Dataset Information

Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes.

ABSTRACT: When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS). Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.

SUBMITTER: Mallanna SK

PROVIDER: S-EPMC5032032 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes.

Mallanna Sunil K SK Cayo Max A MA Twaroski Kirk K Gundry Rebekah L RL Duncan Stephen A SA

Stem cell reports 20160825 3

When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS). Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly en ...[more]

PMID: 27569060

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Project description:The plasma membrane proteome plays a crucial role in inter- and intracellular signaling, cell survival, and cell identity. As such, it is a prominent target for pharmacological intervention. The relatively low abundance of this subproteome in conjunction with challenging extractability and solubility still hampers its comprehensive analysis. Here, we combined a chemical glycoprotein-tagging strategy with mass spectrometry to enable comprehensive analysis of the cell-surface glycoproteome. To benchmark this workflow and to provide guidance for cell line selection for functional experiments, we generated an inventory of the N-linked cell-surface glycoproteomes of 15 standard laboratory human cell lines and three primary lymphocytic cell types. On average, about 900 plasma membrane and secreted proteins were identified per experiment, including more than 300 transporters and ion channels. Primary cells displayed distinct expression of surface markers and transporters underpinning the importance of carefully validating model cell lines selected for the study of cell surface-mediated processes. To monitor dynamic changes of the cell-surface proteome in a highly multiplexed experiment, we employed an isobaric mass tag-based chemical labeling strategy. This enabled the time-resolved analysis of plasma membrane protein presentation during differentiation of the monocytic suspension cell line THP-1 into macrophage-like adherent cells. Time-dependent changes observed in membrane protein presentation reflect functional remodeling during the phenotypic transition in three distinct phases: rapid surface presentation and secretion of proteins from intracellular pools concurrent with rapid internalization of no longer needed proteins and finally delayed presentation of newly synthesized macrophage markers. Perturbation of this process using marketed receptor tyrosine kinase inhibitors revealed dasatinib to severely compromise macrophage differentiation due to an off-target activity. This finding suggests that dynamic processes can be highly vulnerable to drug treatment and should be monitored more rigorously to identify adverse drug effects.

Dataset Information

Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes.

Publications

Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes.

Similar Datasets

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