Summary of Study ST002986

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 PR001858. The data can be accessed directly via it's Project DOI: 10.21228/M8JM83 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 IDST002986
Study TitleDeciphering the metabolic heterogeneity of hematopoietic stem cells with single-cell resolution: Study 2
Study SummaryMetabolic status is crucial for stem cell functions; however, the metabolic heterogeneity of endogenous stem cells has never been directly assessed. Here, we develop a platform for high-throughput single-cell metabolomics (hi-scMet) of hematopoietic stem cells (HSCs). By combining flow cytometric isolation and nanoparticle-enhanced laser desorption/ionization mass spectrometry, we routinely detected >100 features from single cells. We mapped the single-cell metabolomes of all hematopoietic cell populations, and HSC subpopulations with different division times, detecting 33 features whose levels exhibited trending changes during HSC proliferation. We found progressive activation of oxidative pentose phosphate pathway (OxiPPP) from dormant to active HSCs. Genetic or pharmacological interference with OxiPPP increased reactive oxygen species level in HSCs, reducing HSC self-renewal upon oxidative stress. Together, our work uncovers the metabolic dynamics during HSC proliferation, reveals a role of OxiPPP for HSC activation, and illustrates the utility of hi-scMet in dissecting metabolic heterogeneity of immunophenotypically defined cell populations.
Institute
Shanghai Jiao Tong University
Last NameCAO
First NameJING
Address1954 Huashan Road, Shanghai, Shanghai, 200030, China
Emailcaojing1@sjtu.edu.cn
Phone+8615201957271
Submit Date2023-11-21
Raw Data AvailableYes
Raw Data File Type(s).txt
Analysis Type DetailMALDI
Release Date2023-11-29
Release Version1
JING CAO JING CAO
https://dx.doi.org/10.21228/M8JM83
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001858
Project DOI:doi: 10.21228/M8JM83
Project Title:Deciphering the metabolic heterogeneity of hematopoietic stem cells with single-cell resolution
Project Type:Cell Metabolism
Project Summary:Metabolic status is crucial for stem cell functions; however, the metabolic heterogeneity of endogenous stem cells has never been directly assessed. Here, we develop a platform for high-throughput single-cell metabolomics (hi-scMet) of hematopoietic stem cells (HSCs). By combining flow cytometric isolation and nanoparticle-enhanced laser desorption/ionization mass spectrometry, we routinely detected >100 features from single cells. We mapped the single-cell metabolomes of all hematopoietic cell populations, and HSC subpopulations with different division times, detecting 33 features whose levels exhibited trending changes during HSC proliferation. We found progressive activation of oxidative pentose phosphate pathway (OxiPPP) from dormant to active HSCs. Genetic or pharmacological interference with OxiPPP increased reactive oxygen species level in HSCs, reducing HSC self-renewal upon oxidative stress. Together, our work uncovers the metabolic dynamics during HSC proliferation, reveals a role of OxiPPP for HSC activation, and illustrates the utility of hi-scMet in dissecting metabolic heterogeneity of immunophenotypically defined cell populations.
Institute:Shanghai Jiao Tong University
Last Name:CAO
First Name:JING
Address:1954 Huashan Road, Shanghai, Shanghai, 200030, China
Email:caojing1@sjtu.edu.cn
Phone:15201957271
Funding Source:This work was supported by the National Key R&D Program (2022YFE0103500, 2018YFA0107200, 2021YFF0703500, and 2022YFC2502800), Medical-Engineering Joint Funds of Shanghai Jiao Tong University (YG2021ZD09, YG2022QN107, YG2023ZD08), Haihe Laboratory of Cell Ecosystem Innovation Fund (22HHXBSS00016), the National Natural Science Foundation of China (32270785, 31771637, 81730006, 81971771, 22074044, 22122404) and the CAS Youth Interdisciplinary Team (JCTD-2021-12). This work was also sponsored by Shanghai Municipal Science and Technology Major Project Fund, Shanghai Science and Technology Commission (22XD1424000, 22ZR1469000), Shanghai Institutions of Higher Learning (2021-01-07-00-02-E00083), Innovative Research Team of High-Level Local Universities in Shanghai (SHSMU-ZDCX20210700), Innovation Group Project of Shanghai Municipal Health Comission (2019CXJQ03), Innovation Research Plan of Shanghai Municipal Education Commission (ZXWF082101), National Research Center for Translational Medicine Shanghai (TMSK-2021-124, NRCTM(SH)-2021-06), and Fundamental Research Funds for the Central Universities.

Subject:

Subject ID:SU003099
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Celltype
SA323990B_95B
SA323991B_96B
SA323992B_97B
SA323993B_94B
SA323994B_93B
SA323995B_91B
SA323996B_92B
SA323997B_98B
SA323998B_99B
SA323999B_104B
SA324000B_105B
SA324001B_103B
SA324002B_102B
SA324003B_100B
SA324004B_101B
SA324005B_90B
SA324006B_89B
SA324007B_79B
SA324008B_80B
SA324009B_78B
SA324010B_77B
SA324011B_75B
SA324012B_76B
SA324013B_81B
SA324014B_82B
SA324015B_87B
SA324016B_88B
SA324017B_86B
SA324018B_85B
SA324019B_83B
SA324020B_84B
SA324021B_106B
SA324022B_107B
SA324023B_128B
SA324024B_129B
SA324025B_127B
SA324026B_126B
SA324027B_124B
SA324028B_125B
SA324029B_130B
SA324030B_131B
SA324031B_136B
SA324032B_137B
SA324033B_135B
SA324034B_134B
SA324035B_132B
SA324036B_133B
SA324037B_123B
SA324038B_122B
SA324039B_112B
SA324040B_113B
SA324041B_111B
SA324042B_110B
SA324043B_108B
SA324044B_109B
SA324045B_114B
SA324046B_115B
SA324047B_120B
SA324048B_121B
SA324049B_119B
SA324050B_118B
SA324051B_116B
SA324052B_117B
SA324053B_74B
SA324054B_72B
SA324055B_30B
SA324056B_31B
SA324057B_32B
SA324058B_29B
SA324059B_28B
SA324060B_26B
SA324061B_27B
SA324062B_33B
SA324063B_34B
SA324064B_39B
SA324065B_40B
SA324066B_38B
SA324067B_37B
SA324068B_35B
SA324069B_36B
SA324070B_25B
SA324071B_24B
SA324072B_14B
SA324073B_15B
SA324074B_13B
SA324075B_12B
SA324076B_10B
SA324077B_11B
SA324078B_16B
SA324079B_17B
SA324080B_22B
SA324081B_23B
SA324082B_21B
SA324083B_20B
SA324084B_18B
SA324085B_19B
SA324086B_41B
SA324087B_42B
SA324088B_63B
SA324089B_64B
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Collection:

Collection ID:CO003092
Collection Summary:Bones were rapidly dissected and stored on ice in Ca2+- and Mg2+-free HBSS (ThermoFisher) plus 2% heat-inactivated fetal bovine serum (Gibco). Bone marrow cells were flushed out from the bone and then dissociated to single-cell suspension by gently passing through the needle then filtering through a 70-μm nylon mesh. The following antibodies were used to isolate hematopoietic cells: TER-119-FITC, CD3-FITC, CD5-FITC, CD8-FITC, B220-FITC, Gr-1-FITC, TER-119-APC780, CD3e-APC780, CD5-APC780, CD8-APC780, B220-APC780, Gr-1-APC780, TER-119-biotin, CD3-biotin, B220-biotin, Gr-1-biotin, c-kit-biotin, TER-119-PerCP/Cy5.5, Sca-1-PerCP/Cy5.5, MAC-1-APC, CD48-APC, CD16/32-APC, CD135-APC, CD127-PE, CD34-PE and CD3-PE, CD150-PE. FITC streptavidin, APC/Cy7 streptavidin and/or APC-R700 streptavidin were used for biotin-labeled antibodies. All reagents were acquired from BD Biosciences, eBiosciences or BioLegend. For isolation of HSPCs, cells were incubated with c-kit-biotin and paramagnetic microbeads, and then passed through an autoMACS magnetic separator. For isolation of CLPs, lineage was stained with Ter119-biotin, CD3-biotin, B220-biotin and Gr-1-biotin and paramagnetic microbeads. Then an autoMACS magnetic separator was used to enrich lineage- populations. To minimize metabolic changes, antibody-stained cells were fixed with 2% PFA (Sigma) for 15 minutes on ice. Flow cytometric analysis was performed with a BD LSRFortessa cytometer. To measure ROS levels of HSCs, antibody-stained cells were incubated with 5μm DCFDA for 15 minutes at 37°C before flow cytometric analysis. For metabolic detection, cells were sorted with a FACSAria SORP cytometer into 384-well plates containing 2.5-μl 80% methanol (Sigma) per well, and then centrifuged at 1500g for 5 mins at 4°C. Plates were sunk in liquid nitrogen for 10 mins to lyse cells and kept at -80°C before MS analysis.
Sample Type:Stem cells

Treatment:

Treatment ID:TR003108
Treatment Summary:The following antibodies were used to isolate hematopoietic cells: TER-119-FITC, CD3-FITC, CD5-FITC, CD8-FITC, B220-FITC, Gr-1-FITC, TER-119-APC780, CD3e-APC780, CD5-APC780, CD8-APC780, B220-APC780, Gr-1-APC780, TER-119-biotin, CD3-biotin, B220-biotin, Gr-1-biotin, c-kit-biotin, TER-119-PerCP/Cy5.5, Sca-1-PerCP/Cy5.5, MAC-1-APC, CD48-APC, CD16/32-APC, CD135-APC, CD127-PE, CD34-PE and CD3-PE, CD150-PE. FITC streptavidin, APC/Cy7 streptavidin and/or APC-R700 streptavidin were used for biotin-labeled antibodies. All reagents were acquired from BD Biosciences, eBiosciences or BioLegend. For isolation of HSPCs, cells were incubated with c-kit-biotin and paramagnetic microbeads, and then passed through an autoMACS magnetic separator. For isolation of CLPs, lineage was stained with Ter119-biotin, CD3-biotin, B220-biotin and Gr-1-biotin and paramagnetic microbeads. Then an autoMACS magnetic separator was used to enrich lineage- populations.

Sample Preparation:

Sampleprep ID:SP003105
Sampleprep Summary:For metabolic detection, cells were sorted with a FACSAria SORP cytometer into 384-well plates containing 2.5-μl 80% methanol (Sigma) per well, and then centrifuged at 1500g for 5 mins at 4°C. Plates were sunk in liquid nitrogen for 10 mins to lyse cells and kept at -80°C before MS analysis.

Combined analysis:

Analysis ID AN004906
Analysis type MS
Chromatography type
Chromatography system none
Column none
MS Type MALDI
MS instrument type MALDI-TOF-MS
MS instrument name Bruker Autoflex MALDI-TOF (/TOF)-MS
Ion Mode POSITIVE
Units intensity

Chromatography:

Chromatography ID:CH003701
Chromatography Summary:None
Instrument Name:none
Column Name:none
Column Temperature:none
Flow Gradient:none
Flow Rate:none
Solvent A:none
Solvent B:none

MS:

MS ID:MS004649
Analysis ID:AN004906
Instrument Name:Bruker Autoflex MALDI-TOF (/TOF)-MS
Instrument Type:MALDI-TOF-MS
MS Type:MALDI
MS Comments:MS acquisition Comments: MS experiments were conducted on the Autoflex MALDI-TOF (/TOF)-MS (Bruker Autoflex Speed) with Nd:YAG lasers (355 nm) and smart beam system. All MS experiments were performed in the reflector positive ion mode, with 2000 laser shots per analysis and 5 independent experiments per sample. Data processing Comments:MS data preprocessing was carried out on python version 3.8, including peak detection, peak filtration, standardization, and batch effect removal by ComBat algorithm. The heatmap, hierarchical clustering analysis, Pearson correlation analysis and PCA analysis were performed on MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/). Machine learning and t-SNE clustering were conducted on Orange (version 3.25.0, the Bioinformatics Lab at University of Ljubljana, Slovenia). Software/procedures used for feature assignments: Python
Ion Mode:POSITIVE
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