Summary of Study ST001377
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 PR000942. The data can be accessed directly via it's Project DOI: 10.21228/M8XM5C 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 | ST001377 |
| Study Title | Stirred suspension bioreactors maintain naïve pluripotency of human pluripotent stem cells (hPSCs) |
| Study Summary | Although cell therapies require large numbers of quality-controlled hPSCs, existing technologies are limited in their ability to efficiently grow and scale stem cells. We report here that cell-state conversion from primed-to-naïve pluripotency enhances the biomanufacturing of hPSCs. Naïve hPSCs exhibit superior growth kinetics and aggregate formation characteristics in stirred suspension bioreactors compared to their primed counterparts. Moreover, we demonstrate the role of the bioreactor mechanical environment in the maintenance of naïve pluripotency, through transcriptomic enrichment of mechano-sensing signaling for cells in the bioreactor along with a decrease in expression of lineage-specific and primed pluripotency hallmarks. Bioreactor-cultured, naïve hPSCs express epigenetic regulatory transcripts associated with naïve pluripotency, and display hallmarks of X-chromosome reactivation. They exhibit robust production of naïve pluripotency metabolites and display reduced expression of primed pluripotency cell surface markers. We also show that these cells retain the ability to undergo targeted differentiation into beating cardiomyocytes, hepatocytes, and neural rosettes. They additionally display faster kinetics of teratoma formation compared to their primed counterparts. Naïve bioreactor hPSCs also retain structurally stable chromosomes. Our research corroborates that converting hPSCs to the naïve state enhances hPSC manufacturing and indicates a potentially important role for the bioreactor’s mechanical environment in maintaining naïve pluripotency. |
| Institute | University of Calgary |
| Department | Biochemistry and Molecular Biology |
| Laboratory | Stem Cell Research |
| Last Name | Rohani |
| First Name | Leili |
| Address | 405J, 1919 University Drive, NW |
| leili.rohanisarvesta@ucalgary.ca | |
| Phone | +1 5879681647 |
| Submit Date | 2020-05-08 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2020-07-20 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR000942 |
| Project DOI: | doi: 10.21228/M8XM5C |
| Project Title: | Inferring metabolism dynamics between naïve and primed human pluripotent stem cells in the stirred suspension bioreactor |
| Project Type: | Targeted and untargeted MS |
| Project Summary: | We used high-resolution LC-MS targeted metabolomics to screen for extracellular metabolites in the media of bioreactor-cultured, naïve and primed human pluripotent stem cells. |
| Institute: | University of Calgary |
| Department: | Biochemistry and Molecular Biology |
| Laboratory: | Stem Cell Research |
| Last Name: | Rohani |
| First Name: | Leili |
| Address: | 405J, 1919 University Drive, NW |
| Email: | leili.rohanisarvesta@ucalgary.ca |
| Phone: | 5879681647 |
Subject:
| Subject ID: | SU001451 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Cell line | Status |
|---|---|---|---|
| SA100661 | 2019_09_13_RPIP15_Ref-Media-H1-M1_1 | H1 | day 1 |
| SA100662 | 2019_09_13_RPIP15_H1-M1_1 | H1 | day 1 |
| SA100663 | 2019_09_13_RPIP15_H1-M1_2 | H1 | day 2 |
| SA100664 | 2019_09_13_RPIP15_Ref-Media-H1-M1_2 | H1 | day 2 |
| SA100665 | 2019_09_13_RPIP15_H1-M3_1 | H1 | day 3 |
| SA100666 | 2019_09_13_RPIP15_H1-M3_2 | H1 | day 3 |
| SA100667 | 2019_09_13_RPIP15_Ref-Media-H1-M3_1 | H1 | day 3 |
| SA100668 | 2019_09_13_RPIP15_H1-M2_1 | H1 | day 3 |
| SA100669 | 2019_09_13_RPIP15_Ref-Media-H1-M2_1 | H1 | day 3 |
| SA100670 | 2019_09_13_RPIP15_Ref-Media-H1-M3_2 | H1 | day 3 |
| SA100671 | 2019_09_13_RPIP15_Ref-Media-H1-M2_2 | H1 | day 4 |
| SA100672 | 2019_09_13_RPIP15_H1-M2_2 | H1 | day 4 |
| SA100673 | 2019_09_13_RPIP15_Ref-Media-H1-M4_2 | H1 | day 4 |
| SA100674 | 2019_09_13_RPIP15_H1-M4_2 | H1 | day 4 |
| SA100675 | 2019_09_13_RPIP15_Ref-Media-H1-M4_1 | H1 | day 4 |
| SA100676 | 2019_09_13_RPIP15_H1-M4_1 | H1 | day 4 |
| SA100677 | 2019_09_13_RPIP15_Ref-Media-H1-M5_1 | H1 | day 5 |
| SA100678 | 2019_09_13_RPIP15_H1-M5_2 | H1 | day 5 |
| SA100679 | 2019_09_13_RPIP15_Ref-Media-H1-M5_2 | H1 | day 5 |
| SA100680 | 2019_09_13_RPIP15_H1-M5_1 | H1 | day 5 |
| SA100681 | 2019_09_13_RPIP15_Ref-Media-H9-naive-bioreactor-2 | H9 | naive |
| SA100682 | 2019_09_13_RPIP15_H9-naive-bioreactor-2 | H9 | naive |
| SA100683 | 2019_09_13_RPIP15_H9-naive-bioreactor-1- | H9 | naive |
| SA100684 | 2019_09_13_RPIP15_H9-naive-bioreactor-3 | H9 | naive |
| SA100685 | 2019_09_13_RPIP15_Ref-Media-H9-naive-bioreactor-1 | H9 | naive |
| SA100686 | 2019_09_13_RPIP15_Ref-Media-H9-naive-bioreactor-3 | H9 | naive |
| SA100687 | 2019_09_13_RPIP15_Ref-Media-H9-primed-bioreactor-3 | H9 | primed |
| SA100688 | 2019_09_13_RPIP15_Ref-Media-H9-primed-bioreactor-1 | H9 | primed |
| SA100689 | 2019_09_13_RPIP15_Ref-Media-H9-primed-bioreactor-2 | H9 | primed |
| SA100690 | 2019_09_13_RPIP15_H9-primed-bioreactor-1 | H9 | primed |
| SA100691 | 2019_09_13_RPIP15_H9-primed-bioreactor-3 | H9 | primed |
| SA100692 | 2019_09_13_RPIP15_H9-primed-bioreactor-2 | H9 | primed |
| SA100693 | 2019_09_13_RPIP15_Blank_mid8 | none | none |
| SA100694 | 2019_09_13_RPIP15_Blank_mid7 | none | none |
| SA100695 | 2019_09_13_RPIP15_Blank_mid10 | none | none |
| SA100696 | 2019_09_13_RPIP15_Blank_mid6 | none | none |
| SA100697 | 2019_09_13_RPIP15_Blank_mid9 | none | none |
| Showing results 1 to 37 of 37 |
Collection:
| Collection ID: | CO001446 |
| Collection Summary: | Extracellular metabolite dynamics was analyzed during primed to naïve conversion in the bioreactor. For this, the media of H1 hESCs in the bioreactor was gradually switched from mTESR1 to RSeT media within 6 days of culture (D2 to D6) and the media was collected every day for the analysis. For H9 hESCs, the extracellular metabolites were analyzed in the spent media of primed and established naïve (P5) hPSCs in the bioreactor, and the media of each primed or naïve hPSCs was collected at day four of bioreactor culture. |
| Sample Type: | Stem cells |
Treatment:
| Treatment ID: | TR001466 |
| Treatment Summary: | For H1 hESCs, the extracellular metabolite dynamics was analyzed during primed to naïve conversion in the bioreactor. For this, the media of H1 hESCs in the bioreactor was gradually switched from mTESR1 to RSeT media within 6 days of culture (D2 to D6) and the media was collected every day for the analysis. For H9 hESCs, the extracellular metabolites were analyzed in the spent media of primed and established naïve (P5) hPSCs in the bioreactor, and the media of each primed or naïve hPSCs was collected at day four of bioreactor culture. |
Sample Preparation:
| Sampleprep ID: | SP001459 |
| Sampleprep Summary: | The metabolite extraction was started using LC-MS or HPLC grade methanol (Sigma-Aldrich, 1.06035). Briefly, a 950 μL of pre-chilled 50% MeOH/H2O was added to a 50 μL of the bioreactor-collected media (making a D20 dilution), and the samples were incubated on ice for 30 min to allow for full extraction. Macromolecules were then pelleted by centrifugation at max speed (~18,000-21,000 g) for 10 mins in a bench top centrifuge (preferably chilled) to extract the supernatant. Further, the extracted samples were stored in -80 prior to running HPLC-MS. |
| Processing Storage Conditions: | Room temperature |
| Extraction Method: | 50% Methanol in water |
| Extract Storage: | On ice |
Combined analysis:
| Analysis ID | AN002297 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Agilent Zorbax RRHT SB-C18 (50 x 2.1 mm,1.8um) |
| MS Type | ESI |
| MS instrument type | Single quadrupole |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | NEGATIVE |
| Units | relative signal intensity compared to media only |
Chromatography:
| Chromatography ID: | CH001687 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Agilent Zorbax RRHT SB-C18 (50 x 2.1 mm,1.8um) |
| Flow Rate: | 0.6 ml/min |
| Solvent A: | 97% water/3% methanol; 15 mM acetic acid; 10 mM tributylamine |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002140 |
| Analysis ID: | AN002297 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Single quadrupole |
| MS Type: | ESI |
| MS Comments: | The mass spectrometer was run in negative full scan mode at a resolution of 140,000 scanning from 50-750m/z. Data was processed using MAVEN. |
| Ion Mode: | NEGATIVE |
| Capillary Temperature: | 275 |
| Spray Voltage: | 2.50 kV |
| Analysis Protocol File: | MS_Metabolomics.pdf |
