vWF+ cells. Wnt2 mediated sinusoidal-to-hepatic intercellular crosstalk potentiates hepatocyte differentiation and branched endothelial network formation. Intravital imaging revealed iLSEP developed fully patent human vessels with functional sinusoid-like features. Organoid-derived hepatocyte- and sinusoid-derived coagulation factors enabled correction of in vitro clotting time with Factor V, VIII, IX, and XI deficient patients plasma and rescued the severe bleeding phenotype in hemophilia A mice upon transplantation. Advanced organoid vascularization technology allows for interrogating key insights governing organ-specific vessel development, paving the way for coagulation disorder therapeutics.

developmental biology↗

Deciphering Endothelial and Mesenchymal Organ Specification in Vascularized Lung and Intestinal Organoids

To investigate the co-development of vasculature, mesenchyme, and epithelium crucial for organogenesis and the acquisition of organ-specific characteristics, we constructed a human pluripotent stem cell-derived organoid system comprising lung or intestinal epithelium surrounded by organotypic mesenchyme and vasculature. We demonstrated the pivotal role of co-differentiating mesoderm and endoderm via precise BMP regulation in generating multilineage organoids and gut tube patterning. Single-cell RNA-seq analysis revealed organ specificity in endothelium and mesenchyme, and uncovered key ligands driving endothelial specification in the lung (e.g., WNT2B and Semaphorins) or intestine (e.g., GDF15). Upon transplantation under the kidney capsule in mice, these organoids further matured and developed perfusable human-specific sub-epithelial capillaries. Additionally, our model recapitulated the abnormal endothelial-epithelial crosstalk in patients with FOXF1 deletion or mutations. Multilineage organoids provide a unique platform to study developmental cues guiding endothelial and mesenchymal cell fate determination, and investigate intricate cell-cell communications in human organogenesis and disease. HighlightsO_LIBMP signaling fine-tunes the co-differentiation of mesoderm and endoderm. C_LIO_LIThe cellular composition in multilineage organoids resembles that of human fetal organs. C_LIO_LIMesenchyme and endothelium co-developed within the organoids adopt organ-specific characteristics. C_LIO_LIMultilineage organoids recapitulate abnormal endothelial-epithelial crosstalk in FOXF1-associated disorders. C_LI

developmental biology↗

Olfactory ensheathing cells are hybrid glial cells that promote neural repair

Olfactory ensheathing cells (OECs) are unique glial cells found in both central and peripheral nervous systems where they support continuous axonal outgrowth of olfactory sensory neurons to their targets. Previously we reported that following severe spinal cord injury, OECs transplanted near the injury site modify the inhibitory glial scar and facilitate axon regeneration past the scar border and into the lesion. To better understand the mechanisms underlying the reparative properties of OECs, we used single-cell RNA-sequencing of OECs from adult rats to study their gene expression programs. Our analyses revealed five diverse OEC subtypes, each expressing novel marker genes and pathways indicative of progenitor, axonal regeneration, secreted molecules, or microglia-like functions. We found substantial overlap of OEC genes with those of Schwann cells, but also with microglia, astrocytes, and oligodendrocytes. We confirmed established markers on cultured OECs, and localized select top genes of OEC subtypes in olfactory bulb tissue. We also show that OECs secrete Reelin and Connective tissue growth factor, extracellular matrix molecules which are important for neural repair and axonal outgrowth. Our results support that OECs are a unique hybrid glia, some with progenitor characteristics, and that their gene expression patterns indicate functions related to wound healing, injury repair and axonal regeneration.

neuroscience↗

Engraftment and injury repair in regionally conditioned rat lung in vivo by lung progenitors derived from human pluripotent stem cells.

Although lung disease is a major cause of mortality, the mechanisms involved in human lung regeneration are unclear because of the lack of experimental models. Here we report a novel model where human pluripotent stem cell-derived expandable cell lines sharing features of airway secretory and basal cells engraft in the distal rat lung after conditioning by locoregional de-epithelialization followed by irradiation and immunosuppression. The engrafting cells, which we named distal lung epithelial progenitors (DLEPs), contributed to alveolar epithelial cells and generated KRT5-pods, structures involved in distal lung repair after severe injury, but only rarely to distal airways. Most strikingly, however, injury induced by the conditioning regimen was largely prevented by the engrafting DLEPs. The approach described here provides a model to study mechanisms involved in human lung regeneration, and potentially lays the foundation for the preclinical development of cell therapy to treat lung injury and disease.

cell biology↗

An ancient respiratory enzyme metabolically modulates Fusobacterium nucleatum virulence

A prominent oral commensal and opportunistic pathogen, Fusobacterium nucleatum can traverse to extra-oral sites such as placenta and colon, promoting adverse pregnancy outcomes and colorectal cancer, respectively. How this anaerobe sustains many metabolically changing environments enabling its virulence potential remains unclear. Informed by our genome-wide transposon mutagenesis, we report here that the highly conserved Rnf complex, encoded by the rnfCDGEAB gene cluster, is key to fusobacterial metabolic adaptation and virulence. Genetic disruption of the Rnf complex via non-polar, in-frame deletion of rnfC ({Delta}rnfC) abrogates polymicrobial interaction (or coaggregation) associated with adhesin RadD and biofilm formation. The defect in coaggregation is not due to reduced cell surface of RadD, but rather an increased level of extracellular lysine, which binds RadD and inhibits coaggregation. Indeed, removal of extracellular lysine via washing {Delta}rnfC cells restores coaggregation, while addition of lysine inhibits this process. These phenotypes mirror that of a mutant ({Delta}kamA) that fails to metabolize extracellular lysine. Strikingly, the {Delta}rnfC mutant is defective in ATP production, cell growth, cell morphology, and expression of the enzyme MegL that produces hydrogen sulfide from cysteine. Targeted metabolic profiling demonstrated that catabolism of many amino acids, including histidine and lysine, is altered in {Delta}rnfC cells, thereby reducing production of ATP and metabolites including H2S and butyrate. Most importantly, we show that the {Delta}rnfC mutant is severely attenuated in a mouse model of preterm birth. The indispensable function of Rnf complex in fusobacterial pathogenesis via modulation of bacterial metabolism makes it an attractive target for developing therapeutic intervention. IMPORTANCEAlthough viewed as an oral commensal, the Gram-negative F. nucleatum is an opportunistic pathogen that can spread to extra-oral sites such as placenta and colon, promoting adverse pregnancy outcomes and colorectal cancer, respectively. How this anaerobe sustains various metabolically changing environments enabling its virulence potential remains unclear. We demonstrate here that the highly conserved Rnf complex is key to fusobacterial metabolic adaptation and virulence. Genetic disruption of this Rnf complex causes global defects in polymicrobial interaction, biofilm formation, cell growth and morphology, H2S production, and ATP synthesis. Targeted metabolomic profiling demonstrates that the loss of this respiratory enzyme significantly diminishes catabolism of numerous amino acids, which negatively impacts fusobacterial virulence as tested in a preterm birth model in mice.

microbiology↗