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.

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Study IDST001377
Study TitleStirred suspension bioreactors maintain naïve pluripotency of human pluripotent stem cells (hPSCs)
Study SummaryAlthough 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
DepartmentBiochemistry and Molecular Biology
LaboratoryStem Cell Research
Last NameRohani
First NameLeili
Address405J, 1919 University Drive, NW
Emailleili.rohanisarvesta@ucalgary.ca
Phone+1 5879681647
Submit Date2020-05-08
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2020-07-20
Release Version1
Leili Rohani Leili Rohani
https://dx.doi.org/10.21228/M8XM5C
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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
SA1006612019_09_13_RPIP15_Ref-Media-H1-M1_1H1 day 1
SA1006622019_09_13_RPIP15_H1-M1_1H1 day 1
SA1006632019_09_13_RPIP15_H1-M1_2H1 day 2
SA1006642019_09_13_RPIP15_Ref-Media-H1-M1_2H1 day 2
SA1006652019_09_13_RPIP15_H1-M3_1H1 day 3
SA1006662019_09_13_RPIP15_H1-M3_2H1 day 3
SA1006672019_09_13_RPIP15_Ref-Media-H1-M3_1H1 day 3
SA1006682019_09_13_RPIP15_H1-M2_1H1 day 3
SA1006692019_09_13_RPIP15_Ref-Media-H1-M2_1H1 day 3
SA1006702019_09_13_RPIP15_Ref-Media-H1-M3_2H1 day 3
SA1006712019_09_13_RPIP15_Ref-Media-H1-M2_2H1 day 4
SA1006722019_09_13_RPIP15_H1-M2_2H1 day 4
SA1006732019_09_13_RPIP15_Ref-Media-H1-M4_2H1 day 4
SA1006742019_09_13_RPIP15_H1-M4_2H1 day 4
SA1006752019_09_13_RPIP15_Ref-Media-H1-M4_1H1 day 4
SA1006762019_09_13_RPIP15_H1-M4_1H1 day 4
SA1006772019_09_13_RPIP15_Ref-Media-H1-M5_1H1 day 5
SA1006782019_09_13_RPIP15_H1-M5_2H1 day 5
SA1006792019_09_13_RPIP15_Ref-Media-H1-M5_2H1 day 5
SA1006802019_09_13_RPIP15_H1-M5_1H1 day 5
SA1006812019_09_13_RPIP15_Ref-Media-H9-naive-bioreactor-2H9 naive
SA1006822019_09_13_RPIP15_H9-naive-bioreactor-2H9 naive
SA1006832019_09_13_RPIP15_H9-naive-bioreactor-1-H9 naive
SA1006842019_09_13_RPIP15_H9-naive-bioreactor-3H9 naive
SA1006852019_09_13_RPIP15_Ref-Media-H9-naive-bioreactor-1H9 naive
SA1006862019_09_13_RPIP15_Ref-Media-H9-naive-bioreactor-3H9 naive
SA1006872019_09_13_RPIP15_Ref-Media-H9-primed-bioreactor-3H9 primed
SA1006882019_09_13_RPIP15_Ref-Media-H9-primed-bioreactor-1H9 primed
SA1006892019_09_13_RPIP15_Ref-Media-H9-primed-bioreactor-2H9 primed
SA1006902019_09_13_RPIP15_H9-primed-bioreactor-1H9 primed
SA1006912019_09_13_RPIP15_H9-primed-bioreactor-3H9 primed
SA1006922019_09_13_RPIP15_H9-primed-bioreactor-2H9 primed
SA1006932019_09_13_RPIP15_Blank_mid8none none
SA1006942019_09_13_RPIP15_Blank_mid7none none
SA1006952019_09_13_RPIP15_Blank_mid10none none
SA1006962019_09_13_RPIP15_Blank_mid6none none
SA1006972019_09_13_RPIP15_Blank_mid9none 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, 1.8 um)
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, 1.8 um)
Flow Rate:0.6 ml/min
Solvent A:10mM tributylamine, 10mM acetate pH 7.5 in 97/3% (v/v) MS grade water/methanol
Solvent B:MS grade 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
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