Summary of study ST001185

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000796. The data can be accessed directly via it's Project DOI: 10.21228/M8SM3C This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST001185
Study TitleGenetic and metabolic characterization of bioengineered human fatty liver tissue with modified SIRT1 expression
Study SummaryLipidomics and metabolomics was performed three types of tissue samples to compare human normal liver tissue against human NASH liver and the bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1. The purpose of this study was to show that the global lipidomics profile of iPS-derived fatty liver tissue-iKD-SIRT1 was similar to that of patients with NASH
Institute
University of Pittsburgh
DepartmentDepartment of Pathology
Last NameSoto-Gutierrez
First NameAlejandro
Address200 Lothrop Street, 423 Biomedical Science Tower, Pittsburgh, PA 15261, USA
Emailals208@pitt.edu
Phone+14126480064
Submit Date2019-05-20
Num Groups3
Raw Data AvailableYes
Raw Data File Type(s).txt
Analysis Type DetailLC-MS
Release Date2020-01-06
Release Version1
Alejandro Soto-Gutierrez Alejandro Soto-Gutierrez
https://dx.doi.org/10.21228/M8SM3C
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000796
Project DOI:doi: 10.21228/M8SM3C
Project Title:Generation of human fatty liver using custom-engineered induced pluripotent stem cells with modifiable SIRT1 metabolism
Project Summary:The mechanisms by which steatosis of the liver progresses to non-alcoholic steatohepatitis, and endstage liver disease remain elusive. Metabolic derangements in hepatocytes controlled by SIRT1 indicate that this molecule plays a role in the development of fatty liver in inbred animals. The ability to perform similar studies using human tissue has been limited by the genetically variability in baseline SIRT1 expression in man. We now report generation of human induced pluripotent stem (iPS) cells with controlled expression of SIRT1. By differentiating edited iPS cells into hepatocytes and then knocking down (KD) SIRT1, we found that downregulated SIRT1 regulates lipid homeostasis by increasing Srebp1c (a transcription factor driving fatty acid biosynthesis), and by decreasing PPARa and its transcriptional co-activator PGC1a, to exacerbate fat accumulation. To model human fatty livers, we repopulated the parenchyma of decellularized rat livers with human mesenchymal cells, fibroblasts, macrophages, and human SIRT1-knockdown iPS-derived hepatocytes. When SIRT1-metabolism was modified, the human iPS-derived liver tissue developed macrosteatosis and generated cells with a proinflammatory phenotype. Our data indicate that SIRT1 plays an important role in the regulation of hepatic lipid homeostasis and inflammation in the human liver. Given the ability to generate and characterize bioengineered and genetically-edited human liver tissue, we believe that use of genetically modifiable human tissue may become an important tool for investigating human liver biology and disease.
Institute:University of Pittsburgh
Department:Department of Pathology
Last Name:Soto-Gutierrez
First Name:Alejandro
Address:200 Lothrop Street, 423 Biomedical Science Tower, Pittsburgh, PA 15261, USA
Email:als208@pitt.edu
Phone:+14126480064

Subject:

Subject ID:SU001251
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Source
SA082242R3Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1
SA082243R4Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1
SA082244R6Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1
SA082245R2Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1
SA082246R5Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1
SA082247R1Bioengineered human iPS-derived fatty liver tissue-iKD-SIRT1
SA082248N3Healthy Control Liver Tissue
SA082249N2Healthy Control Liver Tissue
SA082250N1Healthy Control Liver Tissue
SA082251F1NASH Liver Tissue from Patient
SA082252F2NASH Liver Tissue from Patient
SA082253F3NASH Liver Tissue from Patient
SA082254NegNegative Control Empty Scaffold
Showing results 1 to 13 of 13

Collection:

Collection ID:CO001245
Collection Summary:De-identified tissues were obtained from Magee Women’s Hospital (Pittsburgh, PA) and the University of Washington Department of Pediatrics, Division of Genetic Medicine, Laboratory of Developmental Biology (Seattle, WA) after obtaining a written informed consent by a protocol approved by the Human Research Review Committee of the University of Pittsburgh (Honest broker approval number HB015 and HB000836). Human fetal liver tissue and/or cells were isolated and culture from fetal livers as previously described (26890260). The de-identified normal human liver tissue and/or cells were obtained through the Liver Tissue Cell Distribution System (Pittsburgh, PA) after obtaining a written informed consent by a protocol approved by the Human Research Review Committee of the University of Pittsburgh, which was funded by NIH Contract # HSN276201200017C. Adult human liver tissue and/or cells were also obtained from Ira J Fox Laboratory at Children’s Hospital of UPMC, after obtaining a written informed consent by a protocol approved by the Human Research Review Committee and the Institutional Review Board (IRB#: PRO12090466) of the University of Pittsburgh.
Sample Type:Liver

Treatment:

Treatment ID:TR001266
Treatment Summary:Normal liver tissue and NASH liver tissue samples were not treated, and were directly analyzed for lipidomics profile. Bioengineered human iPS-derive fatty liver tissue was synthesized as described in the study referenced herein

Sample Preparation:

Sampleprep ID:SP001259
Sampleprep Summary:Human normal liver, human NASH liver and human iPS-derived fatty liver tissueiKD-SIRT1 samples were homogenized using a FastPrep system (MP Bio) with Matrix D ceramic beads in 80% MeOH at a ratio of 15 μL/mg tissue. The homogenate was spiked with isotopically labelled standards, taurine-1,1,2,2-d4 (final concentration 100 μM, Cambridge Isotopes MA) and 10 μL of a 50 μg/mL fatty acid internal standard mix. Chloroform (600 μL) was then added to the homogenate supernatant and the sample was vortexed and centrifuged at 1,500 x g for 5 min. The aqueous phase was taken for polar metabolite analysis and the organic phase was split for targeted free fatty acid analysis and untargeted lipidomics. Polar samples were cleared by centrifugation at 16,000 x g and the supernatant dried under N2. Samples were resuspended in 50 μL of 1.5 mM ammonium fluoride (aq) and 10 μL was injected for separation and analysis. The organic phase was dried under N2 and reconstituted in 100 μL of chloroform:methanol (2:1) and 5 μL was injected for untargeted lipidomics analysis.

Combined analysis:

Analysis ID AN001967 AN001968 AN001969
Analysis type MS MS MS
Chromatography type Reversed phase Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish Thermo Vanquish
Column Thermo Fisher Accucore C18 column (2.1 X 100 mm, 5 μm) Thermo Fisher Accucore C18 column (2.1 X 100 mm, 5 μm) Phenomenex Kinetex C18+ column (2.1 × 100 mm, 1.7 μm)
MS Type ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap
Ion Mode POSITIVE NEGATIVE UNSPECIFIED
Units Peak area normalized to internal standard Peak area normalized to internal standard Peak Area

Chromatography:

Chromatography ID:CH001423
Chromatography Summary:LIPIDOMICS: Samples were separated on a Thermo Fisher Accucore C18 column (2.1 X 100 mm, 5 μ pore size) using solvent A (H2O:ACN (1:1) with 10 mM ammonium acetate + 0.1% formic acid) and solvent B (IPA:ACN (9:1) with 10 mM ammonium acetate + 0.1% formic acid) at a flow rate of 0.2 mL/min. The gradient started at 0%B and increased to 50%B from 2-10 min following a second increase to 95%B from 10-47 min. The gradient was held for 4 min at 95%B before increasing to 100%B at 51 min for a 6 min wash. At 57 min the system was returned to initial conditions to equilibrate before the next injection. Total run time was 60 min.
Instrument Name:Thermo Vanquish
Column Name:Thermo Fisher Accucore C18 column (2.1 X 100 mm, 5 μm)
Chromatography Type:Reversed phase
  
Chromatography ID:CH001424
Chromatography Summary:METABOLOMICS: Briefly, samples were separated over a reversed phase Phenomenex Kinetex C18+ column (2.1 × 100 mm, 1.7 μm particle size) maintained at 40°C. For the 20 minute LC gradient, the mobile phase consisted of the following: solvent A (1.5mM ammonium fluoride) and solvent B (100% acetonitrile). The gradient was the following: 0-12.0 min 5% B, to 100% B, 12.0-15.0 min hold at 100% B, 15.0-15.1 100% to 5% B, 15.1-20.0 min 5%B.
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18+ column (2.1 × 100 mm, 1.7 μm)
Chromatography Type:Reversed phase

MS:

MS ID:MS001821
Analysis ID:AN001967
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:LIPIDOMICS: Samples were analyzed using full scan accurate mass at a resolution of 70K in positive and negative mode and 17.5K for ddMS2. Thermo Fisher LipidSearch 4.2.2 software was used for peak quantification, alignment, and MS2 identification. Peak areas were normalized to internal standard.
Ion Mode:POSITIVE
  
MS ID:MS001822
Analysis ID:AN001968
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:LIPIDOMICS: Samples were analyzed using full scan accurate mass at a resolution of 70K in positive and negative mode and 17.5K for ddMS2. Thermo Fisher LipidSearch 4.2.2 software was used for peak quantification, alignment, and MS2 identification. Peak areas were normalized to internal standard.
Ion Mode:NEGATIVE
  
MS ID:MS001823
Analysis ID:AN001969
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:METABOLOMICS: Metabolites were identified in both positive and negative mode by accurate mass (≤ 10 ppm) and retention time. Integrated peak areas were then extracted manually using Quan Browser (Thermo Fisher Xcalibur ver 2.7) and normalized to internal standard.
Ion Mode:UNSPECIFIED
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