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Proteome analysis of human Wharton's jelly cells during in vitro expansion.

ABSTRACT: BACKGROUND:The human umbilical cord contains mucoid connective tissue and fibroblast-like cells. These cells named Wharton's jelly cells, (WJCs) display properties similar to mesenchymal stem cells therefore representing a rich source of primitive cells to be potentially used in regenerative medicine. RESULTS:To better understand their self-renewal and potential in vitro expansion capacity, a reference 2D map was constructed as a proteomic data set. 158 unique proteins were identified. More than 30% of these proteins belong to cytoskeleton compartment. We also found that several proteins including Shootin1, Adenylate kinase 5 isoenzyme and Plasminogen activator-inhibitor 2 are no longer expressed after the 2nd passage of in vitro replication. This indicates that the proliferative potency of these cells is reduced after the initial stage of in vitro growing. At the end of cellular culturing, new synthesized proteins, including, ERO1-like protein alpha, Aspartyl-tRNA synthetase and Prolyl-4-hydroxylase were identified. It is suggested that these new synthesized proteins are involved in the impairment of cellular surviving during replication and differentiation time. CONCLUSIONS:Our work represents an essential step towards gaining knowledge of the molecular properties of WJCs so as to better understand their possible use in the field of cell therapy and regenerative medicine.

SUBMITTER: Angelucci S

PROVIDER: S-EPMC2867805 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Proteome analysis of human Wharton's jelly cells during in vitro expansion.

Angelucci Stefania S Marchisio Marco M Di Giuseppe Fabrizio F Pierdomenico Laura L Sulpizio Marilisa M Eleuterio Enrica E Lanuti Paola P Sabatino Giuseppe G Miscia Sebastiano S Di Ilio Carmine C

Proteome science 20100326

<h4>Background</h4>The human umbilical cord contains mucoid connective tissue and fibroblast-like cells. These cells named Wharton's jelly cells, (WJCs) display properties similar to mesenchymal stem cells therefore representing a rich source of primitive cells to be potentially used in regenerative medicine.<h4>Results</h4>To better understand their self-renewal and potential in vitro expansion capacity, a reference 2D map was constructed as a proteomic data set. 158 unique proteins were identi ...[more]

PMID: 20346146

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Project description:BackgroundMesenchymal stem/stromal cells (MSCs) are multipotent cells with a promising application potential in regenerative medicine and immunomodulation. However, MSCs cultured in vitro exhibit functional heterogeneity. The underlying molecular mechanisms that define MSC heterogeneity remain unclear.MethodsWe investigated the gene expression profile via single-cell RNA sequencing (scRNA-seq) of human primary Wharton's jelly-derived MSCs (WJMSCs) cultured in vitro from three donors. We also isolated CD142+ and CD142- WJMSCs based on scRNA-seq data and compared their proliferation capacity and "wound healing" potential in vitro. Meanwhile, we analyzed publicly available adipose-derived MSC (ADMSCs) scRNA-seq data and performed transcriptome comparison between WJMSCs and ADMSCs at the single-cell level.ResultsGO enrichment analysis of highly variable genes (HVGs) obtained from WJMSCs revealed that these genes are significantly enriched in extracellular region with binding function, involved in developmental process, signal transduction, cell proliferation, etc. Pathway analysis showed that these HVGs are associated with functional characteristics of classic MSCs, such as inflammation mediated by chemokine and cytokine signaling, integrin signaling, and angiogenesis. After regressing out the batch and cell cycle effects, these HVGs were used for dimension reduction and clustering analysis to identify candidate subpopulations. Differentially expressed gene analysis revealed the existence of several distinct subpopulations of MSCs that exhibit diverse functional characteristics related to proliferation, development, and inflammation response. In line with our data, sorted CD142+ and CD142- WJMSCs showed distinct proliferation capacity as well as "wound healing" potential. Although WJMSCs and ADMSCs were derived from different tissues and were displaying different differentiation potencies, their HVGs were largely overlapped and had similar functional enrichment.ConclusionHVGs identified in MSCs are associated with classic MSC function. Regarding therapeutic potential, these genes are associated with functional characteristics, on which the MSC clinical application were theoretically based, such as development and inflammation response. Altogether, these HVGs hold the potential to be used as candidate markers for further potency association studies.

Project description:Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are a novel and promising strategy for tissue engineering because of their ability to differentiate into many cell types. We characterized the differentiation of WJ-MSCs into endometrial epithelial cell (EEC)-like and endometrial stromal cell (ESC)-like cells and assessed the effect of 17?-estradiol and 8-Br-cAMP on the differentiation system.WJ-MSCs were treated in two ways to differentiate into EEC-like and ESC-like cells respectively: cocultured with ESCs in control/differentiation medium (17?-estradiol, growth factors); and cultured in control/differentiation medium (8-Br-cAMP alone or 8-Br-cAMP plus 17?-estrogen and growth factors). Three signaling pathway inhibitors (SB203580, PD98059, H89) were used to investigate the mechanism of WJ-MSC differentiation into ESC-like cells. Immunofluorescence, western blot and flow cytometry analyses were used to analyze expression of epithelial markers and stromal cell markers. Enzyme-linked immunosorbent assays were used to test the production of secretory proteins associated with the differentiation of ESC-like cells.17?-estradiol at 1 ?M downregulated vimentin and CD13 and upregulated cytokeratin and CD9 proteins, promoting the differentiation of WJ-MSCs into EEC-like cells in the coculture system. 8-Br-cAMP at 0.5 mM upregulated vimentin and CD13 and downregulated CK and CD9, promoting the differentiation of WJ-MSCs into ESC-like cells. Prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) were upregulated and the protein kinase A (PKA) signaling pathway was activated, whereas extracellular signal-regulated (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were not affected.17?-estradiol at 1 ?M is a good inducer for facilitating the differentiation of WJ-MSCs into EEC-like cells. 8-Br-cAMP plus estrogen and growth factors can induce the differentiation of WJ-MSCs into ESC-like cells. During the differentiation of WJ-MSCs into ESC-like cells, PRL and IGFBP1 were upregulated by the treatment and the PKA signaling pathway was activated, whereas ERK1/2 and p38 MAPK were not affected. These findings suggest a promising approach to the treatment of endometrial damage and other endometrial diseases and suggest new applications for WJ-MSCs in clinical practice.

Dataset Information

Proteome analysis of human Wharton's jelly cells during in vitro expansion.

Publications

Proteome analysis of human Wharton's jelly cells during in vitro expansion.

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