Summary of Study ST002055

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 PR001300. The data can be accessed directly via it's Project DOI: 10.21228/M8P69K This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST002055
Study Title	Metabolomic Profiling of Human Pluripotent Stem Cell Differentiation into Lung Progenitors
Study Summary	Metabolism is vital to cellular function and tissue homeostasis during human lung development. In utero, embryonic pluripotent stem cells undergo endodermal differentiation towards a lung progenitor cell fate that can be mimicked in vitro using induced human pluripotent stem cells (hiPSCs) to study genetic mutations. To identify differences between wild type and surfactant protein B (SFTPB)-deficient cell lines during endoderm specification towards lung, we used an untargeted metabolomics approach to evaluate the developmental changes in metabolites. We found that the metabolites most enriched during the differentiation from pluripotent stem cell to lung progenitor cell, regardless of cell line, were sphingomyelins and phosphatidylcholines, two important lipid classes in fetal lung development. The SFTPB mutation had no metabolic impact on early endodermal lung development. The identified metabolite signatures during lung progenitor cell differentiation may be utilized as biomarkers for normal embryonic lung development.
Institute	The Hospital for Sick Children
Last Name	Post
First Name	Martin
Address	555 University Avenue
Email	martin.post@sickkids.ca
Phone	4168136772
Submit Date	2021-06-29
Raw Data Available	Yes
Raw Data File Type(s)	wiff
Analysis Type Detail	LC-MS/MS(Dir. Inf.)
Release Date	2022-01-13
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP002143
Sampleprep Summary:	At each differentiation step, DE, AFE and LPC, cells were analyzed by FACS for stage specific markers. The differentiated cells were harvested with TrypLE at 37 °C for 5-10 minutes and collected by centrifugation at 300 g for 5 minutes at room temperature. The cell pellets were resuspended in 3% (v/v) FBS in PBS, cell aggregates were removed using a cell strainer with a 40-µm pore size and single cells were collected. For surface antigens, the cells were incubated with primary antibodies for 30 minutes on ice with gentle shaking, washed twice with 3% (v/v) FBS in PBS, and if necessary, incubated with the secondary antibodies for 30 minutes followed by two more rinses with 3% (v/v) FBS in PBS. For DE cells, we used PE conjugated mouse anti-human CXCR4 and APC conjugated mouse anti-human cKit antibodies; AFE cells were stained with PE-conjugated mouse anti-human CD56 and APC conjugated mouse anti-human CD271 antibodies and LPCs we identified using mouse anti-human CPM antibody. Alexa Fluor 488 donkey anti-mouse IgG was used as the secondary antibody against primary CPM antibody. The cells were either sorted using the BD aria FACS sorter at the Sick Kids FACS core and/or analyzed using a Beckman Coulter Gallios flow cytometer. Unstained controls were used to set up gates for FSC and SSC and double stained cells underwent fluorescence minus one (FMO) to ensure accurate gating. Sorted cells were collected and the spun down into a cell pellet and stored in -80C degrees for further processing. The endodermal differentiations and cell sorts were repeated at four different times with two biological repeats.

Sampleprep ID:

SP002143

Sampleprep Summary:

At each differentiation step, DE, AFE and LPC, cells were analyzed by FACS for stage specific markers. The differentiated cells were harvested with TrypLE at 37 °C for 5-10 minutes and collected by centrifugation at 300 g for 5 minutes at room temperature. The cell pellets were resuspended in 3% (v/v) FBS in PBS, cell aggregates were removed using a cell strainer with a 40-µm pore size and single cells were collected. For surface antigens, the cells were incubated with primary antibodies for 30 minutes on ice with gentle shaking, washed twice with 3% (v/v) FBS in PBS, and if necessary, incubated with the secondary antibodies for 30 minutes followed by two more rinses with 3% (v/v) FBS in PBS. For DE cells, we used PE conjugated mouse anti-human CXCR4 and APC conjugated mouse anti-human cKit antibodies; AFE cells were stained with PE-conjugated mouse anti-human CD56 and APC conjugated mouse anti-human CD271 antibodies and LPCs we identified using mouse anti-human CPM antibody. Alexa Fluor 488 donkey anti-mouse IgG was used as the secondary antibody against primary CPM antibody. The cells were either sorted using the BD aria FACS sorter at the Sick Kids FACS core and/or analyzed using a Beckman Coulter Gallios flow cytometer. Unstained controls were used to set up gates for FSC and SSC and double stained cells underwent fluorescence minus one (FMO) to ensure accurate gating. Sorted cells were collected and the spun down into a cell pellet and stored in -80C degrees for further processing. The endodermal differentiations and cell sorts were repeated at four different times with two biological repeats.