#METABOLOMICS WORKBENCH abhinavachreja_20190520_200103 DATATRACK_ID:1727 STUDY_ID:ST001185 ANALYSIS_ID:AN001968 PROJECT_ID:PR000796 VERSION 1 CREATED_ON May 22, 2019, 1:52 pm #PROJECT PR:PROJECT_TITLE Generation of human fatty liver using custom-engineered induced pluripotent stem PR:PROJECT_TITLE cells with modifiable SIRT1 metabolism PR:PROJECT_SUMMARY The mechanisms by which steatosis of the liver progresses to non-alcoholic PR:PROJECT_SUMMARY steatohepatitis, and endstage liver disease remain elusive. Metabolic PR:PROJECT_SUMMARY derangements in hepatocytes controlled by SIRT1 indicate that this molecule PR:PROJECT_SUMMARY plays a role in the development of fatty liver in inbred animals. The ability to PR:PROJECT_SUMMARY perform similar studies using human tissue has been limited by the genetically PR:PROJECT_SUMMARY variability in baseline SIRT1 expression in man. We now report generation of PR:PROJECT_SUMMARY human induced pluripotent stem (iPS) cells with controlled expression of SIRT1. PR:PROJECT_SUMMARY By differentiating edited iPS cells into hepatocytes and then knocking down (KD) PR:PROJECT_SUMMARY SIRT1, we found that downregulated SIRT1 regulates lipid homeostasis by PR:PROJECT_SUMMARY increasing Srebp1c (a transcription factor driving fatty acid biosynthesis), and PR:PROJECT_SUMMARY by decreasing PPARa and its transcriptional co-activator PGC1a, to exacerbate PR:PROJECT_SUMMARY fat accumulation. To model human fatty livers, we repopulated the parenchyma of PR:PROJECT_SUMMARY decellularized rat livers with human mesenchymal cells, fibroblasts, PR:PROJECT_SUMMARY macrophages, and human SIRT1-knockdown iPS-derived hepatocytes. When PR:PROJECT_SUMMARY SIRT1-metabolism was modified, the human iPS-derived liver tissue developed PR:PROJECT_SUMMARY macrosteatosis and generated cells with a proinflammatory phenotype. Our data PR:PROJECT_SUMMARY indicate that SIRT1 plays an important role in the regulation of hepatic lipid PR:PROJECT_SUMMARY homeostasis and inflammation in the human liver. Given the ability to generate PR:PROJECT_SUMMARY and characterize bioengineered and genetically-edited human liver tissue, we PR:PROJECT_SUMMARY believe that use of genetically modifiable human tissue may become an important PR:PROJECT_SUMMARY tool for investigating human liver biology and disease. PR:INSTITUTE University of Pittsburgh PR:DEPARTMENT Department of Pathology PR:LAST_NAME Soto-Gutierrez PR:FIRST_NAME Alejandro PR:ADDRESS 200 Lothrop Street, 423 Biomedical Science Tower, Pittsburgh, PA 15261, USA PR:EMAIL als208@pitt.edu PR:PHONE +14126480064 #STUDY ST:STUDY_TITLE Genetic and metabolic characterization of bioengineered human fatty liver tissue ST:STUDY_TITLE with modified SIRT1 expression ST:STUDY_SUMMARY Lipidomics and metabolomics was performed three types of tissue samples to ST:STUDY_SUMMARY compare human normal liver tissue against human NASH liver and the bioengineered ST:STUDY_SUMMARY human iPS-derived fatty liver tissue-iKD-SIRT1. The purpose of this study was to ST:STUDY_SUMMARY show that the global lipidomics profile of iPS-derived fatty liver ST:STUDY_SUMMARY tissue-iKD-SIRT1 was similar to that of patients with NASH ST:INSTITUTE University of Pittsburgh ST:DEPARTMENT Department of Pathology ST:LAST_NAME Soto-Gutierrez ST:FIRST_NAME Alejandro ST:ADDRESS 200 Lothrop Street, 423 Biomedical Science Tower, Pittsburgh, PA 15261, USA ST:EMAIL als208@pitt.edu ST:PHONE +14126480064 ST:NUM_GROUPS 3 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - N1 Source:Healthy Control Liver Tissue SUBJECT_SAMPLE_FACTORS - N2 Source:Healthy Control Liver Tissue SUBJECT_SAMPLE_FACTORS - N3 Source:Healthy Control Liver Tissue SUBJECT_SAMPLE_FACTORS - F1 Source:NASH Liver Tissue from Patient SUBJECT_SAMPLE_FACTORS - F2 Source:NASH Liver Tissue from Patient SUBJECT_SAMPLE_FACTORS - F3 Source:NASH Liver Tissue from Patient SUBJECT_SAMPLE_FACTORS - R1 Source:Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1 SUBJECT_SAMPLE_FACTORS - R2 Source:Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1 SUBJECT_SAMPLE_FACTORS - R3 Source:Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1 SUBJECT_SAMPLE_FACTORS - R4 Source:Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1 SUBJECT_SAMPLE_FACTORS - R5 Source:Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1 SUBJECT_SAMPLE_FACTORS - R6 Source:Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1 SUBJECT_SAMPLE_FACTORS - Neg Source:Negative Control Empty Scaffold #COLLECTION CO:COLLECTION_SUMMARY De-identified tissues were obtained from Magee Women’s Hospital (Pittsburgh, CO:COLLECTION_SUMMARY PA) and the University of Washington Department of Pediatrics, Division of CO:COLLECTION_SUMMARY Genetic Medicine, Laboratory of Developmental Biology (Seattle, WA) after CO:COLLECTION_SUMMARY obtaining a written informed consent by a protocol approved by the Human CO:COLLECTION_SUMMARY Research Review Committee of the University of Pittsburgh (Honest broker CO:COLLECTION_SUMMARY approval number HB015 and HB000836). Human fetal liver tissue and/or cells were CO:COLLECTION_SUMMARY isolated and culture from fetal livers as previously described (26890260). The CO:COLLECTION_SUMMARY de-identified normal human liver tissue and/or cells were obtained through the CO:COLLECTION_SUMMARY Liver Tissue Cell Distribution System (Pittsburgh, PA) after obtaining a written CO:COLLECTION_SUMMARY informed consent by a protocol approved by the Human Research Review Committee CO:COLLECTION_SUMMARY of the University of Pittsburgh, which was funded by NIH Contract # CO:COLLECTION_SUMMARY HSN276201200017C. Adult human liver tissue and/or cells were also obtained from CO:COLLECTION_SUMMARY Ira J Fox Laboratory at Children’s Hospital of UPMC, after obtaining a written CO:COLLECTION_SUMMARY informed consent by a protocol approved by the Human Research Review Committee CO:COLLECTION_SUMMARY and the Institutional Review Board (IRB#: PRO12090466) of the University of CO:COLLECTION_SUMMARY Pittsburgh. CO:SAMPLE_TYPE Liver #TREATMENT TR:TREATMENT_SUMMARY Normal liver tissue and NASH liver tissue samples were not treated, and were TR:TREATMENT_SUMMARY directly analyzed for lipidomics profile. Bioengineered human iPS-derive fatty TR:TREATMENT_SUMMARY liver tissue was synthesized as described in the study referenced herein #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Human normal liver, human NASH liver and human iPS-derived fatty liver SP:SAMPLEPREP_SUMMARY tissueiKD-SIRT1 samples were homogenized using a FastPrep system (MP Bio) with SP:SAMPLEPREP_SUMMARY Matrix D ceramic beads in 80% MeOH at a ratio of 15 μL/mg tissue. The SP:SAMPLEPREP_SUMMARY homogenate was spiked with isotopically labelled standards, taurine-1,1,2,2-d4 SP:SAMPLEPREP_SUMMARY (final concentration 100 μM, Cambridge Isotopes MA) and 10 μL of a 50 μg/mL SP:SAMPLEPREP_SUMMARY fatty acid internal standard mix. Chloroform (600 μL) was then added to the SP:SAMPLEPREP_SUMMARY homogenate supernatant and the sample was vortexed and centrifuged at 1,500 x g SP:SAMPLEPREP_SUMMARY for 5 min. The aqueous phase was taken for polar metabolite analysis and the SP:SAMPLEPREP_SUMMARY organic phase was split for targeted free fatty acid analysis and untargeted SP:SAMPLEPREP_SUMMARY lipidomics. Polar samples were cleared by centrifugation at 16,000 x g and the SP:SAMPLEPREP_SUMMARY supernatant dried under N2. Samples were resuspended in 50 μL of 1.5 mM SP:SAMPLEPREP_SUMMARY ammonium fluoride (aq) and 10 μL was injected for separation and analysis. The SP:SAMPLEPREP_SUMMARY organic phase was dried under N2 and reconstituted in 100 μL of SP:SAMPLEPREP_SUMMARY chloroform:methanol (2:1) and 5 μL was injected for untargeted lipidomics SP:SAMPLEPREP_SUMMARY analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY LIPIDOMICS: Samples were separated on a Thermo Fisher Accucore C18 column (2.1 X CH:CHROMATOGRAPHY_SUMMARY 100 mm, 5 μ pore size) using solvent A (H2O:ACN (1:1) with 10 mM ammonium CH:CHROMATOGRAPHY_SUMMARY acetate + 0.1% formic acid) and solvent B (IPA:ACN (9:1) with 10 mM ammonium CH:CHROMATOGRAPHY_SUMMARY acetate + 0.1% formic acid) at a flow rate of 0.2 mL/min. The gradient started CH:CHROMATOGRAPHY_SUMMARY at 0%B and increased to 50%B from 2-10 min following a second increase to 95%B CH:CHROMATOGRAPHY_SUMMARY from 10-47 min. The gradient was held for 4 min at 95%B before increasing to CH:CHROMATOGRAPHY_SUMMARY 100%B at 51 min for a 6 min wash. At 57 min the system was returned to initial CH:CHROMATOGRAPHY_SUMMARY conditions to equilibrate before the next injection. Total run time was 60 min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Thermo Fisher Accucore C18 column (2.1 X 100 mm, 5 µm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS LIPIDOMICS: Samples were analyzed using full scan accurate mass at a resolution MS:MS_COMMENTS of 70K in positive and negative mode and 17.5K for ddMS2. Thermo Fisher MS:MS_COMMENTS LipidSearch 4.2.2 software was used for peak quantification, alignment, and MS2 MS:MS_COMMENTS identification. Peak areas were normalized to internal standard. MS:MS_RESULTS_FILE ST001185_AN001968_Results.txt UNITS:Peak area normalized to internal standard Has m/z:Yes Has RT:Yes RT units:Minutes #END