Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Secreted footprint of pluripotency in Man-13 HeSCs

ABSTRACT: With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Stem Cell, Cell Culture

SUBMITTER: Phil Lewis

LAB HEAD: Susan Kimber

PROVIDER: PXD045525 | Pride | 2024-03-26

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	11-10-16_LOP1_G_run1_mascot.xml	Xml
	11-10-16_LOP1_G_run1_peptides.csv	Csv
	11-10-16_LOP1_G_run1_proteins.csv	Csv
	11-10-16_LOP1_G_run2_msmsspectra_export.mgf	Mgf
	11-10-16_LOP1_G_run2_peptides.csv	Csv

Items per page:

1 - 5 of 54

Similar Datasets

Secreted footprint of pluripotency in REBL.PAT IPSCs

Project description:With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.

2024-03-26 | PXD045347 | Pride

Multi-Omic Profiling Reveals Dynamics of the Phased Progression of Pluripotency

Project description:Pluripotency is highly dynamic and progresses through a continuum of pluripotent stem-cell states. The two states that bookend the pluripotency continuum, naïve and primed, are well characterized, but our understanding of the intermediate states and transitions between them remain incomplete. Here, we dissect the dynamics of pluripotent state transitions underlying pre- to post-implantation epiblast differentiation. Through comprehensive mapping of the proteome, phosphoproteome, transcriptome, and epigenome of embryonic stem cells transitioning from naïve to primed pluripotency, we find that rapid, acute, and widespread changes to the phosphoproteome precede ordered changes to the epigenome, transcriptome, and proteome. Reconstruction of kinase-substrate networks reveals signaling cascades, dynamics, and crosstalk. Distinct waves of global proteomic changes mark discrete phases of pluripotency, with cell state-specific surface markers tracking pluripotent state transitions. Our data provide new insights into the multi-layered control of the phased progression of pluripotency and a foundation for modeling mechanisms regulating pluripotent state transitions (www.stemcellatlasorg).

2019-05-07 | PXD010621 | Pride

Quantitative subcellular proteomics using SILAC reveals enhanced metabolic buffering in ground state pluripotency

Project description:The ground state of pluripotency is defined as a minimal unrestricted state as present in the Inner Cell Mass (ICM). Mouse embryonic stem cells (ESCs) grown in a defined serum-free medium with two kinase inhibitors (‘2i’) reflect this state, whereas ESCs grown in the presence of serum (‘serum’) share more similarities with post implantation epiblast cells. Pluripotency results from an intricate interplay between cytoplasmic, nuclear and chromatin-associated proteins. Therefore, quantitative information on the (sub)cellular proteome is essential to gain insight in the molecular mechanisms driving different pluripotent states. Here, we describe a full SILAC workflow and quality controls for proteomic comparison of 2i and serum ESCs. We demonstrate that this workflow is applicable for subcellular proteomics of the cytoplasm, nuclear and chromatin. The obtained quantitative information revealed increased levels of naïve pluripotency factors on the chromatin of 2i ESCs. Further, we demonstrate that these pluripotent states are supported by distinct metabolic programs, which include upregulation of free radical buffering by the glutathione pathway in 2i ESCs. Through induction of intracellular radicals, we show that the altered metabolic environment renders 2i ESCs less sensitive to oxidative stress. Altogether, this work provides novel insights into the proteome landscape underlying ground state pluripotency.

2018-11-07 | PXD009217 | Pride

Identification of SUMO targets required to maintain human stem cells in the pluripotent state. - Identification of SUMO modified proteins from hiPS cells

Project description:To determine the role of SUMO modification in the maintenance of pluripotent stem cells we used ML792, a potent and selective inhibitor of SUMO Activating Enzyme. Treatment of human induced pluripotent stem cells with ML792 initiated changes associated with loss of pluripotency such as reduced expression of key pluripotency markers. To identify putative effector proteins and establish sites of SUMO modification, cells were engineered to stably express either SUMO1 or SUMO2 with TGG to KGG mutations that facilitate GlyGly-K peptide immunoprecipitation and identification. A total of 976 SUMO sites were identified in 427 proteins. STRING enrichment created 3 networks of proteins with functions in regulation of gene expression, ribosome biogenesis and RNA splicing, although the latter two categories represented only 5% of the total GGK peptide intensity. The remainder have roles in transcription and chromatin structure regulation. Many of the most heavily SUMOylated proteins form a network of zinc-finger transcription factors centred on TRIM28 and associated with silencing of retroviral elements. At the level of whole proteins there was only limited evidence for SUMO paralogue-specific modification, although at the site level there appears to be a preference for SUMO2 modification over SUMO1 in acidic domains. We show that SUMO is involved in the maintenance of the pluripotent state in hiPSCs and identify many chromatin-associated proteins as bona fide SUMO substrates in human induced pluripotent stem cells.

2021-11-03 | PXD028050 | Pride

Parallel genome-scale CRISPR-Cas9 screens uncouple human pluripotent stem cell identity versus fitness

Project description:Pluripotent stem cells are defined by their self-renewal capacity, which is the ability of the stem cells to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into any somatic cell lineage. However, understanding the mechanisms that control stem cell fitness versus the pluripotent cell identity is challenging. To investigate the interplay between these two aspects of pluripotency, we performed four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSC self-renewal conditions, and the dissolution of the primed pluripotency identity during early differentiation. Comparative analyses led to the discovery of genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further discovered a core set of factors that control both stem cell fitness and pluripotent identity, including a network of chromatin factors that safeguard pluripotency. Our unbiased and systematic screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide rich datasets for exploring pluripotent cell identity versus cell fitness, and offer a valuable model for categorizing gene function in broad biological contexts.

2024-09-05 | PXD055592 | Pride

Human embryonic stem cells in E8 and AKIT culture medium

Project description:Analysis of undifferentiated KhES-1 human embryonic stem cells in growth factors-dependent (E8) and -independent (AKIT) culture medium. Results provide insight into genes differentially expressed in pluripotent states maintained by AKIT and E8 culture medium.

2017-07-01 | GSE92628 | GEO

Transcriptomic profile of human induced pluripotent stem cells at single cell resolution.

Project description:We assessed the pluripotency of human induced pluripotent stem cells (iPSCs) maintained on an automated platform using StemFlexTM and TeSRTM-E8TM media. Single-cell transcriptome sequencing was performed for 20,962 cells from two cell lines grown in the two media. Analysis of transcriptomic profile revealed similar expression of core pluripotency genes, as well as genes associated with naive and primed states of pluripotency. Single cell analysis of the four samples revealed a shared subpopulation structure with three main subpopulations different in pluripotency states. By implementing a machine learning approach, we estimated that 60-96% of the cells in the ground/naive subpopulations and 98-99% of the cells in the primed subpopulations are similar between all four samples. The single cell RNA sequencing analysis of iPSC lines grown in both media is the first report of the molecular pathways modulated in StemFlex medium and how they compare to those modulated in TeSR-E8 medium.

2019-05-08 | E-MTAB-6524 | biostudies-arrayexpress

MPP8 is essential for sustaining self-renewal of ground-state pluripotent stem cells

Project description:Deciphering the mechanisms that control the pluripotent ground state is key for understanding embryonic development. Nonetheless, the epigenetic regulation of ground-state mouse embryonic stem cells (mESCs) is not fully understood. Here, we identify the epigenetic protein MPP8 as being essential for ground-state pluripotency. Its depletion leads to cell cycle arrest and spontaneous differentiation. MPP8 has been suggested to repress LINE1 elements by recruiting the human silencing hub (HUSH) complex to H3K9me3-rich regions. Unexpectedly, we find that LINE1 elements are efficiently repressed by MPP8 lacking the chromodomain, while the unannotated C-terminus is essential for its function. Moreover, we show that SETDB1 recruits MPP8 to its genomic target loci, whereas transcriptional repression of LINE1 elements is maintained without retaining H3K9me3 levels. Taken together our findings demonstrate that MPP8 protects the DNA-hypomethylated pluripotent ground state through its association with the HUSH core complex, however, independently of detectable chromatin binding and maintenance of H3K9me3.

2021-03-21 | PXD019345 | Pride

ERK5 sustains autocrine mitogenic balance for cell fate specification in human pluripotent stem cells [12h XMD RNA-seq]

Project description:To investigate the function ERK5 in the regulation of hPSC pluripotency, we processed ERK5 inhibition (XMD892) treamtent in E8 medium for 12hr in H1 ESCs.

2024-03-01 | GSE234057 | GEO

Expression data from human foreskin fibroblast and human iduced pluripotent stem cell

Project description:Pluripotent cells are important and unique because of unprecedently high proliferation and differentiation potentials. The state of pluripotency is robust enough to sustain years of uninterrupted proliferation in vitro, but, once lost, there is a significant barrier for differentiated cells to regain pluripotency. By now, reprogramming of somatic cells into a pluripotent state is unambiguously proven only for methods that necessarily involve ectopic expression or direct delivery of exogenous factors inside the cells. Molecular mechanisms that provide robustness to the state of pluripotency and sustain the barrier between pluripotent and differentiated cells are not fully understood. We reprogrammed human foreskin fibroblasts into human induced pluripotent stem cells using CoMiP 4in1 plasmid without shRNA p53.

2019-08-06 | GSE135409 | GEO