Positive selection of somatically mutated clones identifies adaptive pathways in metabolic liver disease.

Somatic mutations in nonmalignant tissues accumulate with age and injury, but whether these mutations are adaptive on the cellular or organismal levels is unclear. To interrogate genes in human metabolic disease, we performed lineage tracing in mice harboring somatic mosaicism subjected to nonalcoholic steatohepatitis (NASH). Proof-of-concept studies with mosaic loss of Mboat7, a membrane lipid acyltransferase, showed that increased steatosis accelerated clonal disappearance. Next, we induced pooled mosaicism in 63 known NASH genes, allowing us to trace mutant clones side by side. This in vivo tracing platform, which we coined MOSAICS, selected for mutations that ameliorate lipotoxicity, including mutant genes identified in human NASH. To prioritize new genes, additional screening of 472 candidates identified 23 somatic perturbations that promoted clonal expansion. In validation studies, liver-wide deletion of Tbx3, Bcl6, or Smyd2 resulted in protection against hepatic steatosis. Selection for clonal fitness in mouse and human livers identifies pathways that regulate metabolic disease.

Sprod for de-noising spatially resolved transcriptomics data based on position and image information.

Spatially resolved transcriptomics (SRT) provide gene expression close to, or even superior to, single-cell resolution while retaining the physical locations of sequencing and often also providing matched pathology images. However, SRT expression data suffer from high noise levels, due to the shallow coverage in each sequencing unit and the extra experimental steps required to preserve the locations of sequencing. Fortunately, such noise can be removed by leveraging information from the physical locations of sequencing, and the tissue organization reflected in corresponding pathology images. In this work, we developed Sprod, based on latent graph learning of matched location and imaging data, to impute accurate SRT gene expression. We validated Sprod comprehensively and demonstrated its advantages over previous methods for removing drop-outs in single-cell RNA-sequencing data. We showed that, after imputation by Sprod, differential expression analyses, pathway enrichment and cell-to-cell interaction inferences are more accurate. Overall, we envision de-noising by Sprod to become a key first step towards empowering SRT technologies for biomedical discoveries.

Netie: inferring the evolution of neoantigen-T cell interactions in tumors.

Neoantigens are the key targets of antitumor immune responses from cytotoxic T cells and play a critical role in affecting tumor progressions and immunotherapy treatment responses. However, little is known about how the interaction between neoantigens and T cells ultimately affects the evolution of cancerous masses. Here, we develop a hierarchical Bayesian model, named neoantigen-T cell interaction estimation (netie) to infer the history of neoantigen-CD8+ T cell interactions in tumors. Netie was systematically validated and applied to examine the molecular patterns of 3,219 tumors, compiled from a panel of 18 cancer types. We showed that tumors with an increase in immune selection pressure over time are associated with T cells that have an activation-related expression signature. We also identified a subset of exhausted cytotoxic T cells postimmunotherapy associated with tumor clones that newly arise after treatment. These analyses demonstrate how netie enables the interrogation of the relationship between individual neoantigen repertoires and the tumor molecular profiles. We found that a T cell inflammation gene expression profile (TIGEP) is more predictive of patient outcomes in the tumors with an increase in immune pressure over time, which reveals a curious synergy between T cells and neoantigen distributions. Overall, we provide a new tool that is capable of revealing the imprints left by neoantigens during each tumor's developmental process and of predicting how tumors will progress under further pressure of the host's immune system.

Pkd1l1-deficiency drives biliary atresia through ciliary dysfunction in biliary epithelial cells.

BACKGROUND & AIMS: Syndromic biliary atresia is a cholangiopathy characterized by fibro-obliterative changes in the extrahepatic bile duct (EHBD) and congenital malformations including laterality defects. The etiology remains elusive and faithful animal models are lacking. Genetic syndromes provide important clues regarding the pathogenic mechanisms underlying the disease. We investigated the role of the gene Pkd1l1 in the pathophysiology of syndromic biliary atresia. METHODS: Constitutive and conditional Pkd1l1 knockout mice were generated to explore genetic pathology as a cause of syndromic biliary atresia. We investigated congenital malformations, EHBD and liver pathology, EHBD gene expression, and biliary epithelial cell turnover. Biliary drainage was functionally assessed with cholangiography. Histology and serum chemistries were assessed after DDC (3,5-diethoxycarbony l-1,4-dihydrocollidine) diet treatment and inhibition of the ciliary signaling effector GLI1. RESULTS: Pkd1l1-deficient mice exhibited congenital anomalies including malrotation and heterotaxy. Pkd1l1-deficient EHBDs were hypertrophic and fibrotic. Pkd1l1-deficient EHBDs were patent but displayed delayed biliary drainage. Pkd1l1-deficient livers exhibited ductular reaction and periportal fibrosis. After DDC treatment, Pkd1l1-deficient mice exhibited EHBD obstruction and advanced liver fibrosis. Pkd1l1-deficient mice had increased expression of fibrosis and extracellular matrix remodeling genes (Tgfα, Cdkn1a, Hb-egf, Fgfr3, Pdgfc, Mmp12, and Mmp15) and decreased expression of genes mediating ciliary signaling (Gli1, Gli2, Ptch1, and Ptch2). Primary cilia were reduced on biliary epithelial cells and altered expression of ciliogenesis genes occurred in Pkd1l1-deficient mice. Small molecule inhibition of the ciliary signaling effector GLI1 with Gant61 recapitulated Pkd1l1-deficiency. CONCLUSIONS: Pkd1l1 loss causes both laterality defects and fibro-proliferative EHBD transformation through disrupted ciliary signaling, phenocopying syndromic biliary atresia. Pkd1l1-deficient mice function as an authentic genetic model for study of the pathogenesis of biliary atresia. IMPACT AND IMPLICATIONS: The syndromic form of biliary atresia is characterized by fibro-obliteration of extrahepatic bile ducts and is often accompanied by laterality defects. The etiology is unknown, but Pkd1l1 was identified as a potential genetic candidate for syndromic biliary atresia. We found that loss of the ciliary gene Pkd1l1 contributes to hepatobiliary pathology in biliary atresia, exhibited by bile duct hypertrophy, reduced biliary drainage, and liver fibrosis in Pkd1l1-deficient mice. Pkd1l1-deficient mice serve as a genetic model of biliary atresia and reveal ciliopathy as an etiology of biliary atresia. This model will help scientists uncover new therapeutic approaches for patients with biliary atresia, while pediatric hepatologists should validate the diagnostic utility of PKD1L1 variants.

Spatial molecular profiling: platforms, applications and analysis tools.

Molecular profiling technologies, such as genome sequencing and proteomics, have transformed biomedical research, but most such technologies require tissue dissociation, which leads to loss of tissue morphology and spatial information. Recent developments in spatial molecular profiling technologies have enabled the comprehensive molecular characterization of cells while keeping their spatial and morphological contexts intact. Molecular profiling data generate deep characterizations of the genetic, transcriptional and proteomic events of cells, while tissue images capture the spatial locations, organizations and interactions of the cells together with their morphology features. These data, together with cell and tissue imaging data, provide unprecedented opportunities to study tissue heterogeneity and cell spatial organization. This review aims to provide an overview of these recent developments in spatial molecular profiling technologies and the corresponding computational methods developed for analyzing such data.

Unsupervised gene expression analyses identify IPF-severity correlated signatures, associated genes and biomarkers.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a fatal fibrotic lung disease occurring predominantly in middle-aged and older adults. The traditional diagnostic classification of IPF is based on clinical, radiological, and histopathological features. However, the considerable heterogeneity in IPF presentation suggests that differences in gene expression profiles can help to characterize and distinguish disease severity. METHODS: We used data-driven unsupervised clustering analysis, combined with a knowledge-based approach to identify and characterize IPF subgroups. RESULTS: Using transcriptional profiles on lung tissue from 131 patients with IPF/UIP and 12 non-diseased controls, we identified six subgroups of IPF that generally correlated with the disease severity and lung function decline. Network-informed clustering identified the most severe subgroup of IPF that was enriched with genes regulating inflammatory processes, blood pressure and branching morphogenesis of the lung. The differentially expressed genes in six subgroups of IPF compared to healthy control include transcripts of extracellular matrix, epithelial-mesenchymal cell cross-talk, calcium ion homeostasis, and oxygen transport. Further, we compiled differentially expressed gene signatures to identify unique gene clusters that can segregate IPF from normal, and severe from mild IPF. Additional validations of these signatures were carried out in three independent cohorts of IPF/UIP. Finally, using knowledge-based approaches, we identified several novel candidate genes which may also serve as potential biomarkers of IPF. CONCLUSIONS: Discovery of unique and redundant gene signatures for subgroups in IPF can be greatly facilitated through unsupervised clustering. Findings derived from such gene signatures may provide insights into pathogenesis of IPF and facilitate the development of clinically useful biomarkers.

A Single-Short Partial Reprogramming of the Endothelial Cells decreases Blood Pressure via attenuation of EndMT in Hypertensive Mice.

Background: Small artery remodeling and endothelial dysfunction are hallmarks of hypertension. Growing evidence supports a likely causal association between cardiovascular diseases and the presence of endothelial-to-mesenchymal transition (EndMT), a cellular transdifferentiation process in which endothelial cells (ECs) partially lose their identity and acquire additional mesenchymal phenotypes. EC reprogramming represents an innovative strategy in regenerative medicine to prevent deleterious effects induced by cardiovascular diseases. Methods: Using a partial reprogramming of ECs, via overexpression of Oct-3/4, Sox-2, and Klf-4 (OSK) transcription factors, we aimed to bring ECs back to a youthful phenotype in hypertensive mice. Primary ECs were infected with lentiviral vectors (LV) containing the specific EC marker cadherin 5 (Cdh5) and the fluorescent reporter enhanced green fluorescence protein (EGFP) with empty vector (LVCO) or with OSK (LV-OSK). Confocal microscopy and western blotting analysis were used to confirm the OSK overexpression. Cellular migration, senescence, and apoptosis were evaluated. Human aortic ECs (HAoECs) from male and female normotensive and hypertensive patients were analyzed after OSK or control treatments for their endothelial nitric oxide synthase (eNOS) levels, nitric oxide (NO), and genetic profile. Male and female normotensive (BPN/3J) and hypertensive (BPH/2J) mice were treated with an intravenous (i.v.) injection of LVCO or LV-OSK and evaluated 10 days post-infection. The blood pressure, cardiac function, vascular reactivity of small arteries, in vivo EGFP signal and EndMT inhibition were analyzed. Results: OSK overexpression induced partial EC reprogramming in vitro , and these cells showed endothelial progenitor cell (EPC)-like features with lower migratory capability. OSK treatment of hypertensive BPH/2J mice normalized blood pressure and resistance arteries hypercontractility, via the attenuation of EndMT and elastin breaks. EGFP signal was detected in vivo in the prefrontal cortex of both BPN/3J and BPH/2J-treated mice, but OSK induced angiogenesis only in male BPN/3J mice. OSK-treated human ECs from hypertensive patients showed high eNOS activation and NO production, with low ROS formation. Single-cell RNA analysis showed that OSK alleviated EC senescence and EndMT, restoring their phenotypes in human ECs from hypertensive patients. Conclusion: Overall, these data indicate that OSK treatment and EC reprogramming can decrease blood pressure and reverse hypertension-induced vascular damage.

UBE2J1 is the E2 ubiquitin-conjugating enzyme regulating androgen receptor degradation and antiandrogen resistance.

Prostate cancer (PCa) is primarily driven by aberrant Androgen Receptor (AR) signaling. Although there has been substantial advancement in antiandrogen therapies, resistance to these treatments remains a significant obstacle, often marked by continuous or enhanced AR signaling in resistant tumors. While the dysregulation of the ubiquitination-based protein degradation process is instrumental in the accumulation of oncogenic proteins, including AR, the molecular mechanism of ubiquitination-driven AR degradation remains largely undefined. We identified UBE2J1 as the critical E2 ubiquitin-conjugating enzyme responsible for guiding AR ubiquitination and eventual degradation. The absence of UBE2J1, found in 5-15% of PCa patients, results in disrupted AR ubiquitination and degradation. This disruption leads to an accumulation of AR proteins, promoting resistance to antiandrogen treatments. By employing a ubiquitination-based AR degrader to adeptly restore AR ubiquitination, we reestablished AR degradation and inhibited the proliferation of antiandrogen-resistant PCa tumors. These findings underscore the fundamental role of UBE2J1 in AR degradation and illuminate an uncharted mechanism through which PCa maintains heightened AR protein levels, fostering resistance to antiandrogen therapies.

PKD1 mutant clones within cirrhotic livers inhibit steatohepatitis without promoting cancer.

Somatic mutations in non-malignant tissues are selected for because they confer increased clonal fitness. However, it is uncertain whether these clones can benefit organ health. Here, ultra-deep targeted sequencing of 150 liver samples from 30 chronic liver disease patients revealed recurrent somatic mutations. PKD1 mutations were observed in 30% of patients, whereas they were only detected in 1.3% of hepatocellular carcinomas (HCCs). To interrogate tumor suppressor functionality, we perturbed PKD1 in two HCC cell lines and six in vivo models, in some cases showing that PKD1 loss protected against HCC, but in most cases showing no impact. However, Pkd1 haploinsufficiency accelerated regeneration after partial hepatectomy. We tested Pkd1 in fatty liver disease, showing that Pkd1 loss was protective against steatosis and glucose intolerance. Mechanistically, Pkd1 loss selectively increased mTOR signaling without SREBP-1c activation. In summary, PKD1 mutations exert adaptive functionality on the organ level without increasing transformation risk.

Mapping Cellular Interactions from Spatially Resolved Transcriptomics Data.

Cell-cell communication (CCC) is essential to how life forms and functions. However, accurate, high-throughput mapping of how expression of all genes in one cell affects expression of all genes in another cell is made possible only recently, through the introduction of spatially resolved transcriptomics technologies (SRTs), especially those that achieve single cell resolution. However, significant challenges remain to analyze such highly complex data properly. Here, we introduce a Bayesian multi-instance learning framework, spacia, to detect CCCs from data generated by SRTs, by uniquely exploiting their spatial modality. We highlight spacia's power to overcome fundamental limitations of popular analytical tools for inference of CCCs, including losing single-cell resolution, limited to ligand-receptor relationships and prior interaction databases, high false positive rates, and most importantly the lack of consideration of the multiple-sender-to-one-receiver paradigm. We evaluated the fitness of spacia for all three commercialized single cell resolution ST technologies: MERSCOPE/Vizgen, CosMx/Nanostring, and Xenium/10X. Spacia unveiled how endothelial cells, fibroblasts and B cells in the tumor microenvironment contribute to Epithelial-Mesenchymal Transition and lineage plasticity in prostate cancer cells. We deployed spacia in a set of pan-cancer datasets and showed that B cells also participate in PDL1/PD1 signaling in tumors. We demonstrated that a CD8+ T cell/PDL1 effectiveness signature derived from spacia analyses is associated with patient survival and response to immune checkpoint inhibitor treatments in 3,354 patients. We revealed differential spatial interaction patterns between γδ T cells and liver hepatocytes in healthy and cancerous contexts. Overall, spacia represents a notable step in advancing quantitative theories of cellular communications.

PRMT5 is an actionable therapeutic target in CDK4/6 inhibitor-resistant ER+/RB-deficient breast cancer.

CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with estrogen receptor-positive (ER+) breast cancer. However, patients treated with CDK4/6i eventually develop drug resistance and progress. RB1 loss-of-function alterations confer resistance to CDK4/6i, but the optimal therapy for these patients is unclear. Through a genome-wide CRISPR screen, we identify protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in ER+/RB1-knockout breast cancer cells. Inhibition of PRMT5 blocks the G1-to-S transition in the cell cycle independent of RB, leading to growth arrest in RB1-knockout cells. Proteomics analysis uncovers fused in sarcoma (FUS) as a downstream effector of PRMT5. Inhibition of PRMT5 results in dissociation of FUS from RNA polymerase II, leading to hyperphosphorylation of serine 2 in RNA polymerase II, intron retention, and subsequent downregulation of proteins involved in DNA synthesis. Furthermore, treatment with the PRMT5 inhibitor pemrametostat and a selective ER degrader fulvestrant synergistically inhibits growth of ER+/RB-deficient cell-derived and patient-derived xenografts. These findings highlight dual ER and PRMT5 blockade as a potential therapeutic strategy to overcome resistance to CDK4/6i in ER+/RB-deficient breast cancer.

Comparative genomics incorporating translocation renal cell carcinoma mouse model reveals molecular mechanisms of tumorigenesis.

Translocation renal cell carcinoma (tRCC) most commonly involves an ASPSCR1-TFE3 fusion, but molecular mechanisms remain elusive and animal models are lacking. Here, we show that human ASPSCR1-TFE3 driven by Pax8-Cre (a credentialed clear cell RCC driver) disrupted nephrogenesis and glomerular development, causing neonatal death, while the clear cell RCC failed driver, Sglt2-Cre, induced aggressive tRCC (as well as alveolar soft part sarcoma) with complete penetrance and short latency. However, in both contexts, ASPSCR1-TFE3 led to characteristic morphological cellular changes, loss of epithelial markers, and an epithelial-mesenchymal transition. Electron microscopy of tRCC tumors showed lysosome expansion, and functional studies revealed simultaneous activation of autophagy and mTORC1 pathways. Comparative genomic analyses encompassing an institutional human tRCC cohort (including a hitherto unreported SFPQ-TFEB fusion) and a variety of tumorgraft models (ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3, RBM10-TFE3, and MALAT1-TFEB) disclosed significant convergence in canonical pathways (cell cycle, lysosome, and mTORC1) and less established pathways such as Myc, E2F, and inflammation (IL-6/JAK/STAT3, interferon-γ, TLR signaling, systemic lupus, etc.). Therapeutic trials (adjusted for human drug exposures) showed antitumor activity of cabozantinib. Overall, this study provides insight into MiT/TFE-driven tumorigenesis, including the cell of origin, and characterizes diverse mouse models available for research.

ZNF397 Deficiency Triggers TET2-driven Lineage Plasticity and AR-Targeted Therapy Resistance in Prostate Cancer.

Cancer cells exhibit phenotypical plasticity and epigenetic reprogramming, which allows them to evade lineage-dependent targeted treatments by adopting lineage plasticity. The underlying mechanisms by which cancer cells exploit the epigenetic regulatory machinery to acquire lineage plasticity and therapy resistance remain poorly understood. We identified Zinc Finger Protein 397 (ZNF397) as a bona fide coactivator of the androgen receptor (AR), essential for the transcriptional program governing AR-driven luminal lineage. ZNF397 deficiency facilitates the transition of cancer cell from an AR-driven luminal lineage to a Ten-Eleven Translocation 2 (TET2)-driven lineage plastic state, ultimately promoting resistance to therapies inhibiting AR signaling. Intriguingly, our findings indicate that a TET2 inhibitor can eliminate the resistance to AR targeted therapies in ZNF397-deficient tumors. These insights uncover a novel mechanism through which prostate cancer acquires lineage plasticity via epigenetic rewiring and offer promising implications for clinical interventions designed to overcome therapy resistance dictated by lineage plasticity.

Image-based quantification of histological features as a function of spatial location using the Tissue Positioning System.

BACKGROUND: Tissues such as the liver lobule, kidney nephron, and intestinal gland exhibit intricate patterns of zonated gene expression corresponding to distinct cell types and functions. To quantitatively understand zonation, it is important to measure cellular or genetic features as a function of position along a zonal axis. While it is possible to manually count, characterize, and locate features in relation to the zonal axis, it is labor-intensive and difficult to do manually while maintaining precision and accuracy. METHODS: We addressed this challenge by developing a deep-learning-based quantification method called the "Tissue Positioning System" (TPS), which can automatically analyze zonation in the liver lobule as a model system. FINDINGS: By using algorithms that identified vessels, classified vessels, and segmented zones based on the relative position along the portal vein to central vein axis, TPS was able to spatially quantify gene expression in mice with zone specific reporters. INTERPRETATION: TPS could discern expression differences between zonal reporter strains, ages, and disease states. TPS could also reveal the zonal distribution of cells previously thought to be positioned randomly. The design principles of TPS could be generalized to other tissues to explore the biology of zonation. FUNDING: CPRIT (RP190208, RP220614, RP230330) and NIH (P30CA142543, R01AA028791, R01CA251928, R01DK1253961, R01GM140012, 1R01GM141519, 1R01DE030656, 1U01CA249245). The Pollack Foundation, Simmons Comprehensive Cancer Center Cancer & Obesity Translational Pilot Award, and the Emerging Leader Award from the Mark Foundation For Cancer Research (#21-003-ELA).

Perturbations of glutathione and sphingosine metabolites in Port Wine Birthmark patient-derived induced pluripotent stem cells.

Port Wine Birthmark (PWB) is a congenital vascular malformation in the skin, occurring in 1-3 per 1,000 live births. We recently generated PWB-derived induced pluripotent stem cells (iPSCs) as clinically relevant disease models. The metabolites associated with the pathological phenotypes of PWB-derived iPSCs are unknown, which we aimed to explore in this study. Metabolites were separated by ultra-performance liquid chromatography and were screened with electrospray ionization mass spectrometry. Orthogonal partial least-squares discriminant analysis, multivariate and univariate analysis were used to identify differential metabolites (DMs). KEGG analysis was used for the enrichment of metabolic pathways. A total of 339 metabolites were identified. There were 22 DMs confirmed with 9 downregulated DMs including sphingosine and 13 upregulated DMs including glutathione in PWB iPSCs as compared to controls. Pathway enrichment analysis confirmed the upregulation of glutathione and downregulation of sphingolipid metabolism in PWB-derived iPSCs as compared to normal ones. We next examined the expression patterns of the key factors associated with glutathione metabolism in PWB lesions. We found that hypoxia-inducible factor 1α (HIF1α), glutathione S-transferase Pi 1 (GSTP1), γ-glutamyl transferase 7 (GGT7), and glutamate cysteine ligase modulatory subunit (GCLM) were upregulated in PWB vasculatures as compared to blood vessels in normal skins. Our data demonstrate that there are perturbations in sphingolipid and cellular redox homeostasis in the PWB vasculature, which may facilitate cell survival and pathological progression. Our data imply that upregulation of glutathione may contribute to laser-resistant phenotypes in the PWB vasculature.

Perturbations of Glutathione and Sphingosine Metabolites in Port Wine Birthmark Patient-Derived Induced Pluripotent Stem Cells.

Port Wine Birthmarks (PWBs) are a congenital vascular malformation on the skin, occurring in 1-3 per 1000 live births. We have recently generated PWB-derived induced pluripotent stem cells (iPSCs) as clinically relevant disease models. The metabolites associated with the pathological phenotypes of PWB-derived iPSCs are unknown, and so we aim to explore them in this study. Metabolites were separated by ultra-performance liquid chromatography and screened with electrospray ionization mass spectrometry. Orthogonal partial least-squares discriminant, multivariate, and univariate analyses were used to identify differential metabolites (DMs). KEGG analysis was used to determine the enrichment of metabolic pathways. A total of 339 metabolites was identified. There were 22 DMs, among which nine were downregulated-including sphingosine-and 13 were upregulated, including glutathione in PWB iPSCs, as compared to controls. Pathway enrichment analysis confirmed the upregulation of glutathione and the downregulation of sphingolipid metabolism in PWB-derived iPSCs as compared to normal ones. We next examined the expression patterns of the key molecules associated with glutathione metabolism in PWB lesions. We found that hypoxia-inducible factor 1α (HIF1α), glutathione S-transferase Pi 1 (GSTP1), γ-glutamyl transferase 7 (GGT7), and glutamate cysteine ligase modulatory subunit (GCLM) were upregulated in PWB vasculatures as compared to blood vessels in normal skin. Other significantly affected metabolic pathways in PWB iPSCs included pentose and glucuronate interconversions; amino sugar and nucleotide sugars; alanine, aspartate, and glutamate; arginine, purine, D-glutamine, and D-glutamate; arachidonic acid, glyoxylate, and dicarboxylate; nitrogen, aminoacyl-tRNA biosynthesis, pyrimidine, galactose, ascorbate, and aldarate; and starch and sucrose. Our data demonstrated that there were perturbations in sphingolipid and cellular redox homeostasis in PWB vasculatures, which could facilitate cell survival and pathological progression. Our data implied that the upregulation of glutathione could contribute to laser-resistant phenotypes in some PWB vasculatures.

IGFBP2 expressing midlobular hepatocytes preferentially contribute to liver homeostasis and regeneration.

Although midlobular hepatocytes in zone 2 are a recently identified cellular source for liver homeostasis and regeneration, these cells have not been exclusively fate mapped. We generated an Igfbp2-CreER knockin strain that specifically labels midlobular hepatocytes. During homeostasis over 1 year, zone 2 hepatocytes increased in abundance from occupying 21%-41% of the lobular area. After either pericentral injury with carbon tetrachloride or periportal injury with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), IGFBP2+ cells replenished lost hepatocytes in zones 3 and 1, respectively. IGFBP2+ cells also preferentially contributed to regeneration after 70% partial hepatectomy, as well as liver growth during pregnancy. Because IGFBP2 labeling increased substantially with fasting, we used single nuclear transcriptomics to explore zonation as a function of nutrition, revealing that the zonal division of labor shifts dramatically with fasting. These studies demonstrate the contribution of IGFBP2-labeled zone 2 hepatocytes to liver homeostasis and regeneration.

Supporting materials: Endothelial cells differentiated from patient dermal fibroblast-derived induced pluripotent stem cells resemble vascular malformations of Port Wine Birthmark.

Background: Port wine birthmark (PWB) is a congenital vascular malformation resulting from developmentally defective endothelial cells (ECs). Developing clinically relevant disease models for PWB studies is currently an unmet need. Objective: Our study aims to generate PWB-derived induced pluripotent stem cells (iPSCs) and iPSC-derived ECs that preserve disease-related phenotypes. Methods: PWB iPSCs were generated by reprogramming lesional dermal fibroblasts and differentiated into ECs. RNA-seq was performed to identify differentially expressed genes (DEGs) and enriched pathways. The functional phenotypes of iPSC-derived ECs were characterized by capillary-like structure (CLS) formation in vitro and Geltrex plug-in assay in vivo . Results: Human PWB and control iPSC lines were generated through reprogramming of dermal fibroblasts by introducing the "Yamanaka factors" (Oct3/4, Sox2, Klf4, c-Myc) into them; the iPSCs were successfully differentiated into ECs. These iPSCs and their derived ECs were validated by expression of a series of stem cell and EC biomarkers, respectively. PWB iPSC-derived ECs showed impaired CLS in vitro with larger perimeters and thicker branches as compared to control iPSC-derived ECs. In the plug-in assay, perfused human vasculature formed by PWB iPSC- derived ECs showed bigger perimeters and greater densities than those formed by control iPSC- derived ECs in severe combined immune deficient (SCID) mice. The transcriptome analysis showed that dysregulated pathways of stem cell differentiation, Hippo, Wnt, and focal adhesion persisted through differentiation of PWB iPSCs to ECs. Functional enrichment analysis showed that Hippo and Wnt pathway-related PWB DEGs are enriched for vasculature development, tube morphology, endothelium development, and EC differentiation. Further, members of the zinc finger (ZNF) gene family were overrepresented among the DEGs in PWB iPSCs. ZNF DEGs confer significant functions in transcriptional regulation, chromatin remodeling, protein ubiquitination, and retinoic acid receptor signaling. Furthermore, NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways were dysregulated in PWB ECs as readouts of impaired differentiation. Conclusions: PWB iPSC-derived ECs render a novel and clinically-relevant disease model by retaining pathological phenotypes. Our data demonstrate multiple pathways, such as Hippo and Wnt, NF-kappa B, TNF, MAPK, and cholesterol metabolism, are dysregulated, which may contribute to the development of differentiation-defective ECs in PWB. Bulleted statements: What is already known about this topic?: Port Wine Birthmark (PWB) is a congenital vascular malformation with an incidence rate of 0.1 - 0.3 % per live births.PWB results from developmental defects in the dermal vasculature; PWB endothelial cells (ECs) have differentiational impairments.Pulse dye laser (PDL) is currently the preferred treatment for PWB; unfortunately, the efficacy of PDL treatment of PWB has not improved over the past three decades.What does this study add?: Induced pluripotent stem cells (iPSCs) were generated from PWB skin fibroblasts and differentiated into ECs.PWB ECs recapitulated their pathological phenotypes such as forming enlarged blood vessels in vitro and in vivo.Hippo and Wnt pathways were dysregulated in PWB iPSCs and ECs.Zinc-finger family genes were overrepresented among the differentially expressed genes in PWB iPSCs.Dysregulated NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways were enriched in PWB ECs.What is the translational message?: Targeting Hippo and Wnt pathways and Zinc-finger family genes could restore the physiological differentiation of ECs.Targeting NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways could mitigate the pathological progression of PWB.These mechanisms may lead to the development of paradigm-shifting therapeutic interventions for PWB.

Positive selection of somatically mutated clones identifies adaptive pathways in metabolic liver disease.

Somatic mutations in non-malignant tissues accumulate with age and insult, but whether these mutations are adaptive on the cellular or organismal levels is unclear. To interrogate mutations found in human metabolic disease, we performed lineage tracing in mice harboring somatic mosaicism subjected to non-alcoholic steatohepatitis (NASH). Proof-of-concept studies with mosaic loss of Mboat7 , a membrane lipid acyltransferase, showed that increased steatosis accelerated clonal disappearance. Next, we induced pooled mosaicism in 63 known NASH genes, allowing us to trace mutant clones side-by-side. This in vivo tracing platform, which we coined MOSAICS, selected for mutations that ameliorate lipotoxicity, including mutant genes identified in human NASH. To prioritize new genes, additional screening of 472 candidates identified 23 somatic perturbations that promoted clonal expansion. In validation studies, liver-wide deletion of Bcl6, Tbx3, or Smyd2 resulted in protection against NASH. Selection for clonal fitness in mouse and human livers identifies pathways that regulate metabolic disease. Highlights: Mosaic Mboat7 mutations that increase lipotoxicity lead to clonal disappearance in NASH. In vivo screening can identify genes that alter hepatocyte fitness in NASH. Mosaic Gpam mutations are positively selected due to reduced lipogenesis. In vivo screening of transcription factors and epifactors identified new therapeutic targets in NASH.