ABSTRACT
Genotype-phenotype associations for common diseases are often compounded by pleiotropy and metabolic state. Here, we devised a pooled human organoid-panel of steatohepatitis to investigate the impact of metabolic status on genotype-phenotype association. En masse population-based phenotypic analysis under insulin insensitive conditions predicted key non-alcoholic steatohepatitis (NASH)-genetic factors including the glucokinase regulatory protein (GCKR)-rs1260326:C>T. Analysis of NASH clinical cohorts revealed that GCKR-rs1260326-T allele elevates disease severity only under diabetic state but protects from fibrosis under non-diabetic states. Transcriptomic, metabolomic, and pharmacological analyses indicate significant mitochondrial dysfunction incurred by GCKR-rs1260326, which was not reversed with metformin. Uncoupling oxidative mechanisms mitigated mitochondrial dysfunction and permitted adaptation to increased fatty acid supply while protecting against oxidant stress, forming a basis for future therapeutic approaches for diabetic NASH. Thus, "in-a-dish" genotype-phenotype association strategies disentangle the opposing roles of metabolic-associated gene variant functions and offer a rich mechanistic, diagnostic, and therapeutic inference toolbox toward precision hepatology. VIDEO ABSTRACT.
Subject(s)
Genetic Predisposition to Disease , Non-alcoholic Fatty Liver Disease , Humans , Non-alcoholic Fatty Liver Disease/genetics , Organoids , Genetic Association Studies , Alleles , LiverABSTRACT
Organogenesis is a complex and interconnected process that is orchestrated by multiple boundary tissue interactions1-7. However, it remains unclear how individual, neighbouring components coordinate to establish an integral multi-organ structure. Here we report the continuous patterning and dynamic morphogenesis of hepatic, biliary and pancreatic structures, invaginating from a three-dimensional culture of human pluripotent stem cells. The boundary interactions between anterior and posterior gut spheroids differentiated from human pluripotent stem cells enables retinoic acid-dependent emergence of hepato-biliary-pancreatic organ domains specified at the foregut-midgut boundary organoids in the absence of extrinsic factors. Whereas transplant-derived tissues are dominated by midgut derivatives, long-term-cultured microdissected hepato-biliary-pancreatic organoids develop into segregated multi-organ anlages, which then recapitulate early morphogenetic events including the invagination and branching of three different and interconnected organ structures, reminiscent of tissues derived from mouse explanted foregut-midgut culture. Mis-segregation of multi-organ domains caused by a genetic mutation in HES1 abolishes the biliary specification potential in culture, as seen in vivo8,9. In sum, we demonstrate that the experimental multi-organ integrated model can be established by the juxtapositioning of foregut and midgut tissues, and potentially serves as a tractable, manipulatable and easily accessible model for the study of complex human endoderm organogenesis.
Subject(s)
Biliary Tract/embryology , Intestines/embryology , Liver/embryology , Models, Biological , Morphogenesis , Pancreas/embryology , Animals , Biliary Tract/cytology , Biomarkers/analysis , Biomarkers/metabolism , Body Patterning , Endoderm/cytology , Endoderm/embryology , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Intestines/cytology , Liver/cytology , Male , Mice , Organoids/cytology , Organoids/embryology , Pancreas/cytology , Spheroids, Cellular/cytology , Spheroids, Cellular/metabolism , Spheroids, Cellular/transplantation , Transcription Factor HES-1/analysis , Transcription Factor HES-1/metabolismABSTRACT
BACKGROUND: The aim of the present study was to clarify the roles of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6) in immunocompetent children with acute liver dysfunction not resulting from hepatitis virus. METHODS: Sixty-eight children (median age, 3 years) hospitalized as a result of acute liver dysfunction were enrolled in this study. Hepatitis A, B, and C were excluded. The prevalence of CMV, EBV, and HHV-6 and viral DNA load in whole blood was prospectively evaluated on multiplex real-time polymerase chain reaction (PCR). RESULTS: Of the 68 children with acute liver dysfunction, multiplex real-time PCR was positive in 30 (44%). CMV, EBV, and HHV-6 DNA were detected in 13 (19%), 14 (21%), and seven (10%), respectively. Serum CMV immunoglobulin (Ig)G/IgM and EBV viral capsid antigen IgG/IgM were measured in 40 (CMV DNA positive, n = 10; negative, n = 30) and 45 (EBV DNA positive, n = 14; negative, n = 31) of the 68 children, respectively. Eighteen percent (CMV, 7/40) and 9% (EBV, 4/45) were positive for both PCR and viral-specific IgM. There was no significant difference in CMV and EBV viral load between IgM-positive and -negative children with viremia. CONCLUSIONS: CMV, EBV, and HHV-6 DNA were frequently detected in immunocompetent children with acute liver dysfunction, but primary CMV and EBV infection were confirmed in 10-20% of the children with acute liver dysfunction. The combination of PCR assay and serology is necessary to make a diagnosis of acute liver dysfunction due to primary CMV, EBV and/or HHV-6 infection in immunocompetent children.
Subject(s)
Cytomegalovirus Infections/complications , Epstein-Barr Virus Infections/complications , Hepatic Insufficiency/virology , Herpesvirus 6, Human/isolation & purification , Immunocompetence , Roseolovirus Infections/complications , Acute Disease , Adolescent , Child , Child, Preschool , Cytomegalovirus Infections/diagnosis , Cytomegalovirus Infections/epidemiology , Cytomegalovirus Infections/immunology , Epstein-Barr Virus Infections/diagnosis , Epstein-Barr Virus Infections/epidemiology , Epstein-Barr Virus Infections/immunology , Female , Hepatic Insufficiency/immunology , Humans , Infant , Infant, Newborn , Male , Multiplex Polymerase Chain Reaction , Prevalence , Prospective Studies , Real-Time Polymerase Chain Reaction , Roseolovirus Infections/diagnosis , Roseolovirus Infections/epidemiology , Roseolovirus Infections/immunology , Viral LoadABSTRACT
BACKGROUND AND AIM: To assess the feasibility and safety of same-day regimen of low-volume polyethylene glycol solution with ascorbic acid for bowel cleansing before colonoscopy in children. METHODS: Data on children who received polyethylene glycol solution with ascorbic acid for bowel cleansing in our department were retrospectively analyzed. On the day before the procedure, patients ate a low-residue diet and received sodium picosulfate in the evening. The following day, patients took polyethylene glycol solution with ascorbic acid in the morning; the procedure was carried out in the afternoon. Dosages of sodium picosulfate and polyethylene glycol solution with ascorbic acid were adjusted based on bodyweight. Bowel cleansing efficacy was rated on a scale of 1-5 by the colonoscopist. RESULTS: Between July 2013 and November 2014, polyethylene glycol solution with ascorbic acid was used in 112 cases (96 patients; male : female 73:39; median age 10.9 years, range 4-19 years). Ninety-one cases (81%) were able to orally ingest the prescribed amount of polyethylene glycol solution with ascorbic acid. Satisfactory bowel cleansing (cleansing grade ≥3) was attained in 87% on intention-to-treat analysis and 85% on per-protocol analysis. Cleansing grade was significantly better in children who ingested polyethylene glycol solution with ascorbic acid within 60 min (P < 0.05). There were no serious adverse events. CONCLUSION: Same-day regimen of polyethylene glycol solution with ascorbic acid is effective and safe for bowel cleansing in children.
Subject(s)
Ascorbic Acid/therapeutic use , Balloon Enteroscopy , Colonoscopy , Polyethylene Glycols/therapeutic use , Preoperative Care , Adolescent , Age Factors , Antioxidants/therapeutic use , Child , Child, Preschool , Feasibility Studies , Female , Humans , Male , Pilot Projects , Retrospective Studies , Surface-Active Agents/therapeutic use , Young AdultABSTRACT
BACKGROUND: Vaccination against hepatitis B virus (HBV) infection in infants born to hepatitis B surface antigen (HBsAg)-positive mothers using HB immunoglobulin (HBIG) and hepatitis B (HB) vaccine was launched in Japan in 1985. Infants testing positive for HBsAg at 1 month of age are considered to have prenatally acquired the infection and are usually excluded from the prevention program. Infants born to HB e antigen (HBeAg)-positive mothers are at a high risk of perinatally acquiring the infection. In this study, long-term outcome was evaluated in children with prenatal HBV infection who received the HBIG and HB vaccine in Japan. METHODS: Newborns of both HBsAg- and HBeAg-positive carrier mothers received HBIG within 48 h of birth and at 2 months of age. Subsequently, three doses of recombinant HB vaccine were given at 2, 3, and 5 months of age. Outcome was compared between the following two groups: infants who completed the vaccination program, even if they were HBsAg positive at 1 month of age (n = 15), and infants who did not (n = 51). RESULTS: Seroconversion from HBeAg to anti-HBe antibody (HBeAb) before 3 years of age was observed in five children (33%) who completed the vaccination program and in two (4%) who did not (P = 0.005). In 2/5 children who completed the vaccination program and achieved HBeAb seroconversion, seroconversion from HBsAg to anti-HBs antibody was also noted. CONCLUSION: This specific vaccination program for children with prenatal HBV infection has the potential to alter immune tolerance to HBV.
Subject(s)
Hepatitis B Vaccines/administration & dosage , Hepatitis B virus/immunology , Hepatitis B/therapy , Immunoglobulins/administration & dosage , Uterine Diseases/drug therapy , DNA, Viral/analysis , Female , Follow-Up Studies , Hepatitis B/virology , Hepatitis B Antibodies/analysis , Hepatitis B Vaccines/genetics , Hepatitis B Vaccines/immunology , Humans , Immunization, Passive , Incidence , Infant , Japan/epidemiology , Male , Retrospective Studies , Treatment Outcome , Uterine Diseases/epidemiology , Uterine Diseases/virologyABSTRACT
BACKGROUND AND AIMS: We previously identified small molecules predicted to reverse an ileal gene signature for future Crohn's Disease (CD) strictures. Here we used a new human intestinal organoid (HIO) model system containing macrophages to test a lead candidate, eicosatetraynoic acid (ETYA). METHODS: Induced pluripotent stem cell lines (iPSC) were derived from CD patients and differentiated into macrophages and HIOs. Macrophages and macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment. Cytospin and flow cytometry characterized macrophage morphology and activation markers, and RNA sequencing defined the global pattern of macrophage gene expression. TaqMan Low Density Array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to measure macrophage cytokine production and HIO pro-fibrotic gene expression and collagen content. RESULTS: iPSC-derived macrophages exhibited morphology similar to primary macrophages and expressed inflammatory macrophage cell surface markers including CD64 and CD68. LPS-stimulated macrophages expressed a global pattern of gene expression enriched in CD ileal inflammatory macrophages and matrisome secreted products, and produced cytokines and chemokines including CCL2, IL1B, and OSM implicated in refractory disease. ETYA suppressed CD64 abundance and pro-fibrotic gene expression pathways in LPS stimulated macrophages. Co-culture of LPS-primed macrophages with HIO led to up-regulation of fibroblast activation genes including ACTA2 and COL1A1, and an increase in HIO collagen content. ETYA pre-treatment prevented pro-fibrotic effects of LPS-primed macrophages. CONCLUSIONS: ETYA inhibits pro-fibrotic effects of LPS-primed macrophages upon co-cultured HIO. This model may be used in future untargeted screens for small molecules to treat refractory CD.
ABSTRACT
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.
ABSTRACT
Background and Aims: We previously identified small molecules predicted to reverse an ileal gene signature for future Crohn's Disease (CD) strictures. Here we used a new human intestinal organoid (HIO) model system containing macrophages to test a lead candidate, eicosatetraynoic acid (ETYA). Methods: Induced pluripotent stem cell lines (iPSC) were derived from CD patients and differentiated into macrophages and HIOs. Macrophages and macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment. Cytospin and flow cytometry characterized macrophage morphology and activation markers, and RNA sequencing defined the global pattern of macrophage gene expression. TaqMan Low Density Array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to measure macrophage cytokine production and HIO pro-fibrotic gene expression and collagen content. Results: iPSC-derived macrophages exhibited morphology similar to primary macrophages and expressed inflammatory macrophage cell surface markers including CD64 and CD68. LPS-stimulated macrophages expressed a global pattern of gene expression enriched in CD ileal inflammatory macrophages and matrisome secreted products, and produced cytokines and chemokines including CCL2, IL1B, and OSM implicated in refractory disease. ETYA suppressed CD64 abundance and pro-fibrotic gene expression pathways in LPS stimulated macrophages. Co-culture of LPS-primed macrophages with HIO led to up-regulation of fibroblast activation genes including ACTA2 and COL1A1 , and an increase in HIO collagen content. ETYA pre-treatment prevented pro-fibrotic effects of LPS-primed macrophages. Conclusions: ETYA inhibits pro-fibrotic effects of LPS-primed macrophages upon co-cultured HIO. This model may be used in future untargeted screens for small molecules to treat refractory CD.
ABSTRACT
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. Highlights: BMP signaling fine-tunes the co-differentiation of mesoderm and endoderm.The cellular composition in multilineage organoids resembles that of human fetal organs.Mesenchyme and endothelium co-developed within the organoids adopt organ-specific characteristics.Multilineage organoids recapitulate abnormal endothelial-epithelial crosstalk in FOXF1-associated disorders.
ABSTRACT
UGT1A1 (UDP glucuronosyltransferase family 1 member A1) is the primary enzyme required for bilirubin conjugation, which is essential for preventing hyperbilirubinemia. Animal models lack key human organic anion transporting polypeptides with distinct epigenetic control over bilirubin metabolism, necessitating a human model to interrogate the regulatory mechanism behind UGT1A1 function. Here, we use induced pluripotent stem cells to develop human liver organoids that can emulate conjugation failure phenotype. Bilirubin conjugation assays, chromatin immunoprecipitation, and transcriptome analysis elucidated the role of glucocorticoid antagonism in UGT1A1 activation. This antagonism prevents the binding of transcriptional repressor MECP2 at the expense of NRF2 with associated off-target effects. Therefore, we introduced functional GULO (L-gulonolactone oxidase) in human organoids to augment intracellular ascorbate for NRF2 reactivation. This engineered organoid conjugated more bilirubin and protected against hyperbilirubinemia when transplanted in immunosuppressed Crigler-Najjar syndrome rat model. Collectively, we demonstrate that our organoid system serves as a manipulatable model for interrogating hyperbilirubinemia and potential therapeutic development.
Subject(s)
Crigler-Najjar Syndrome , Pluripotent Stem Cells , Humans , Animals , Rats , Bilirubin/pharmacology , Bilirubin/metabolism , NF-E2-Related Factor 2/metabolism , Liver/metabolism , Crigler-Najjar Syndrome/genetics , Crigler-Najjar Syndrome/therapy , Hyperbilirubinemia/genetics , Hyperbilirubinemia/metabolism , Hyperbilirubinemia/therapy , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Pluripotent Stem Cells/metabolismABSTRACT
Development of visceral organs such as the esophagus, lung, liver and stomach are coordinated by reciprocal signaling interactions between the endoderm and adjacent mesoderm cells in the fetal foregut. Although the recent successes in recapitulating developmental signaling in vitro has enabled the differentiation of human pluripotent stem cells (hPSCs) into various types of organ-specific endodermal epithelium, the generation of organ-specific mesenchyme has received much less attention. This is a major limitation in ongoing efforts to engineer complex human tissue. Here, we describe a protocol to differentiate hPSCs into different types of organ-specific mesoderm, leveraging signaling networks and molecular markers elucidated from single-cell transcriptomics of mouse foregut organogenesis. Building on established methods, hPSC-derived lateral plate mesoderm treated with either retinoic acid (RA) or RA together with a Hedgehog (HH) agonist generates posterior or anterior foregut splanchnic mesoderm, respectively, after 4-d cultures. These are directed into organ-specific mesenchyme lineages by the combinatorial activation or inhibition of WNT, BMP, RA or HH pathways from days 4 to 7 in cultures. By day 7, the cultures are enriched for different types of mesoderm with distinct molecular signatures: 60-90% pure liver septum transversum/mesothelium-like, 70-80% pure liver-like fibroblasts and populations of ~35% respiratory-like mesoderm, gastric-like mesoderm or esophageal-like mesoderm. This protocol can be performed by anyone with moderate experience differentiating hPSCs, provides a novel platform to study human mesoderm development and can be used to engineer more complex foregut tissue for disease modeling and regenerative medicine.
Subject(s)
Hedgehog Proteins , Pluripotent Stem Cells , Humans , Mice , Animals , Hedgehog Proteins/metabolism , Mesoderm , Endoderm , Cell Differentiation , Tretinoin/pharmacology , LungABSTRACT
Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct the 140 kb inversion of the F8 gene frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we develop a coupled heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells. Transient RecF8 treatment of endothelial cells, differentiated from patient-derived induced pluripotent stem cells (iPSCs) of a hemophilic donor, results in 12% correction of the inversion and restores Factor VIII mRNA expression. In this work, we present designer-recombinases as an efficient and specific means towards treatment of monogenic diseases caused by large gene inversions.
Subject(s)
Chromosome Inversion/genetics , Factor VIII/genetics , Recombinases/metabolism , Amino Acid Sequence , Base Sequence , Cell Differentiation , Clone Cells , Directed Molecular Evolution , Endothelial Cells/cytology , Endothelial Cells/metabolism , Exons/genetics , HEK293 Cells , HeLa Cells , Humans , Induced Pluripotent Stem Cells/metabolism , Inverted Repeat Sequences/genetics , Recombination, Genetic/genetics , Substrate Specificity , Whole Genome SequencingABSTRACT
BACKGROUND: Perturbagen analysis of Crohn's disease (CD) ileal gene expression data identified small molecules including eicosatetraynoic acid (ETYA), which may exert an antifibrotic effect. We developed a patient-specific human intestinal organoid (HIO) model system to test small molecule regulation of mitochondrial and wound-healing functions implicated in stricturing behavior. METHODS: HIOs were made from CD induced pluripotent stem cells with and without a loss-of-function haplotype in the DUOX2 gene implicated in ileal homeostasis and characterized under basal conditions and following exposure to butyrate and ETYA using RNA sequencing, flow cytometry, and immunofluorescent and polarized light microscopy. Mitochondrial activity was measured using high-resolution respirometry and tissue stiffness using atomic force microscopy. RESULTS: HIOs expressed core mitochondrial and extracellular matrix (ECM) genes and enriched biologic functions implicated in CD ileal strictures; ECM gene expression was suppressed by both butyrate and ETYA, with butyrate also suppressing genes regulating epithelial proliferation. Consistent with this, butyrate, but not ETYA, exerted a profound effect on HIO epithelial mitochondrial function, reactive oxygen species production, and cellular abundance. Butyrate and ETYA suppressed HIO expression of alpha smooth muscle actin expressed by myofibroblasts, type I collagen, and collagen protein abundance. HIOs exhibited tissue stiffness comparable to normal human ileum; this was reduced by chronic ETYA exposure in HIOs carrying the DUOX2 loss-of-function haplotype. CONCLUSIONS: ETYA regulates ECM genes implicated in strictures and suppresses collagen content and tissue stiffness in an HIO model. HIOs provide a platform to test personalized therapeutics, including small molecules prioritized by perturbagen analysis.
A subset of pediatric Crohn's disease patients develop intestinal strictures requiring surgery. The microbial metabolite butyrate and eicosatetraynoic acid regulate pathways implicated in stricture formation in a human intestinal organoid model system, which may be used to test new therapies.
Subject(s)
Crohn Disease , Butyrates/metabolism , Butyrates/pharmacology , Collagen/metabolism , Constriction, Pathologic/metabolism , Crohn Disease/genetics , Dual Oxidases/metabolism , Extracellular Matrix/metabolism , Humans , Intestinal Mucosa/metabolism , Mitochondria/metabolism , Organoids/metabolismABSTRACT
Organoids are three-dimensional structures that self-organize from human pluripotent stem cells or primary tissue, potentially serving as a traceable and manipulatable platform to facilitate our understanding of organogenesis. Despite the ongoing advancement in generating organoids of diverse systems, biological applications of in vitro generated organoids remain as a major challenge in part due to a substantial lack of intricate complexity. The studies of development and regeneration enumerate the essential roles of highly diversified nonepithelial populations such as mesenchyme and endothelium in directing fate specification, morphogenesis, and maturation. Furthermore, organoids with physiological and homeostatic functions require direct and indirect inter-organ crosstalk recapitulating what is seen in organogenesis. We herein review the evolving organoid technology at the cell, tissue, organ, and system level with a main emphasis on endoderm derivatives.
Subject(s)
Organogenesis , Pluripotent Stem Cells , Endoderm , Humans , Morphogenesis , OrganoidsABSTRACT
Detailed herein is the protocol for synthesis, characterization, and application of POLYseq for cell pooling in single-cell sequencing runs. POLYseq is synthesized through commercially available reagents and is highly tailorable. Synthesis is easily performed in a two-step protocol, utilizing Michael addition only requiring a temperature-stable hot bath capable of holding 90°C. However, care must be taken when mixing reagents for synthesis, as the final product is sensitive to initial mixing ratios of POLYseq reagents. For complete details on the use and execution of this protocol, please refer to Dunn et al. (2021).
Subject(s)
Liver/cytology , Optical Imaging/methods , Organoids/cytology , Sequence Analysis, DNA/methods , Single-Cell Analysis/methods , HumansABSTRACT
Despite evolving biological application of next-generation sequencing (NGS) at single-cell level, current techniques in NGS library preparation restrict multiplexing, necessitating the costly preparation of distinct libraries for each sample. Here, we report the development of a novel poly(ß-amino) ester labeling system synthesized with inexpensive, common reagents, termed POLYseq, capable of efficiently delivering fluorescent molecules or sample-distinguishing DNA barcodes through non-covalent binding enabling rapid creation of custom sample pools. Chemical formulation was found to determine cellular labeling propensity. Live image-based tracking of fluorescent conjugated POLYseq vectors demonstrated lysosomal compartmentalization. Barcode labeling was uniformly detected across 90% of cells by single-cell RNA sequencing, allowing for the successful identification of human and mouse cultured cell lines from a single pool. These findings highlight the multifunctional applications of POLYseq in live cell imaging and NGS in a scalable and cost-effective manner.
Subject(s)
DNA Barcoding, Taxonomic/methods , High-Throughput Nucleotide Sequencing/methods , Polymers/chemistry , Staining and Labeling/methods , Cell Culture Techniques , Cell Differentiation/genetics , Flow Cytometry , Fluorescent Dyes , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Organoids , Single-Cell Analysis/methodsABSTRACT
Human organoids are emerging as a valuable resource to investigate human organ development and disease. The applicability of human organoids has been limited, partly due to the oversimplified architecture of the current technology, which generates single-tissue organoids that lack inter-organ structural connections. Thus, engineering organoid systems that incorporate connectivity between neighboring organs is a critical unmet challenge in an evolving organoid field. Here, we describe a protocol for the continuous patterning of hepatic, biliary and pancreatic (HBP) structures from a 3D culture of human pluripotent stem cells (PSCs). After differentiating PSCs into anterior and posterior gut spheroids, the two spheroids are fused together in one well. Subsequently, self-patterning of multi-organ (i.e., HBP) domains occurs within the boundary region of the two spheroids, even in the absence of any extrinsic factors. Long-term culture of HBP structures induces differentiation of the domains into segregated organs complete with developmentally relevant invagination and epithelial branching. This in-a-dish model of human hepato-biliary-pancreatic organogenesis provides a unique platform for studying human development, congenital disorders, drug development and therapeutic transplantation. More broadly, our approach could potentially be used to establish inter-organ connectivity models for other organ systems derived from stem cell cultures.
Subject(s)
Cell Culture Techniques/methods , Organoids/cytology , Tissue Engineering/methods , Bile Ducts/cytology , Cell Differentiation/physiology , Humans , Liver/cytology , Organogenesis/physiology , Organoids/metabolism , Pancreas/cytology , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolismABSTRACT
The host response to SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, demonstrates significant interindividual variability. In addition to showing more disease in males, the elderly, and individuals with underlying comorbidities, SARS-CoV-2 can seemingly afflict healthy individuals with profound clinical complications. We hypothesize that, in addition to viral load and host antibody repertoire, host genetic variants influence vulnerability to infection. Here we apply human induced pluripotent stem cell (hiPSC)-based models and CRISPR engineering to explore the host genetics of SARS-CoV-2. We demonstrate that a single-nucleotide polymorphism (rs4702), common in the population and located in the 3' UTR of the protease FURIN, influences alveolar and neuron infection by SARS-CoV-2 in vitro. Thus, we provide a proof-of-principle finding that common genetic variation can have an impact on viral infection and thus contribute to clinical heterogeneity in COVID-19. Ongoing genetic studies will help to identify high-risk individuals, predict clinical complications, and facilitate the discovery of drugs.
Subject(s)
COVID-19/genetics , Genetic Predisposition to Disease/genetics , Polymorphism, Single Nucleotide/genetics , 3' Untranslated Regions/genetics , Adolescent , Adult , Animals , COVID-19/virology , Cell Line , Chlorocebus aethiops , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Female , Furin/genetics , Host-Pathogen Interactions/genetics , Humans , Induced Pluripotent Stem Cells/virology , Male , Neurons/virology , Peptide Hydrolases/genetics , SARS-CoV-2/pathogenicity , Vero CellsABSTRACT
Visceral organs, such as the lungs, stomach and liver, are derived from the fetal foregut through a series of inductive interactions between the definitive endoderm (DE) and the surrounding splanchnic mesoderm (SM). While DE patterning is fairly well studied, the paracrine signaling controlling SM regionalization and how this is coordinated with epithelial identity is obscure. Here, we use single cell transcriptomics to generate a high-resolution cell state map of the embryonic mouse foregut. This identifies a diversity of SM cell types that develop in close register with the organ-specific epithelium. We infer a spatiotemporal signaling network of endoderm-mesoderm interactions that orchestrate foregut organogenesis. We validate key predictions with mouse genetics, showing the importance of endoderm-derived signals in mesoderm patterning. Finally, leveraging these signaling interactions, we generate different SM subtypes from human pluripotent stem cells (hPSCs), which previously have been elusive. The single cell data can be explored at: https://research.cchmc.org/ZornLab-singlecell .