Dataset Information

Identification of mouse embryonic fibroblast secreted protein factors that are responsible for reprogramming human pluripotent stem cell metabolism

ABSTRACT: Previously we found that human pluripotent stem cells (hPSCs) utilize glucose differently depending on the presence of the feeder cells, which are mouse embryonic fibroblasts, or MEFs. More specifically, feeder-free cultured hPSCs are more reliant on glycolysis for proliferation. Therefore, we hypothesized that secreted factors by MEFs might be responsible for reprogramming the metabolism of hPSCs. To test this hypothesis, we separated the components in the MEF-conditioned medium by using size-based fractionation columns, and tested whether each fraction alters the reliance of feeder-free hPSCs on glucose. We concluded that it was the protein fraction of the MEF-conditioned medium potentially responsible for reprogramming glycolytic metabolism in hPSCs. To further understand which specific protein(s) could alter the metabolism of hPSCs, we here conduct mass spectrometry based proteomics experiment.

INSTRUMENT(S): LTQ

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Wen Gu

LAB HEAD: Wen Gu

PROVIDER: PXD013662 | Pride | 2022-04-01

REPOSITORIES: Pride

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Publications

Fast Deisotoping Algorithm and Its Implementation in the MSFragger Search Engine.

Teo Guo Ci GC Polasky Daniel A DA Yu Fengchao F Nesvizhskii Alexey I AI

Journal of proteome research 20201217 1

Deisotoping, or the process of removing peaks in a mass spectrum resulting from the incorporation of naturally occurring heavy isotopes, has long been used to reduce complexity and improve the effectiveness of spectral annotation methods in proteomics. We have previously described MSFragger, an ultrafast search engine for proteomics, that did not utilize deisotoping in processing input spectra. Here, we present a new, high-speed parallelized deisotoping algorithm, based on elements of several ex ...[more]

PMID: 33332123

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Project description:These are 3 TMT10 samples. The indicated "standard" sub-samples all refer to the same biological sample (obtained as a mixture of all other samples), split and tagged as indicated, to play as a reference for normalization. TMT10 sample1 contains the following sub-samples: -TAG 126: Standard. -TAG 127N: Day 3 conditioned medium of replication-incompetent MEFs cultured on VTN coating in RSeT medium. -TAG 127C: Day 3 conditioned medium of naive human pluripotent stem cells cultured on a MEF feeder-layer in RSeT medium. -TAG 128N: Day 3 conditioned medium of naive human pluripotent stem cells cultured on a VTN coating in RSeT medium. -TAG 128C: Not relevant. -TAG 129N: Not relevant. -TAG 129C: Not relevant. -TAG 130N: Not relevant. -TAG 130C: RSet medium. -TAG 131: Standard. TMT10 sample2 contains the following sub-samples: -TAG 126: Standard -TAG 127N: Day 3 conditioned medium of replication-incompetent MEFs cultured on VTN coating in RSeT medium. -TAG 127C: Day 3 conditioned medium of naive human pluripotent stem cells cultured on a MEF feeder-layer in RSeT medium. -TAG 128N: Day 3 conditioned medium of naive human pluripotent stem cells cultured on a VTN coating in RSeT medium. -TAG 128C: Not relevant. -TAG 129N: Day 2 conditioned medium of naive human pluripotent stem cells cultured on a MEF feeder-layer in RSeT medium. -TAG 129C: Day 2 conditioned medium of naive human pluripotent stem cells cultured on a VTN coating in RSeT medium. -TAG 130N: Not relevant. -TAG 130C: RSet medium. -TAG 131: Standard. TMT10 sample3 contains the following sub-samples: -TAG 126: Standard. -TAG 127N: Day 3 conditioned medium of replication-incompetent MEFs cultured on VTN coating in RSeT medium. -TAG 127C: Day 3 conditioned medium of naive human pluripotent stem cells cultured on a MEF feeder-layer in RSeT medium. -TAG 128N: Day 2 conditioned medium of replication-incompetent MEFs cultured on VTN coating in RSeT medium. -TAG 128C: Day 2 conditioned medium of replication-incompetent MEFs cultured on VTN coating in RSeT medium. -TAG 129N: Day 2 conditioned medium of naive human pluripotent stem cells cultured on a MEF feeder-layer in RSeT medium. -TAG 129C: Day 2 conditioned medium of naive human pluripotent stem cells cultured on a VTN coating in RSeT medium. -TAG 130N: Not relevant. -TAG 130C: RSet medium. -TAG 131: Standard. Detailed culture and processing methods are described in Cesare et al, under submission.

Project description:Background: The hES-T3 cell line with normal female karyotype was used to differentiate into autogeneic fibroblast-like cells (T3HDF) as feeder to support the undifferentiated growth of hES-T3 cells (T3/HDF) for 14 passages. A feeder-free culture on Matrigel in hES medium conditioned by these autogeneic feeder cells (T3HDF) was established to maintain the undifferentiated growth of hES-T3 cells (T3/CMHDF) for 8 passages. Results: The gene expression profiles of mRNAs, microRNAs and proteins between the undifferentiated T3/HDF and T3/CMHDF cells were shown to be very similar, and their expression profiles were also found to be similar to those of T3/MEF and T3/CMMEF cells grown on MEF feeder and feeder-free Matrigel in MEF-conditioned medium, respectively. The abundantly expressed genes of T3/HDF, T3/CMHDF, T3/MEF and T3/CMMEF cells were found to play prominent roles in signaling pathways and GO process networks. The miR-302/367 cluster and miR-371/372/373 cluster were extremely abundantly expressed in undifferentiated T3/HDF and T3/CMHDF as well as T3/MEF andT3/CMMEF cells. The undifferentiated state of T3/HDF and T3/CMHDF cells was evidenced by the very high expression levels of ¡§stemness¡¨ genes and low expression levels of differentiation markers of ectoderm, mesoderm and endoderm in addition to the strong staining of OCT4 and NANOG. Conclusion: The T3HDF feeder and T3HDF-conditioned medium were able to support the undifferentiated growth of hES cells, and they would be useful for drug development and toxicity testing in addition to the reduced risks of xenogeneic pathogens when used for medical applications such as cell therapies. Keywords: genetic modification In this investigation, both miRNA and mRNA expression profiles from human embryonic stem cells (hES-T3) grown on MEF feeder (T3_MEF), feeder-free Matrigel in MEF-conditioned medium (T3_CMMEF), T3HDF (hES-T3 differentiated fibroblast-like cells) feeder and feeder-free Matrigel in T3HDF-conditioned medium were quantitatively determined. Several target genes of T3BA and T3CM cells-specific miRNAs were identified. ***This submission represents the mRNA expression component of the study only***

Dataset Information

Identification of mouse embryonic fibroblast secreted protein factors that are responsible for reprogramming human pluripotent stem cell metabolism

Publications

Fast Deisotoping Algorithm and Its Implementation in the MSFragger Search Engine.

Similar Datasets

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