Summary of Study ST002009

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 PR001272. The data can be accessed directly via it's Project DOI: 10.21228/M89402 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	ST002009
Study Title	Metabolomics analysis of stress erythroid progenitors
Study Summary	A time course study to assess the intracellular metabolic changes of progenitors isolated from stress erythropoiesis expansion culture.
Institute	Pennsylvania State University
Department	Veterinary and Biomedical Sciences
Laboratory	Paulson Lab
Last Name	Paulson
First Name	Robert
Address	228 AVBS Building Shortlidge Road University Park, PA 16802
Email	rfp5@psu.edu
Phone	814-863-6306
Submit Date	2021-12-05
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2022-03-11
Release Version	1

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Project:

Project ID:	PR001272
Project DOI:	doi: 10.21228/M89402
Project Title:	Metabolic profiles of murine stress erythroid progenitors
Project Summary:	Inflammation alters hematopoiesis, skewing production to generate myeloid effector cells at the expense of steady state erythropoiesis. To compensate, stress erythropoiesis is induced to maintain homeostasis until the inflammation is resolved. Unlike the constant production of steady state erythropoiesis, stress erythropoiesis generates a bolus of new erythrocytes by first producing immature progenitor cells, which then transition to committed erythroid progenitors and differentiate. We hypothesize that the proliferation of early progenitor cells and their transition to differentiation is regulated by changes in metabolism. Metabolomics and isotope tracing analysis was performed to assess the intracellular metabolic profiles in proliferating progenitors isolated from in vitro stress erythropoiesis cultures. We observed an active engagement of glucose metabolism in glycolysis and anabolic biosynthesis, while the levels of TCA intermediates suggested that TCA cycle and mitochondrial respiration were blocked. Concomitantly, inducible nitric oxide synthase (iNOS) was induced in progenitor cells to increase the production of nitric oxide (NO), which was demonstrated to be crucial for proliferating progenitor metabolism. Inhibition or genetic mutation of iNOS decreased NO levels resulting in the suppression of progenitor proliferation in vitro and in vivo. As evaluated by RNA-seq, inhibition of iNOS suppressed cell proliferation-related pathways including cell cycle and nucleotide metabolism, while upregulating erythroid differentiation genes. These data suggest that iNOS-derived NO production establishes a metabolism that promotes the proliferation of progenitor cells while inhibiting their differentiation. In contrast, the transition to differentiation is marked by decreased Nos2 expression and a change in metabolism to support induction of the erythroid gene expression program. These data support a model where increased pro-inflammatory signals inhibit steady state erythropoiesis, while at the same time promoting stress erythropoiesis to maintain homeostasis.
Institute:	Pennsylvania State University
Department:	Veterinary and Biomedical Sciences
Laboratory:	Paulson Lab
Last Name:	Paulson
First Name:	Robert
Address:	228 AVBS Building, Shortlidge Road, University Park, PA 16802
Email:	rfp5@psu.edu
Phone:	814-863-6306

Subject:

Subject ID:	SU002090
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

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

mb_sample_id	local_sample_id	Mouse_strain	Culture_stage	Time_point(days)
SA188279	D1_1	wild type C57BL/6	Expansion stage	1
SA188280	D1_5	wild type C57BL/6	Expansion stage	1
SA188281	D1_4	wild type C57BL/6	Expansion stage	1
SA188282	D1_2	wild type C57BL/6	Expansion stage	1
SA188283	D1_3	wild type C57BL/6	Expansion stage	1
SA188284	D3_4	wild type C57BL/6	Expansion stage	3
SA188285	D3_5	wild type C57BL/6	Expansion stage	3
SA188286	D3_3	wild type C57BL/6	Expansion stage	3
SA188287	D3_2	wild type C57BL/6	Expansion stage	3
SA188288	D3_1	wild type C57BL/6	Expansion stage	3
SA188289	D5_4	wild type C57BL/6	Expansion stage	5
SA188290	D5_5	wild type C57BL/6	Expansion stage	5
SA188291	D5_3	wild type C57BL/6	Expansion stage	5
SA188292	D5_1	wild type C57BL/6	Expansion stage	5
SA188293	D5_2	wild type C57BL/6	Expansion stage	5

Showing results 1 to 15 of 15

Showing results 1 to 15 of 15

Collection:

Collection ID:	CO002083
Collection Summary:	Unfractionated wild-type bone marrow cells were cultured in stress erythropoiesis expansion medium (SEEM). At indicated time point, stress erythroid progenitor cells were counted, washed with PBS and snap frozen in liquid nitrogen.
Sample Type:	Stem cells

Treatment:

Treatment ID:	TR002102
Treatment Summary:	All cells are untreated.

Sample Preparation:

Sampleprep ID:	SP002096
Sampleprep Summary:	Cell pellets were extracted with 1 ml pre-chilled 50:50 HPLC-grade water:methanol (v/v) containing 1 µM chlorpropamide as the internal standard. The samples were vortexed briefly followed by thorough homogenization. The samples were then snap frozen with liquid nitrogen and immediately thawed at room temperature. This step was repeated for three times followed by centrifuging for 10 min at 12,000 x g and 4 °C. The supernatants were transferred into fresh microfuge tubes. The remaining cell pellets were re-extracted with 0.5 ml 50% methanol containing 1 µM chlorpropamide, homogenized, frozen and thawed three times, spun down, and the supernatants were combined with the first extraction. Metabolites-containing supernatants were concentrated to dryness at room temperature in a SpeedVac concentrator and re-dissolved in 100 µl 97:3 water:methanol (v/v). After centrifuging for 10 min at 13000 × g and 4°C, 70 µl of supernatants were transferred into autosampler vials for LC-MS analysis. Two types of control were prepared in triplicates to run in concert with the experimental samples: the process blank control, and the pooled control containing an equal volume from each experimental sample.

Sampleprep ID:

SP002096

Sampleprep Summary:

Cell pellets were extracted with 1 ml pre-chilled 50:50 HPLC-grade water:methanol (v/v) containing 1 µM chlorpropamide as the internal standard. The samples were vortexed briefly followed by thorough homogenization. The samples were then snap frozen with liquid nitrogen and immediately thawed at room temperature. This step was repeated for three times followed by centrifuging for 10 min at 12,000 x g and 4 °C. The supernatants were transferred into fresh microfuge tubes. The remaining cell pellets were re-extracted with 0.5 ml 50% methanol containing 1 µM chlorpropamide, homogenized, frozen and thawed three times, spun down, and the supernatants were combined with the first extraction. Metabolites-containing supernatants were concentrated to dryness at room temperature in a SpeedVac concentrator and re-dissolved in 100 µl 97:3 water:methanol (v/v). After centrifuging for 10 min at 13000 × g and 4°C, 70 µl of supernatants were transferred into autosampler vials for LC-MS analysis. Two types of control were prepared in triplicates to run in concert with the experimental samples: the process blank control, and the pooled control containing an equal volume from each experimental sample.

Combined analysis:

Analysis ID	AN003275
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Dionex Ultimate 3000
Column	Waters SSelect HSS C18
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Exactive Plus Orbitrap
Ion Mode	NEGATIVE
Units	Normalized peak area

Chromatography:

Chromatography ID:	CH002417
Chromatography Summary:	The sample run order was randomized to reduce bias from instrument drift. 10 µl sample was subjected to LC-MS analysis on a Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to an Ultimate 3000 UHPLC system (Thermo Fisher Scientific). Reversed-phase chromatography mode was used to separate compounds on a Xselect C18 HSS column (Waters) with solvent A (97:3 water:methanol (v/v), 10 mM tributylamine, and 15 mM acetic acid ) and solvent B (methanol). The flow rate was 200 µl/min, and the total run time was 25 min. The gradient was 0 min, 0% B; 5 min, 20% B; 7.5 min, 55% B; 15 min, 65% B; 17.5 min, 95% B; and 21 min, 0% B. The mass spectrometer was operated in a negative-ion mode at a resolution of 140,000 at m/z 200 and with a scan range of 85 to 1000 m/z.
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Waters SSelect HSS C18
Flow Gradient:	0 min, 0% B; 5 min, 20% B; 7.5 min, 55% B; 15 min, 65% B; 17.5 min, 95% B; and 21 min, 0% B.
Flow Rate:	200 µl/min
Solvent A:	97% water/3% methanol; 15 mM acetic acid; 10 mM tributylamine
Solvent B:	100% methanol
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003047
Analysis ID:	AN003275
Instrument Name:	Thermo Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Raw data files were converted to .mzML file format using the ProteoWizard software, and they were analyzed by the MS-DIAL software. Metabolites were identified by comparison to an in-house reference library of pure metabolite standards which included mass-to-charge ratio (m/z) and retention time. For quantification of metabolite abundance, peak areas of identified metabolites were first normalized to the internal standard chlorpropamide, and then normalized to cell numbers from each sample. Data were analyzed using R and Cytoscape software.
Ion Mode:	NEGATIVE