Summary of Study ST003544
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 PR002180. The data can be accessed directly via it's Project DOI: 10.21228/M8TV67 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 | ST003544 |
| Study Title | Metabolomics of Ndufs4 KO human induced pluripotent stem cells (iPSCs) |
| Study Summary | Mitochondrial diseases, often linked to complex I (CI) defects, lack curative treatments. High-throughput drug screening using human-relevant platforms is crucial for identifying new therapeutics. Induced pluripotent stem cell (iPSC) and CRISPR technologies offer a powerful tool for this purpose. While typically differentiated into disease-relevant cell types, recent studies support the use of undifferentiated iPSCs for drug discovery. Here, we developed and characterized NDUFS4 KO iPSCs in their pluripotent state. Metabolomic profiling revealed a distinct phenotype in NDUFS4 KO iPSCs, predominantly associated with an elevated NADH/NAD+ ratio, consistent with alterations observed in other models of mitochondrial dysfunction. These findings underscore the potential of iPSCs for early-stage, high-throughput therapeutic screening in mitochondrial diseases. |
| Institute | North-West University |
| Last Name | Louw |
| First Name | Roan |
| Address | Hofman Street, Potchefstroom, North-West, 2520, South Africa |
| Roan.Louw@nwu.ac.za | |
| Phone | +27182994074 |
| Submit Date | 2024-10-06 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML,cdf,d |
| Analysis Type Detail | GC-MS/LC-MS |
| Release Date | 2025-10-06 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002180 |
| Project DOI: | doi: 10.21228/M8TV67 |
| Project Title: | Metabolomics of Ndufs4 KO human induced pluripotent stem cells (iPSCs) |
| Project Type: | Multi-platform metabolomics analysis |
| Project Summary: | Mitochondrial diseases, often linked to complex I (CI) defects, lack curative treatments. High-throughput drug screening using human-relevant platforms is crucial for identifying new therapeutics. Induced pluripotent stem cell (iPSC) and CRISPR technologies offer a powerful tool for this purpose. While typically differentiated into disease-relevant cell types, recent studies support the use of undifferentiated iPSCs for drug discovery. The aim of this project was to develop and characterize NDUFS4 KO iPSCs in their pluripotent state. The metabolic profile of Ndufs4 KO human induced pluripotent stem cells (iPSCs) were compared to that of isogenic controls using multi-platform metabolomics, consisting of targeted LC-MS/MS, targeted GC-MS/MS, and untargeted GC-TOFMS analyses. Metabolic profiling revealed a distinct phenotype in NDUFS4 KO iPSCs, predominantly associated with an elevated NADH/NAD+ ratio, consistent with alterations observed in other models of mitochondrial dysfunction. These findings underscore the potential of iPSCs for early-stage, high-throughput therapeutic screening in mitochondrial diseases. |
| Institute: | North-West University |
| Last Name: | Louw |
| First Name: | Roan |
| Address: | Hofman Street, Potchefstroom, North-West, 2520, South Africa |
| Email: | Roan.Louw@nwu.ac.za |
| Phone: | +27182994074 |
Subject:
| Subject ID: | SU003673 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Species Group: | Mammals |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype |
|---|---|---|
| SA387723 | KO5_2 | KO |
| SA387724 | KO5_1 | KO |
| SA387725 | KO5_3 | KO |
| SA387726 | KO5_4 | KO |
| SA387727 | KO5_5 | KO |
| SA387728 | WT7_1 | WT |
| SA387729 | WT7_2 | WT |
| SA387730 | WT7_3 | WT |
| SA387731 | WT7_4 | WT |
| SA387732 | WT7_5 | WT |
| Showing results 1 to 10 of 10 |
Collection:
| Collection ID: | CO003666 |
| Collection Summary: | Five replicate samples per genotype (i.e. distinct cultures in parallel) were prepared for each metabolic analysis. Each cell pellet, harvested from a T25 culture flasks, was washed three times with chilled PBS before being quenched in cold HPLC-grade methanol, with subsequent addition of an internal standard mixture in cold HPLC-grade water. Thereafter, samples were homogenized using a vibration mill (30 Hz, 1 min) and incubated on ice (10 min) after the addition of cold HPLC-grade chloroform. Solvents were added in a ratio of 3:1:1, methanol/water/chloroform, as required for a modified monophasic Bligh-Dyer extraction. After centrifugation at 12 000 ×g (10 min, 4°C) supernatants were aliquoted into 2 mL glass vials. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR003682 |
| Treatment Summary: | N/A |
Sample Preparation:
| Sampleprep ID: | SP003680 |
| Sampleprep Summary: | Samples were derivatized by butylation on the day of analysis. To butylate the dried extracts, 300 μL of freshly prepared 1-butanol:acetyl chloride (4:1, v/v) was added and samples were incubated at 50°C for 60 min. Thereafter, the samples were evaporated to dryness under a gentle stream of nitrogen at 37 °C. The samples were then reconstituted in 100 μL of water:acetonitrile (50:50, v/v), containing 0.1% formic acid and vortex mixed. Finally, the total volume was transferred to 250 μL tapered glass inserts placed in 2 mL vials and loaded onto the autosampler for analysis. |
Chromatography:
| Chromatography ID: | CH004421 |
| Instrument Name: | Agilent 1200 Infinity |
| Column Name: | Agilent C18 Zorbax SB-AQ (100 x 2.1mm, 1.8um) |
| Column Temperature: | 45°C |
| Flow Gradient: | 95 % of solvent A held for 0.2 min, increased to 25 % of solvent B over 1.8 min, held for 5 min, increased to 90 % of solvent B over 0.5 min, held for 1.6 min, increased to 95 % of solvent B over 2.9 min, decreased to 5 % of solvent B over 1 min, and re-equilibrated for 3 min to give a total run time of 16 min. |
| Flow Rate: | For the first 9 min of the run, the mobile phase flow rate was set at 0.3 mL/min, after which it was increased to 0.4 mL/min in a span of 0.1 min and maintained at this level for the subsequent 3.9 min of the run |
| Solvent A: | 100% Water; 0.1 % Formic acid |
| Solvent B: | 100% Acetonitrile; 0.1 % Formic acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH004422 |
| Instrument Name: | Agilent 7890B |
| Column Name: | Restek Rxi-1MS (30m x 0.32mm x 0.25μm) |
| Column Temperature: | 70°C for 1 min; followed by a ramp at 12°C/min until reaching 135°C, held for 1.5 min; a ramp at 2°C/min until reaching 145°C, held for 1.5 min; and a ramp of 23°C/min until reaching a final temperature of 300°C, held for 1 min |
| Flow Gradient: | N/A |
| Flow Rate: | 1 mL/min |
| Solvent A: | N/A |
| Solvent B: | N/A |
| Chromatography Type: | GC |
| Chromatography ID: | CH004423 |
| Instrument Name: | Agilent 8890 |
| Column Name: | Restek Rxi-5MS (29.69m x 0.25mm,0.25um) |
| Column Temperature: | 70°C for 1 min, followed by a ramp at 10°C/min until reaching a final temperature of 320°C, held for 3 min |
| Flow Gradient: | N/A |
| Flow Rate: | 1 mL/min |
| Solvent A: | N/A |
| Solvent B: | N/A |
| Chromatography Type: | GC |
Analysis:
| Analysis ID: | AN005820 |
| Analysis Type: | MS |
| Chromatography ID: | CH004421 |
| Num Factors: | 2 |
| Num Metabolites: | 42 |
| Units: | Normalised peak areas |
| Analysis ID: | AN005821 |
| Analysis Type: | MS |
| Chromatography ID: | CH004422 |
| Num Factors: | 2 |
| Num Metabolites: | 16 |
| Analysis ID: | AN005822 |
| Analysis Type: | MS |
| Chromatography ID: | CH004423 |
| Has Mz: | 1 |
| Has Rt: | 1 |
| Rt Units: | Seconds |
| Results File: | ST003544_AN005822_Results.txt |
| Units: | Normalised peak areas |
