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 |
| 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 |
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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 |
