#METABOLOMICS WORKBENCH abhinavachreja_20190520_200103 DATATRACK_ID:1727 STUDY_ID:ST001185 ANALYSIS_ID:AN001969 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 METABOLOMICS: Briefly, samples were separated over a reversed phase Phenomenex CH:CHROMATOGRAPHY_SUMMARY Kinetex C18+ column (2.1 × 100 mm, 1.7 μm particle size) maintained at 40°C. CH:CHROMATOGRAPHY_SUMMARY For the 20 minute LC gradient, the mobile phase consisted of the following: CH:CHROMATOGRAPHY_SUMMARY solvent A (1.5mM ammonium fluoride) and solvent B (100% acetonitrile). The CH:CHROMATOGRAPHY_SUMMARY gradient was the following: 0-12.0 min 5% B, to 100% B, 12.0-15.0 min hold at CH:CHROMATOGRAPHY_SUMMARY 100% B, 15.0-15.1 100% to 5% B, 15.1-20.0 min 5%B. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18+ column (2.1 × 100 mm, 1.7 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 UNSPECIFIED MS:MS_COMMENTS METABOLOMICS: Metabolites were identified in both positive and negative mode by MS:MS_COMMENTS accurate mass (≤ 10 ppm) and retention time. Integrated peak areas were then MS:MS_COMMENTS extracted manually using Quan Browser (Thermo Fisher Xcalibur ver 2.7) and MS:MS_COMMENTS normalized to internal standard. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples N1 N2 N3 F1 F2 F3 R1 R2 R3 R4 R5 R6 Factors Source:Healthy Control Liver Tissue Source:Healthy Control Liver Tissue Source:Healthy Control Liver Tissue Source:NASH Liver Tissue from Patient Source:NASH Liver Tissue from Patient Source:NASH Liver Tissue from Patient Source:Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1 Source:Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1 Source:Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1 Source:Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1 Source:Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1 Source:Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1 Aspartic acid 1424.176465 4508.442663 2288.947546 2950.338909 1967.465406 3034.113558 1481.396135 3715.999464 2701.764303 2572.191186 445.5967253 328.9367329 Alanine 774.3639101 6924.49552 1324.709917 2526.633827 2122.223742 2775.450311 1191.22904 1447.392229 390.0973716 795.2065001 752.3773785 650.2042584 Serine 142.8815832 553.3631629 386.1622378 823.5484338 578.6046444 370.9040017 2026.971372 2351.35514 1382.972673 2963.262641 317.1591059 714.5148768 Arginine 47.54567788 279.9589255 525.294541 1227.564606 484.2619503 398.2466176 26125.03112 98079.49032 45801.12346 42910.24026 101377.5454 26696.38537 Valine 6486.653458 38511.77101 2710.994926 20225.96699 26108.22033 26324.31055 12846.09825 66201.07293 123166.9397 15201.77107 115391.7803 14610.21339 Lysine 218.9291362 6860.793212 1509.868999 6468.14712 0 0 1542.559869 5069.02367 4175.645728 2564.529574 2158.106187 1773.190471 Creatinine 2086.726047 6181.820356 1691.145259 747.3710115 1353.898986 4052.881284 0 0 0 0 0 0 Histidine 4476.330171 31853.60193 10709.0007 23464.71279 10529.43388 21996.27993 25544.61042 99398.60708 63054.67742 31712.7911 95252.34045 31389.49202 Leucine 35725.94265 215962.1528 13404.92391 100069.8945 142676.5587 166747.5916 42619.57053 212011.7284 401723.1506 47731.94537 370107.5525 47122.65352 Methionine 374.5242132 1335.124358 1007.515097 0 1591.713646 1217.194206 2153.920941 2135.637126 1693.22094 4015.461223 1695.249859 1962.919632 Tyrosine 319.3998518 2687.022068 3016.094745 7840.579691 1417.913783 1462.105426 4547.409265 13736.54297 4834.099105 7611.918264 9863.006551 3709.894307 Ornithine 226.3115715 2492.983183 395.9936212 2659.75445 4053.142267 1181.458473 364.6256063 0 1099.862178 283.9215978 1198.618935 117.5291032 Adenosine 25910.49853 387617.2494 13415.46792 148537.2452 79858.12583 49513.9355 0 22328.96153 21574.11055 975.4840489 92436.60938 3316.6781 Tryptophan 11348.89594 60759.30861 3990.078845 36790.39404 47317.04922 69113.36673 13417.61322 80346.45021 135851.7031 11205.58662 92516.42971 7814.183517 Succinic acid 303.7648201 3729.648921 1187.834532 452.2815827 254.5923022 935.7676259 847.6636815 1408.360578 139.7446807 1766.282541 158.3330668 979.8537727 Hexose 3963.160432 12974.20144 3133.765468 4924.059712 2919.141727 4858.372662 365.2896013 459.6012741 1569.53385 957.7227983 2349.996757 915.5254178 Itaconic acid 2264.156835 5120.013669 733.0741007 2490.42518 2714.220863 2169.23741 549.1288108 414.0137294 311.3466068 517.3307934 277.9989695 400.3776948 Oxoglutaric acid 216.0285612 655.4620144 148.2156835 601.4465468 327.478705 296.787482 172.5894829 634.0037227 60.64774682 114.7913317 1430.273322 333.1663015 Fructose-1,6-biphosphate 531.4866906 2011.023741 191.7999281 236.9853957 351.6920144 535.2071942 0 134.5245186 248.2698227 0 109.9205927 0 Glucose-6-phosphate 10623.7554 91699.46043 12832.72662 12149.27338 10283.64029 7922.079137 662.2896245 2028.852881 1840.164461 0 1908.015682 0 Fumaric acid 7981.122302 15503.1223 1846.851799 3596.009353 4101.233813 7530.028777 359.1684152 1195.678832 736.5252191 657.1972817 2531.2216 208.6118698 Citric acid 0 0 161.2580576 876.7489209 0 0 48.05691645 19.09509786 24.13493729 0 33.2520981 69.73154977 Glutamic acid 10159.30935 16029.80576 9026.705036 18185.02878 11164.48201 16087.39568 8459.586873 21288.27287 10958.56575 6623.250166 13192.80483 3899.897866 Glutamine 1608.633094 3124.291367 3503.876978 1290.656115 796 1864.1 8288.403716 12022.47435 7699.397843 16191.35808 9567.967204 9636.161426 CAR(2:0) 9505.899281 26066.10072 6305.629496 4840.823741 4856.310072 9821.28777 0 0 0 0 0 0 Cis-Aconitic acid 2420.204317 4642.860432 581.0433813 1093.901439 2395.315827 1926.984892 3.492999907 6.345401216 25.56015204 0 55.20095076 12.56735935 Phosphoenolpyruvic acid 0 0 0 0 0 0 0 2086.804004 5620.469891 0 0 0 Lactic acid 55910.65468 262717.1583 21406.7554 48909.56835 64138.60432 208399.6259 65315.29695 130059.4699 96928.86361 33358.86874 59023.24026 28047.75278 Pyruvic acid 975.6136691 3107.589209 0 788.0805755 1100.61295 2457.440288 0 0 0 0 0 0 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Formula REGNO Retention Times (min) Aspartic acid C4H7NO4 37126 1.11 Alanine C3H7NO2 37109 1.25 Serine C3H7NO3 37123 1.24 Arginine C6H14N4O2 37289 1.38 Valine C5H11NO2 37484 1.31 Lysine C6H14N2O2 37121 1.32 Creatinine C4H7N3O 37310 1.47 Histidine C6H9N3O2 37119 1.49 Leucine C6H13NO2 42493 1.55 Methionine C5H11NO2S 37371 1.45 Tyrosine C9H11NO3 37107 1.52 Ornithine C5H12N2O2 37139 1.48 Adenosine C10H13N5O4 37045 2.57 Tryptophan C11H12N2O2 37505 3.02 Succinic acid C4H6O4 1966 1.22 Hexose C6H12O6 71389 1.25 Itaconic acid C5H6O4 1984 1.26 Oxoglutaric acid C5H6O5 37135 1.2 Fructose-1,6-biphosphate C6H14O12P2 49892 1.09 Glucose-6-phosphate C6H13O9P 37776 1.12 Fumaric acid C4H4O4 37095 1.13 Citric acid C6H8O7 37071 1.14 Glutamic acid C5H9NO4 37101 1.11 Glutamine C5H10N2O3 37346 1.25 CAR(2:0) C9H17NO4 71521 1.49 Cis-Aconitic acid C6H6O6 37059 1.34 Phosphoenolpyruvic acid C3H5O6P 37169 1.36 Lactic acid C3H6O3 37125 1.24 Pyruvic acid C3H4O3 1584 1.33 METABOLITES_END #END