Summary of Study ST003331
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 PR002070. The data can be accessed directly via it's Project DOI: 10.21228/M81Z4C 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.
| Study ID | ST003331 |
| Study Title | Increased Cholesterol Synthesis Drives Neurotoxicity in Patient Stem Cell-Derived Model of Multiple Sclerosis - cellular metabolomics |
| Study Summary | Fibroblasts from skin biopsies from patients with progressive multiple sclerosis (PMS) and age-matched controls (AMC) were derived and reprogrammed to yield induced neural stem cells (iNSCs). The resulting iNSCs were cultured in the presence of 13C6 glucose for 24h to assess changes in the intracellular metabolome, in particular changes in the flux of glucose-derived carbon into the TCA cycle along with steady state levels of compounds in other areas of the metabolome. |
| Institute | University of Colorado Denver |
| Last Name | Haines |
| First Name | Julie |
| Address | 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA |
| julie.haines@cuanschutz.edu | |
| Phone | 3037243339 |
| Submit Date | 2024-07-19 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-08-08 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002070 |
| Project DOI: | doi: 10.21228/M81Z4C |
| Project Title: | Increased Cholesterol Synthesis Drives Neurotoxicity in Patient Stem Cell-Derived Model of Multiple Sclerosis |
| Project Summary: | Senescent neural progenitor cells have been identified in brain lesions of people with progressive multiple sclerosis (PMS). However, their role in disease pathobiology and contribution to the lesion environment remains unclear. By establishing directly induced neural stem/progenitor cell (iNSC) lines from PMS patient fibroblasts, we studied their senescent phenotype in vitro. Senescence was strongly associated with inflammatory signaling, hypermetabolism, and the senescence associated secretory phenotype (SASP). PMS-derived iNSCs displayed increased glucose-dependent fatty acid and cholesterol synthesis, which resulted in the accumulation of cholesteryl ester-enriched lipid droplets. An HMG-CoA reductase-mediated lipogenic state was found to induce secretion of the SASP in PMS iNSC conditioned media via transcriptional regulation by cholesterol-dependent transcription factors. SASP from PMS iNSCs induced neurotoxicity. Chemical targeting of HMG-CoA reductase using the cholesterol-lowering drug simvastatin (SV) reprogrammed the SASP and rescued neurotoxicity. Our findings suggest a disease-associated, cholesterol-related, hypermetabolic phenotype of PMS iNSCs that leads to neurotoxic signaling and is rescuable pharmacologically. |
| Institute: | University of Colorado Denver |
| Laboratory: | Lab of Angelo D'Alessandro in collaboration with lab of Stefano Pluchino (Univ of Cambridge) |
| Last Name: | Haines |
| First Name: | Julie |
| Address: | 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA |
| Email: | julie.haines@cuanschutz.edu |
| Phone: | 3037243339 |
Subject:
| Subject ID: | SU003452 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Female |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Disease factor | Sample source |
|---|---|---|---|
| SA362439 | DN47-10 | AMC | induced neural stem cell |
| SA362440 | DN47-02 | AMC | induced neural stem cell |
| SA362441 | DN47-12 | AMC | induced neural stem cell |
| SA362442 | DN47-11 | AMC | induced neural stem cell |
| SA362443 | DN47-01 | AMC | induced neural stem cell |
| SA362444 | DN47-06 | AMC | induced neural stem cell |
| SA362445 | DN47-05 | AMC | induced neural stem cell |
| SA362446 | DN47-04 | AMC | induced neural stem cell |
| SA362447 | DN47-03 | AMC | induced neural stem cell |
| SA362448 | DN47-13 | PMS | induced neural stem cell |
| SA362449 | DN47-14 | PMS | induced neural stem cell |
| SA362450 | DN47-16 | PMS | induced neural stem cell |
| SA362451 | DN47-17 | PMS | induced neural stem cell |
| SA362452 | DN47-18 | PMS | induced neural stem cell |
| SA362453 | DN47-19 | PMS | induced neural stem cell |
| SA362454 | DN47-20 | PMS | induced neural stem cell |
| SA362455 | DN47-21 | PMS | induced neural stem cell |
| SA362456 | DN47-22 | PMS | induced neural stem cell |
| SA362457 | DN47-23 | PMS | induced neural stem cell |
| SA362458 | DN47-24 | PMS | induced neural stem cell |
| SA362459 | DN47-15 | PMS | induced neural stem cell |
| Showing results 1 to 21 of 21 |
Collection:
| Collection ID: | CO003445 |
| Collection Summary: | 3-5 mm skin biopsies were collected in Biopsy Collection Medium (RPMI 1460 [Thermo Fisher] with 1X Antibiotic-Antimycotic [Thermo Fisher]), cut into smaller pieces (<1 mm) and plated onto a TC-treated 35 mm dish in Biopsy Plating Medium, composed by Knockout DMEM (Thermo Fisher), 2 mM GlutaMax (Thermo Fisher), 0.1 mM non-essential amino acids (Thermo Fisher), 0.1 mM β-Mercaptoethanol (Thermo Fisher), 10% Fetal Bovine Serum (FBS) (Thermo Fisher), 1X Penicillin-Streptomycin (P/S) (Thermo Fisher) and 1% Nucleosides (Millipore). Once the first fibroblasts migrated out of the biopsies, the cultures were maintained in growth medium (DMEM Glutamax I [Thermo Fisher] supplemented with 10% fetal bovine serum, 1% non-essential amino acids and 1 mM sodium pyruvate (Thermo Fisher) at 37°C with 5 % CO2 and fed every 3-4 days. After reaching 90% confluency the fibroblasts were detached with trypsin-EDTA 0.05% for 5 min followed by neutralization in DMEM and spun down at 300xg for 5 min. They were split 1:4 into growth media. Fibroblasts at passage 3-5 were reprogrammed to iPSCs using the integration free technology based on non-modified RNA plus microRNA (kit from REPROCELL, formerly Stemgent), following manufacturer’s instructions. About 25x10^3 fibroblasts/well were plated onto Matrigel-coated 12-well plates in culture medium for 24 hours and then in NuFF-conditioned Pluriton reprogramming medium with B18R. Cells were transfected for 11 consecutive days using Stemfect as following: day 0 microRNA only, days 1-3 RNA only, day 4 microRNA plus RNA, days 5-11 RNA only. From day 11, TRA-1-60+ colonies (live stained) were manually picked and re-plated on mouse embryonic fibroblasts in HUESM medium (Knockout-DMEM, 20% knock-out serum, glutamax 2mM, NEAA 0.1mM, 1X P/S and β-mercaptoethanol 0.1mM [Thermo Fisher]). Pluripotent colonies were passaged and adapted to feeder-free conditions with hESC matrigel (Corning) and mTeSR1 (STEMCELL Technologies) medium. The WIBJ iPSC line, obtained from Cambridge BioResource as a gift from Alessandra Granada, was reprogrammed from fibroblasts using the CytoTune 1 non-integrating kit (Thermo Fisher). iPSC media was changed every day. When confluent, cells were lifted using accutase (Thermo Fisher), spun at 300xg for 5 min, and split 1:10-1:20 onto hESC-matrigel coated plates with Y-27632 (10 uM) (Thermo Fisher) in mTeSR1 media. To generate directly induced neural stem cells (iNSCs) from fibroblasts we used a nonintegrating Sendai virus-based direct conversion strategy, as previously described (https://doi.org/10.3791/52831). Briefly, fibroblasts were seeded at 75,000/well in fibroblast media in a non-coated 12-well. On the next day the fibroblasts were transduced using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher) with hKOS (MOI: 3), hc-Myc (MOI: 3), hKlf4 (MOI: 3). The day following transfection, the medium was switched to neural induction medium (NIM) [DMEM:F12 and Neurobasal (1:1), supplemented with N2 supplement (1x, ThermoFisher), 1% glutamax, B27 supplement (1x, ThermoFisher), CHIR99021 (3 µM, Cell Guidance Systems), SB-431542 (2 µM, Cayman Chemicals) and hLIF (10 ng/ml, Cell Signaling Technology)], and cells were moved to 39°C with 5% CO2 to achieve viral clearance over 14 days. A few samples were collected here to generate positive controls for quality control assays. Medium changes were performed every other day. Following 25 days of transfection, iNSC colonies were manually selected, seeded onto Growth Factor Reduced (GFR) Matrigel Matrix (1:20 in DMEM/F12) coated plates for expansion, and subjected to quality control assays. iNSCs were maintained in NIM media until 70% confluent, then lifted using accutase (ThermoFisher), spun at 300xg for 3 mins, and plated onto GFR-Matrigel coated plates with Y-27632 (10 µM, Miltenyi Biotec) between 1:3-1:5 in NIM media. Media was changed every second day as needed. Experiments were performed on cells from passages 20-40. |
| Sample Type: | Stem cells |
Treatment:
| Treatment ID: | TR003461 |
| Treatment Summary: | iNSCs were seeded in NIM with Y-27632 (10 µM) at a density of 100,000 cells/cm2 GFR-matrigel coated wells, in technical triplicates per line. Medium was changed the next day. Glucose tracer experiments were run on day 2 after seeding by removing the culture medium and adding glucose-free NIM [DMEM:F12 w/o glucose and Neurobasal (1:1), supplemented with N2 supplement (1x, ThermoFisher), Glutamax (Thermo Fisher, 3.2 mM), pyruvate (Agilent, 0.35 mM), B27 supplement (1x, ThermoFisher), CHIR99021 (3 µM, Cell Guidance Systems), SB-431542 (2 µM, Cayman Chemicals) and hLIF (10 ng/ml, Cell Signaling Technology)] supplemented with D-Glucose-13C6 (ThermoFisher, 20.4 mM). Samples were collected after 24 hours for optimal resolution into TCA cycle and anabolic metabolic pathways. Sample collection was performed on ice. Conditioned culture medium was collected, spun at 300 x g for 5 minutes to remove cellular debris and 100 µL was stored at -80 oC until sample processing. Cells were then lifted using accutase and spun at 300 x g for 5 mins. Cell pellets were resuspended in PBS for counting and spun once more at 300 x g for 5 mins. Supernatants were aspirated and cell pellets were stored at -80oC until sample processing. |
Sample Preparation:
| Sampleprep ID: | SP003459 |
| Sampleprep Summary: | Metabolites from frozen pellets were extracted at 2e6 cells per mL using ice cold 5:3:2 methanol:acetonitrile:water (v/v/v) with vigorous vortexing at 4 degrees C followed by centrifugation as described for 10 min at 18,000 g at 4°C and stored at −80°C until analysis. |
| Processing Storage Conditions: | 4℃ |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN005456 | AN005457 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | NEGATIVE | POSITIVE |
| Units | peak area | peak area |
Chromatography:
| Chromatography ID: | CH004143 |
| Chromatography Summary: | Negative C18 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min 100-0% B, 3-5 min hold at 0% B |
| Flow Rate: | 450 uL/min |
| Sample Injection: | 6 uL |
| Solvent A: | 95% water/5% acetonitrile; 1 mM ammonium acetate |
| Solvent B: | 95% acetonitrile/5% water; 1 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH004144 |
| Chromatography Summary: | Positive C18 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min 95-5% B, 3-5 min hold at 5% B |
| Flow Rate: | 450 uL/min |
| Sample Injection: | 6 uL |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100%acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS005182 |
| Analysis ID: | AN005456 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS005183 |
| Analysis ID: | AN005457 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database. |
| Ion Mode: | POSITIVE |
