Summary of Study ST003328

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	ST003328
Study Title	Increased Cholesterol Synthesis Drives Neurotoxicity in Patient Stem Cell-Derived Model of Multiple Sclerosis - cellular lipidomics
Study Summary	Lipidomics analysis was performed on directly induced neural stem/progenitor cell (iNSC) lines derived from fibroblasts of patients with progressive multiple sclerosis (PMS) versus age matched controls (AMC) treated or untreated with cholesterol synthesis inhibitor simvastatin.
Institute	University of Colorado Denver
Last Name	Haines
First Name	Julie
Address	12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
Email	julie.haines@cuanschutz.edu
Phone	3037243339
Submit Date	2024-07-17
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:	SU003449
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	25-63
Gender:	Male and female

Factors:

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

mb_sample_id	local_sample_id	Disease status	Treatment	Sample source
SA362361	AMC A SV P3	AMC	SV	induced neural stem cell
SA362362	AMC C SV P3	AMC	SV	induced neural stem cell
SA362363	AMC C SV P2	AMC	SV	induced neural stem cell
SA362364	AMC C SV P1	AMC	SV	induced neural stem cell
SA362365	AMC B SV P3	AMC	SV	induced neural stem cell
SA362366	AMC B SV P2	AMC	SV	induced neural stem cell
SA362367	AMC B SV P1	AMC	SV	induced neural stem cell
SA362368	AMC A SV P1	AMC	SV	induced neural stem cell
SA362369	AMC A SV P2	AMC	SV	induced neural stem cell
SA362370	AMC A P2	AMC	untreated	induced neural stem cell
SA362371	AMC A P1	AMC	untreated	induced neural stem cell
SA362372	AMC C P3	AMC	untreated	induced neural stem cell
SA362373	AMC B P1	AMC	untreated	induced neural stem cell
SA362374	AMC C P2	AMC	untreated	induced neural stem cell
SA362375	AMC A P3	AMC	untreated	induced neural stem cell
SA362376	AMC B P2	AMC	untreated	induced neural stem cell
SA362377	AMC B P3	AMC	untreated	induced neural stem cell
SA362378	AMC C P1	AMC	untreated	induced neural stem cell
SA362379	PMS D SV P3	PMS	SV	induced neural stem cell
SA362380	PMS D SV P2	PMS	SV	induced neural stem cell
SA362381	PMS D SV P1	PMS	SV	induced neural stem cell
SA362382	PMA A SV P2	PMS	SV	induced neural stem cell
SA362383	PMS C SV P3	PMS	SV	induced neural stem cell
SA362384	PMS C SV P2	PMS	SV	induced neural stem cell
SA362385	PMS C SV P1	PMS	SV	induced neural stem cell
SA362386	PMA B SV P3	PMS	SV	induced neural stem cell
SA362387	PMA B SV P2	PMS	SV	induced neural stem cell
SA362388	PMA B SV P1	PMS	SV	induced neural stem cell
SA362389	PMA A SV P3	PMS	SV	induced neural stem cell
SA362390	PMA A SV P1	PMS	SV	induced neural stem cell
SA362391	PMS D P1	PMS	untreated	induced neural stem cell
SA362392	PMS D P3	PMS	untreated	induced neural stem cell
SA362393	PMS D P2	PMS	untreated	induced neural stem cell
SA362394	PMS C P3	PMS	untreated	induced neural stem cell
SA362395	PMS C P2	PMS	untreated	induced neural stem cell
SA362396	PMS C P1	PMS	untreated	induced neural stem cell
SA362397	PMA B P3	PMS	untreated	induced neural stem cell
SA362398	PMA B P2	PMS	untreated	induced neural stem cell
SA362399	PMA A P3	PMS	untreated	induced neural stem cell
SA362400	PMA A P2	PMS	untreated	induced neural stem cell
SA362401	PMA A P1	PMS	untreated	induced neural stem cell
SA362402	PMA B P1	PMS	untreated	induced neural stem cell

Showing results 1 to 42 of 42

Showing results 1 to 42 of 42

Collection:

Collection ID:	CO003442
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 2 mM, NEAA 0.1 mM, 1X P/S and β-mercaptoethanol 0.1 mM [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:	TR003458
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. Simvastatin-treated cells were exposed to 10 uM simvastatin for 48 hours. Sample collection was performed on ice. Conditioned culture medium was removed then cells were 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 -80 degrees C until sample processing.

Treatment ID:

TR003458

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. Simvastatin-treated cells were exposed to 10 uM simvastatin for 48 hours. Sample collection was performed on ice. Conditioned culture medium was removed then cells were 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 -80 degrees C until sample processing.

Sample Preparation:

Sampleprep ID:	SP003456
Sampleprep Summary:	Lipids from frozen cell pellets were extracted with cold methanol at a concentration of 2 million cells/mL. Samples were vortexed 30 min at 4 degrees C then supernatants clarified by centrifugation (10 min, 10,000 g, 4 degrees C) and transferred to autosampler vials.
Processing Storage Conditions:	4℃
Extract Storage:	-80℃

Combined analysis:

Analysis ID	AN005452	AN005453
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	Phenomenex Kinetex C18 (30 x 2.1mm,1.7um)	Phenomenex Kinetex C18 (30 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:	CH004139
Chromatography Summary:	Negative C18
Instrument Name:	Thermo Vanquish
Column Name:	Phenomenex Kinetex C18 (30 x 2.1mm,1.7um)
Column Temperature:	45
Flow Gradient:	0-3 min 10-95% B at 0.3 mL/min, 3-4.2 min hold at 95% B at 0.3 mL/min, 4.2-4.3 min drop to 10% B at 0.45 mL/min, 4.3-4.9 min hold at 10%B while lowering flow rate to 0.4 mL/min, 4.9-5 min hold at 10%B while lowering flow rate to 0.3 mL/min.
Flow Rate:	0.3 mL/min - 0.45 mL/min
Sample Injection:	10 uL
Solvent A:	75% water/25% acetonitrile; 5 mM ammonium acetate
Solvent B:	90% isopropanol/10% acetonitrile; 5 mM ammonium acetate
Chromatography Type:	Reversed phase

Chromatography ID:	CH004140
Chromatography Summary:	Positive C18
Instrument Name:	Thermo Vanquish
Column Name:	Phenomenex Kinetex C18 (30 x 2.1mm,1.7um)
Column Temperature:	45
Flow Gradient:	0-3 min 30-100% B at 0.3 mL/min, 3-4.2 min hold at 100% B at 0.3 mL/min, 4.2-4.3 min 100-30% B at 0.4 mL/min, 4.3-4.9 min hold at 30%B and 0.4 mL/min, 4.9-5 min hold at 30%B while lowering flow rate to 0.3 mL/min
Flow Rate:	0.3 mL/min - 0.4 mL/min
Sample Injection:	10 uL
Solvent A:	75% water/25% acetonitrile; 5 mM ammonium acetate
Solvent B:	90% isopropanol/10% acetonitrile; 5 mM ammonium acetate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005178
Analysis ID:	AN005452
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The Q Exactive was run independently in positive and negative ion mode, scanning using full MS from 125-1500 m/z at 70,000 resolution and top 10 data-dependent MS2 at 17,500 resolution. Electrospray ionization was achieved with 45 Arb sheath gas, 25 Arb auxiliary gas, and 4 kV spray voltage. Calibration was performed prior to the run using the PierceTM Positive and Negative Ion Calibration Solutions (Thermo Fisher Scientific). Run order of samples was randomized and technical replicates were injected after every 4 samples to assess quality control.
Ion Mode:	NEGATIVE

MS ID:	MS005179
Analysis ID:	AN005453
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The Q Exactive was run independently in positive and negative ion mode, scanning using full MS from 125-1500 m/z at 70,000 resolution and top 10 data-dependent MS2 at 17,500 resolution. Electrospray ionization was achieved with 45 Arb sheath gas, 25 Arb auxiliary gas, and 4 kV spray voltage. Calibration was performed prior to the run using the PierceTM Positive and Negative Ion Calibration Solutions (Thermo Fisher Scientific). Run order of samples was randomized and technical replicates were injected after every 4 samples to assess quality control.
Ion Mode:	POSITIVE