ABSTRACT
Carbon-rich peat soils have been drained and used extensively for agriculture throughout human history, leading to significant losses of their soil carbon. One solution for rewetting degraded peat is wet crop cultivation. Crops such as rice, which can grow in water-saturated conditions, could enable agricultural production to be maintained whilst reducing CO2 and N2O emissions from peat. However, wet rice cultivation can release considerable methane (CH4). Water table and soil management strategies may enhance rice yield and minimize CH4 emissions, but they also influence plant biomass allocation strategies. It remains unclear how water and soil management influences rice allocation strategies and how changing plant allocation and associated traits, particularly belowground, influence CH4-related processes. We examined belowground biomass (BGB), aboveground biomass (AGB), belowground:aboveground ratio (BGB:ABG), and a range of root traits (root length, root diameter, root volume, root area, and specific root length) under different soil and water treatments; and evaluated plant trait linkages to CH4. Rice (Oryza sativa L.) was grown for six months in field mesocosms under high (saturated) or low water table treatments, and in either degraded peat soil or degraded peat covered with mineral soil. We found that BGB and BGB:AGB were lowest in water saturated conditions where mineral soil had been added to the peat, and highest in low-water table peat soils. Furthermore, CH4 and BGB were positively related, with BGB explaining 60% of the variation in CH4 but only under low water table conditions. Our results suggest that a mix of low water table and mineral soil addition could minimize belowground plant allocation in rice, which could further lower CH4 likely because root-derived carbon is a key substrate for methanogenesis. Minimizing root allocation, in conjunction with water and soil management, could be explored as a strategy for lowering CH4 emissions from wet rice cultivation in degraded peatlands.
Subject(s)
Biomass , Methane , Oryza , Plant Roots , Soil , Oryza/metabolism , Oryza/growth & development , Methane/metabolism , Soil/chemistry , Plant Roots/metabolism , Plant Roots/growth & development , Agriculture/methods , Crops, Agricultural/metabolism , Crops, Agricultural/growth & development , Water/metabolismABSTRACT
Rationale: Pancreatic lineage specification follows the formation of tripotent pancreatic progenitors (PPs). Current protocols rebuilding PPs in vitro have an endocrine lineage bias and are mostly based on PDX1/NKX6-1 coexpression neglecting other markers decisive for PP heterogeneity and lineage potential. However, true tripotent PPs are of utmost interest to study also exocrine disorders such as pancreatic cancer and to simultaneously generate all three pancreatic lineages from the same ancestor. Methods: Here, we performed a comprehensive compound testing to advance the generation of multipotent progenitors, which were further characterized for their trilineage potential in vitro and in vivo. The heterogeneity and cell-cell communication across the PP subpopulations were analyzed via single-cell transcriptomics. Results: We introduce a novel PP differentiation platform based on a comprehensive compound screening with an advanced design of experiments computing tool to reduce impurities and to increase Glycoprotein-2 expression and subsequent trilineage potential. Superior PP tripotency was proven in vitro by the generation of acinar, endocrine, and ductal cells as well as in vivo upon orthotopic transplantation revealing all three lineages at fetal maturation level. GP2 expression levels at PP stage ascribed varying pancreatic lineage potential. Intermediate and high GP2 levels were superior in generating endocrine and duct-like organoids (PDLO). FACS-based purification of the GP2high PPs allowed the generation of pancreatic acinar-like organoids (PALO) with proper morphology and expression of digestive enzymes. scRNA-seq confirmed multipotent identity, positioned the GP2/PDX1/NKX6-1high population next to human fetal tip and trunk progenitors and identified novel ligand-receptor (LR) interactions in distinct PP subpopulations. LR validation experiments licensed midkine and VEGF signaling to increase markers labelling the single cell clusters with high GP2 expression. Conclusion: In this study, we guide human pluripotent stem cells into multipotent pancreatic progenitors. This common precursor population, which has the ability to mature into acinar, ductal and functional ß-cells, serves as a basis for studying developmental processes and deciphering early cancer formation in a cell type-specific context. Using single-cell RNA sequencing and subsequent validation studies, we were able to dissect PP heterogeneity and specific cell-cell communication signals.
Subject(s)
Insulin-Secreting Cells , Pluripotent Stem Cells , Humans , Pancreas/metabolism , Cell Differentiation/physiology , Insulin-Secreting Cells/metabolism , OrganoidsABSTRACT
Human pluripotent stem cells, with their ability to proliferate indefinitely and to differentiate into virtually all cell types of the human body, provide a novel resource to study human development and to implement relevant disease models. Here, we employed a human pancreatic differentiation platform complemented with an shRNA screen in human pluripotent stem cells (PSCs) to identify potential drivers of early endoderm and pancreatic development. Deep sequencing followed by abundancy ranking pinpointed six top hit genes potentially associated with either improved or impaired endodermal differentiation, which were selected for functional validation in CRISPR-Cas9 mediated knockout (KO) lines. Upon endoderm differentiation (DE), particularly the loss of SLC22A1 and DSC2 led to impaired differentiation efficiency into CXCR4/KIT-positive DE cells. qPCR analysis also revealed changes in differentiation markers CXCR4, FOXA2, SOX17, and GATA6. Further differentiation of PSCs to the pancreatic progenitor (PP) stage resulted in a decreased proportion of PDX1/NKX6-1-positive cells in SLC22A1 KO lines, and in DSC2 KO lines when differentiated under specific culture conditions. Taken together, our study reveals novel genes with potential roles in early endodermal development.
Subject(s)
Endoderm , Pluripotent Stem Cells , Cell Differentiation/genetics , Genomics , Humans , Pancreas/metabolism , Pluripotent Stem Cells/metabolismABSTRACT
The recapitulation of human developmental processes and pathological manifestations requires access to specific cell types and precursor stages during embryogenesis and disease. Here, we describe a scalable in vitro differentiation protocol to guide human pluripotent stem cells stepwise into pancreatic duct-like organoids. The protocol mimics pancreatic duct development and was successfully used to model the onset and progression of pancreatic ductal adenocarcinoma; the approach is suitable for multiple downstream applications. However, the protocol is cost- and time-intensive. For complete details on the use and execution of this protocol, please refer to Breunig et al. (2021).
Subject(s)
Cell Culture Techniques/methods , Pancreatic Ducts/cytology , Pluripotent Stem Cells/cytology , Cell Differentiation/physiology , Cell Line , Humans , Organoids/cytologyABSTRACT
The bat sarbecovirus RaTG13 is a close relative of SARS-CoV-2, the cause of the COVID-19 pandemic. However, this bat virus was most likely unable to directly infect humans since its Spike (S) protein does not interact efficiently with the human ACE2 receptor. Here, we show that a single T403R mutation increases binding of RaTG13 S to human ACE2 and allows VSV pseudoparticle infection of human lung cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S reduces pseudoparticle infection and viral replication. The T403R RaTG13 S is neutralized by sera from individuals vaccinated against COVID-19 indicating that vaccination might protect against future zoonoses. Our data suggest that a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2 by S proteins of bat coronaviruses. This finding could help to better predict the zoonotic potential of animal coronaviruses.
Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Protein Binding , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Animals , COVID-19/virology , COVID-19 Vaccines , Caco-2 Cells , Cloning, Molecular , HEK293 Cells , Humans , Molecular Dynamics Simulation , Mutation , Replicon , Species Specificity , Stem Cells , ZoonosesABSTRACT
Cell type specification during pancreatic development is tightly controlled by a transcriptional and epigenetic network. The precise role of most transcription factors, however, has been only described in mice. To convey such concepts to human pancreatic development, alternative model systems such as pancreatic in vitro differentiation of human pluripotent stem cells can be employed. Here, we analyzed stage-specific RNA-, ChIP-, and ATAC-sequencing data to dissect transcriptional and regulatory mechanisms during pancreatic development. Transcriptome and open chromatin maps of pancreatic differentiation from human pluripotent stem cells provide a stage-specific pattern of known pancreatic transcription factors and indicate ONECUT1 as a crucial fate regulator in pancreas progenitors. Moreover, our data suggest that ONECUT1 is also involved in preparing pancreatic progenitors for later endocrine specification. The dissection of the transcriptional and regulatory circuitry revealed an important role for ONECUT1 within such network and will serve as resource to study human development and disease.
Subject(s)
Hepatocyte Nuclear Factor 6/genetics , Pancreas/physiology , Cell Differentiation , Cell Line , Gene Expression Regulation, Developmental , Hepatocyte Nuclear Factor 6/metabolism , Human Embryonic Stem Cells , Humans , Transcription, GeneticABSTRACT
Patient-derived induced pluripotent stem cells (iPSCs) provide a unique platform to study hereditary disorders and predisposition syndromes by resembling germline mutations of affected individuals and by their potential to differentiate into nearly every cell type of the human body. We employed plucked human hair from two siblings with a family history of cancer carrying a pathogenic CDKN2A variant, P16-p.G101W/P14-p.R115L, to generate patient-specific iPSCs in a cancer-prone ancestry for downstream analytics. The differentiation capacity to pancreatic progenitors and to pancreatic duct-like organoids (PDLOs) according to a recently developed protocol remained unaffected. Upon inducible expression of KRASG12Dusing a piggyBac transposon system in CDKN2A-mutated PDLOs, we revealed structural and molecular changes in vitro, including disturbed polarity and epithelial-to-mesenchymal (EMT) transition. CDKN2A-mutated KRASG12DPDLO xenotransplants formed either a high-grade precancer lesion or a partially dedifferentiated PDAC-like tumor. Intriguingly, P14/P53/P21 and P16/RB cell-cycle checkpoint controls have been only partly overcome in these grafts, thereby still restricting the tumorous growth. Hereby, we provide a model for hereditary human pancreatic cancer that enables dissection of tumor initiation and early development starting from patient-specific CDKN2A-mutated pluripotent stem cells.
ABSTRACT
Genes involved in distinct diabetes types suggest shared disease mechanisms. Here we show that One Cut Homeobox 1 (ONECUT1) mutations cause monogenic recessive syndromic diabetes in two unrelated patients, characterized by intrauterine growth retardation, pancreas hypoplasia and gallbladder agenesis/hypoplasia, and early-onset diabetes in heterozygous relatives. Heterozygous carriers of rare coding variants of ONECUT1 define a distinctive subgroup of diabetic patients with early-onset, nonautoimmune diabetes, who respond well to diabetes treatment. In addition, common regulatory ONECUT1 variants are associated with multifactorial type 2 diabetes. Directed differentiation of human pluripotent stem cells revealed that loss of ONECUT1 impairs pancreatic progenitor formation and a subsequent endocrine program. Loss of ONECUT1 altered transcription factor binding and enhancer activity and NKX2.2/NKX6.1 expression in pancreatic progenitor cells. Collectively, we demonstrate that ONECUT1 controls a transcriptional and epigenetic machinery regulating endocrine development, involved in a spectrum of diabetes, encompassing monogenic (recessive and dominant) as well as multifactorial inheritance. Our findings highlight the broad contribution of ONECUT1 in diabetes pathogenesis, marking an important step toward precision diabetes medicine.
Subject(s)
Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/pathology , Hepatocyte Nuclear Factor 6/genetics , Pancreas/embryology , Cell Differentiation/genetics , Congenital Abnormalities/genetics , Fetal Growth Retardation/genetics , Gallbladder/abnormalities , Homeobox Protein Nkx-2.2/biosynthesis , Homeodomain Proteins/biosynthesis , Humans , Infant , Infant, Newborn , Male , Multifactorial Inheritance/genetics , Organogenesis/genetics , Pancreas/abnormalities , Pancreatic Diseases/congenital , Pancreatic Diseases/genetics , Pluripotent Stem Cells/cytology , Transcription, Genetic/geneticsABSTRACT
Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) can restrict viral pathogens, but pro- and anti-viral activities have been reported for coronaviruses. Here, we show that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. However, endogenous IFITM expression supports efficient infection of SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral infection. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. IFITM-derived peptides and targeting antibodies inhibit SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are cofactors for efficient SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and are potential targets for therapeutic approaches.
Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Antigens, Differentiation/genetics , Membrane Proteins/genetics , RNA-Binding Proteins/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/pharmacology , Antigens, Differentiation/metabolism , Binding Sites , COVID-19/virology , Gene Expression Regulation , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/genetics , Humans , Interferon-beta/pharmacology , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Protein Binding , Protein Interaction Domains and Motifs , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Spike Glycoprotein, Coronavirus/metabolism , Virus Attachment/drug effectsABSTRACT
Diabetes, as one of the major diseases in industrial countries, affects over 350 million people worldwide. Type 1 (T1D) and type 2 diabetes (T2D) are the most common forms with both types having invariable genetic influence. It is accepted that a subset of all diabetes patients, generally estimated to account for 1-2% of all diabetic cases, is attributed to mutations in single genes. As only a subset of these genes has been identified and fully characterized, there is a dramatic need to understand the pathophysiological impact of genetic determinants on ß-cell function and pancreatic development but also on cell replacement therapies. Pluripotent stem cells differentiated along the pancreatic lineage provide a valuable research platform to study such genes. This review summarizes current perspectives in applying this platform to study monogenic diabetes variants.
Subject(s)
Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/metabolism , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Mutation , Pluripotent Stem Cells/cytology , Animals , CRISPR-Cas Systems , Cell Differentiation , Cell Lineage , Embryonic Stem Cells/cytology , Epigenesis, Genetic , Gene Editing , Genetic Variation , Heterozygote , Humans , Insulin/metabolism , Insulin-Secreting Cells/metabolism , Mice , Mice, Knockout , Pancreas/embryology , Pancreas/pathology , Phenotype , RegenerationABSTRACT
Genetic risk variants that have been identified in genome-wide association studies of complex diseases are primarily non-coding1. Translating these risk variants into mechanistic insights requires detailed maps of gene regulation in disease-relevant cell types2. Here we combined two approaches: a genome-wide association study of type 1 diabetes (T1D) using 520,580 samples, and the identification of candidate cis-regulatory elements (cCREs) in pancreas and peripheral blood mononuclear cells using single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) of 131,554 nuclei. Risk variants for T1D were enriched in cCREs that were active in T cells and other cell types, including acinar and ductal cells of the exocrine pancreas. Risk variants at multiple T1D signals overlapped with exocrine-specific cCREs that were linked to genes with exocrine-specific expression. At the CFTR locus, the T1D risk variant rs7795896 mapped to a ductal-specific cCRE that regulated CFTR; the risk allele reduced transcription factor binding, enhancer activity and CFTR expression in ductal cells. These findings support a role for the exocrine pancreas in the pathogenesis of T1D and highlight the power of large-scale genome-wide association studies and single-cell epigenomics for understanding the cellular origins of complex disease.
Subject(s)
Diabetes Mellitus, Type 1/genetics , Epigenomics , Genetic Predisposition to Disease , Single-Cell Analysis , Chromatin/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Female , Gene Expression Regulation , Genome-Wide Association Study , Humans , Immunity/genetics , Male , Pancreatic Ducts/metabolism , Pancreatic Ducts/pathologyABSTRACT
Personalized in vitro models for dysplasia and carcinogenesis in the pancreas have been constrained by insufficient differentiation of human pluripotent stem cells (hPSCs) into the exocrine pancreatic lineage. Here, we differentiate hPSCs into pancreatic duct-like organoids (PDLOs) with morphological, transcriptional, proteomic, and functional characteristics of human pancreatic ducts, further maturing upon transplantation into mice. PDLOs are generated from hPSCs inducibly expressing oncogenic GNAS, KRAS, or KRAS with genetic covariance of lost CDKN2A and from induced hPSCs derived from a McCune-Albright patient. Each oncogene causes a specific growth, structural, and molecular phenotype in vitro. While transplanted PDLOs with oncogenic KRAS alone form heterogenous dysplastic lesions or cancer, KRAS with CDKN2A loss develop dedifferentiated pancreatic ductal adenocarcinomas. In contrast, transplanted PDLOs with mutant GNAS lead to intraductal papillary mucinous neoplasia-like structures. Conclusively, PDLOs enable in vitro and in vivo studies of pancreatic plasticity, dysplasia, and cancer formation from a genetically defined background.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Pluripotent Stem Cells , Animals , Humans , Mice , Mutation , Organoids , Pancreatic Ducts , Pancreatic Neoplasms/genetics , ProteomicsABSTRACT
Infection-related diabetes can arise as a result of virus-associated ß-cell destruction. Clinical data suggest that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the coronavirus disease 2019 (COVID-19), impairs glucose homoeostasis, but experimental evidence that SARS-CoV-2 can infect pancreatic tissue has been lacking. In the present study, we show that SARS-CoV-2 infects cells of the human exocrine and endocrine pancreas ex vivo and in vivo. We demonstrate that human ß-cells express viral entry proteins, and SARS-CoV-2 infects and replicates in cultured human islets. Infection is associated with morphological, transcriptional and functional changes, including reduced numbers of insulin-secretory granules in ß-cells and impaired glucose-stimulated insulin secretion. In COVID-19 full-body postmortem examinations, we detected SARS-CoV-2 nucleocapsid protein in pancreatic exocrine cells, and in cells that stain positive for the ß-cell marker NKX6.1 and are in close proximity to the islets of Langerhans in all four patients investigated. Our data identify the human pancreas as a target of SARS-CoV-2 infection and suggest that ß-cell infection could contribute to the metabolic dysregulation observed in patients with COVID-19.
Subject(s)
Islets of Langerhans/virology , SARS-CoV-2/growth & development , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2/biosynthesis , Angiotensin-Converting Enzyme 2/genetics , COVID-19/physiopathology , Cells, Cultured , Diabetes Mellitus , Female , Humans , Islets of Langerhans/cytology , Islets of Langerhans/physiopathology , Male , Pancreas, Exocrine/cytology , Pancreas, Exocrine/physiopathology , Pancreas, Exocrine/virology , Pancreatic Diseases/etiology , Pancreatic Diseases/virology , Serine Endopeptidases/biosynthesis , Serine Endopeptidases/genetics , Virus Internalization , Virus ReplicationABSTRACT
BACKGROUND AND AIMS: The COVID-19 pandemic has spread worldwide and poses a severe health risk. While most patients present mild symptoms, descending pneumonia can lead to severe respiratory insufficiency. Up to 50% of patients show gastrointestinal symptoms like diarrhea or nausea, intriguingly associating with prolonged symptoms and increased severity. Thus, models to understand and validate drug efficiency in the gut of COVID-19 patients are of urgent need. METHODS: Human intestinal organoids derived from pluripotent stem cells (PSC-HIOs) have led, due to their complexity in mimicking human intestinal architecture, to an unprecedented number of successful disease models including gastrointestinal infections. Here, we employed PSC-HIOs to dissect SARS-CoV-2 pathogenesis and its inhibition by remdesivir, one of the leading drugs investigated for treatment of COVID-19. RESULTS: Immunostaining for viral entry receptor ACE2 and SARS-CoV-2 spike protein priming protease TMPRSS2 showed broad expression in the gastrointestinal tract with highest levels in the intestine, the latter faithfully recapitulated by PSC-HIOs. Organoids could be readily infected with SARS-CoV-2 followed by viral spread across entire PSC-HIOs, subsequently leading to organoid deterioration. However, SARS-CoV-2 spared goblet cells lacking ACE2 expression. Importantly, we challenged PSC-HIOs for drug testing capacity. Specifically, remdesivir effectively inhibited SARS-CoV-2 infection dose-dependently at low micromolar concentration and rescued PSC-HIO morphology. CONCLUSIONS: Thus, PSC-HIOs are a valuable tool to study SARS-CoV-2 infection and to identify and validate drugs especially with potential action in the gut.
Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , COVID-19 Drug Treatment , COVID-19/metabolism , Human Embryonic Stem Cells , Intestinal Mucosa , Organoids , SARS-CoV-2/physiology , Virus Replication/drug effects , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Caco-2 Cells , Human Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/pathology , Human Embryonic Stem Cells/virology , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Intestinal Mucosa/virology , Organoids/metabolism , Organoids/pathology , Organoids/virologyABSTRACT
Pandemic SARS-CoV-2 infection has rapidly developed into a socioeconomic and humanitarian catastrophe. Basic principles to prevent SARS-CoV-2 transmission are social distancing, face masks, contact tracing and early detection of SARS-CoV-2. To meet these requirements, virtually unlimited test capacities delivering results in a rapid and reliable manner are a prerequisite. Here, we provide and validate such a rapid, convenient and efficient kit-independent detection of SARS-CoV-2 RNA, termed COVID-quick-DET. This straightforward method operates with simple proteinase K treatment and repetitive heating steps with a sensitivity of 94.6% in head-to-head comparisons with kit-based isolation methods. This result is supported by data obtained from serially diluted SARS-CoV-2 virus stocks. Given its cost- and time-effective operation, COVID-quick-DET might be best suited for countries with general shortage or temporary acute scarcity of resources and equipment.
Subject(s)
Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , RNA, Viral/isolation & purification , Reagent Kits, Diagnostic , COVID-19 , COVID-19 Testing , Diagnostic Tests, Routine , Endopeptidase K/chemistry , Heating , Humans , Pandemics , RNA, Viral/genetics , SARS-CoV-2 , Sensitivity and SpecificityABSTRACT
Inflammatory bowel disease (IBD) has been linked to active signaling with bacterial components and reduced mitochondrial ATP production; however, synergism between both of these disease characteristics remains unclear. We aimed to determine in human IBD transcriptomes the link between a transcriptional signature unique to intestinal cells (ICs) with reduced mitochondrial ATP production (Mito-0) and bacteria triggered signaling using a bioinformatics approach. We generated an IC Mito-0 panel comprised of 199 differentially expressed (DE) transcripts mediated by reduced mitochondrial ATP function (DEGseq, log2 fold-change > |2|, p < .001). Transcripts from this panel were involved in diverse biological functions including regulation of mitochondrial energy (lower ATP), extracellular matrix, cell-cell contact, cytoskeleton, growth, metabolism, and inflammation. Next, unsupervised hierarchical clustering showed that the Mito-0 panel distinctly separated inflamed IBD from non-inflamed transcriptomes, which was also supported by principal component analysis (PCA) revealing distinct variation between sample types based on presence of the Mito-0 signature (PCA, p = 8.77e-09). Utilizing three independent IBD cohorts, we validated that 60 novel transcripts from the Mito-0 panel were significantly increased in inflamed tissue. Subsequently, KEGG generated bacterial TLR4 and NOD2 transcriptional signatures strongly associated with inflamed IBD transcriptomes and with the Mito-0 signature as determined by Spearman's analysis (coefficient of correlation, r = 0.92, p < .05). Herein, using a comprehensive analysis we demonstrated existence of an axis between bacteria triggered signaling and reduced mitochondrial energy function. Furthermore, we identified and validated novel transcripts within this axis as potential drivers and therapeutic targets for human IBD.
Subject(s)
Bacteria/metabolism , Inflammatory Bowel Diseases/metabolism , Mitochondria/metabolism , Nod2 Signaling Adaptor Protein/metabolism , Toll-Like Receptor 4/metabolism , Adenosine Triphosphate/metabolism , Computational Biology , Humans , Inflammation , Inflammatory Bowel Diseases/genetics , Inflammatory Bowel Diseases/pathology , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Nod2 Signaling Adaptor Protein/genetics , Signal Transduction , Toll-Like Receptor 4/genetics , TranscriptomeABSTRACT
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are characterized by their unique capacity to stepwise differentiate towards any particular cell type in an adult organism. Pluripotent stem cells provide a beneficial platform to model hereditary diseases and even cancer development. While the incidence of pancreatic diseases such as diabetes and pancreatitis is increasing, the understanding of the underlying pathogenesis of particular diseases remains limited. Only a few recent publications have contributed to the characterization of human pancreatic development in the fetal stage. Hence, most knowledge of pancreatic specification is based on murine embryology. Optimizing and understanding current in vitro protocols for pancreatic differentiation of ESCs and iPSCs constitutes a prerequisite to generate functional pancreatic cells for better disease modeling and drug discovery. Moreover, human pancreatic organoids derived from pluripotent stem cells, organ-restricted stem cells, and tumor samples provide a powerful technology to model carcinogenesis and hereditary diseases independent of genetically engineered mouse models. Herein, we summarize recent advances in directed differentiation of pancreatic organoids comprising endocrine cell types. Beyond that, we illustrate up-and-coming applications for organoid-based platforms.
ABSTRACT
BACKGROUND & AIMS: Diminished forkhead box O3 (FOXO3) function drives inflammation and cancer growth; however, mechanisms fostering these pathobiologies are unclear. Here, we aimed to identify in colon loss of FOXO3-dependent cellular and molecular changes that facilitate inflammation-mediated tumor growth. METHODS: FOXO3 knockout (KO) and wild-type (WT) mice were used in the AOM/DSS model of inflammation-mediated colon cancer. Bioinformatics were used for profiling of mRNA sequencing data from human and mouse colon and tumors; specific targets were validated in human colon cancer cells (shFOXO3). RESULTS: In mice, FOXO3 deficiency led to significantly elevated colonic tumor burden (incidence and size) compared with WT (P < .05). In FOXO3 KO colon, activated molecular pathways overlapped with those associated with mouse and human colonic inflammation and cancer, especially human colonic tumors with inflammatory microsatellite instability (false discovery rate < 0.05). FOXO3 KO colon, similar to tumors, had increased neutrophils, macrophages, B cells, T cells, and decreased natural killer cells (false discovery rate < 0.05). Moreover, in KO colon differentially expressed transcripts were linked to activation of inflammatory nuclear factor kappa B, tumorigenic cMyc, and bacterial Toll-like receptor signaling. Among differentially expressed transcripts, we validated altered expression of integrin subunit alpha 2 (ITGA2), ADAM metallopeptidase with thrombospondin type 1 motif 12, and ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 5 in mouse WT and FOXO3 KO colon and tumors (P < .05). Similarly, their altered expression was found in human inflammatory bowel disease and colon cancer tissues and linked to poor patient survival. Ultimately, in human colon cancer cells, FOXO3 knockdown (shFOXO3) led to significantly increased ITGA2, and silencing ITGA2 (siRNA) alone diminished cell growth. CONCLUSIONS: We identified the loss of FOXO3-mediated immune landscape, pathways, and transcripts that could serve as biomarkers and new targets for inflammatory colon cancer treatment.
Subject(s)
Colonic Neoplasms/genetics , Colonic Neoplasms/immunology , Forkhead Box Protein O3/deficiency , Gene Expression Profiling , Inflammation/genetics , Inflammation/immunology , Animals , Carcinogenesis/genetics , Cell Proliferation , Colon/microbiology , Colon/pathology , Colonic Neoplasms/pathology , Disease Progression , Forkhead Box Protein O3/genetics , Forkhead Box Protein O3/metabolism , Gene Expression Regulation, Neoplastic , HCT116 Cells , Humans , Inflammation/pathology , Inflammatory Bowel Diseases/genetics , Inflammatory Bowel Diseases/pathology , Mice, Inbred C57BL , Mice, Knockout , Phosphatidylinositol 3-Kinases/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Survival Analysis , Tumor Burden , Tumor Microenvironment/geneticsABSTRACT
Obesity, an immense epidemic affecting approximately half a billion adults, has doubled in prevalence in the last several decades. Epidemiological data support that obesity, due to intake of a high-fat, western diet, increases the risk of colon cancer; however, the mechanisms underlying this risk remain unclear. Here, utilizing next generation RNA sequencing, we aimed to determine the high-fat diet (HFD) mediated expression profile in mouse colon and the azoxymethane/dextran sulfate sodium model of colon cancer. Mice on HFD had significantly higher colonic inflammation, tumor burden, and a number of differentially expressed transcripts compared to mice on regular diet (RD). We identified 721 transcripts differentially expressed in mouse HFD colon that were in a shared pattern with colonic tumors (RD and HFD). Importantly, in mouse colon, HFD stimulated an expression signature strikingly similar to human colon cancer, especially those with inflammatory microsatellite instability. Furthermore, pathway analysis of these transcripts demonstrated their association with active inflammation and colon cancer signaling, with leptin and Wnt as the top two transcripts elevated in mouse HFD colon shared with tumors. Moreover, in mouse colon, HFD-stimulated tumorigenic Wnt pathway activation was further validated by upregulation of ß-catenin transcriptional targets. Finally, in human colon cancer, upregulation of leptin pathway members was shown with a large network of dysregulated transcripts being linked with worse overall survival.