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Stem cell research & therapy2018; 9(1); 288; doi: 10.1186/s13287-018-1041-8

Mapping of equine mesenchymal stromal cell surface proteomes for identification of specific markers using proteomics and gene expression analysis: an in vitro cross-sectional study.

Abstract: Stem cells have great potential for tissue regeneration, but before stem cell populations can be used in the clinic, it is crucial that the stem cells have been definitely characterized by a set of specific markers. Although there have been attempts to identify a set of immunophenotypic markers to characterize equine mesenchymal stromal cells (MSCs), immunophenotyping of equine MSCs is still challenging due to the limited availability of suitable antibodies of high quality and consistent performance across different laboratories. The aim of this study was to evaluate a strategy for mapping the equine MSCs surface proteome by use of biotin-enrichment and mass spectrometry (MS) analysis and mine the proteome for potential equine MSCs surface markers belonging to the cluster of differentiation protein group. Gene expression analysis was used for verification. Equine MSCs derived from bone marrow (BM) (n = 3) and adipose tissue (AT) (n = 3) were expanded to P3 and either used for (1) cell differentiation into mesodermal lineages (chondrogenic and osteogenic), (2) enrichment of the MSCs surface proteins by biotinylation followed by in-gel digest of the isolated proteins and nanoLC-MS/MS analysis to unravel the enriched cell surface proteome, and (3) RNA isolation and quantitative real-time reverse transcriptase PCR analysis of the CD29, CD44, CD90, CD105, CD166, CD34, CD45, and CD79a gene expression. A total of 1239 proteins at 1% FDR were identified by MS analysis of the enriched MSCs surface protein samples. Of these proteins, 939 were identified in all six biological samples. The identified proteins included 19 proteins appointed to the cluster of differentiation classification system as potential cell surface targets. The protein and gene expression pattern was positive for the commonly used positive MSCs markers CD29, CD44, CD90, CD105, and CD166, and lacked the negative MSCs markers CD34, CD45, and CD79a. The findings of this study show that enrichment of the MSCs surface proteome by biotinylation followed by MS analysis is a valuable alternative to immunophenotyping of surface markers, when suitable antibodies are not available. Further, they support gene expression analysis as a valuable control analysis to verify the data.
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Publication Date: 2018-10-25 PubMed ID: 30359315PubMed Central: PMC6202851DOI: 10.1186/s13287-018-1041-8Google Scholar: Lookup
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  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article focuses on an innovative approach for identifying immunophenotypic markers to characterize equine mesenchymal stromal cells (MSCs). The method involves mapping the equine MSCs surface proteome using biotin-enrichment, mass spectrometry analysis, and gene expression analysis.

Objective of the Study

  • The primary objective of the researchers was to develop a dependable method for characterizing equine MSCs. This was necessitated by the challenges faced during immunophenotyping due to insufficient high quality and consistent performing antibodies.

Methodology of the Study

  • Equine MSCs were derived from bone marrow and adipose tissue and were taken through different processes such as cell differentiation into mesodermal lineages (osteogenic and chondrogenic).
  • An enrichment process of the MSCs surface proteins was conducted through biotinylation. This was then followed by in-gel digest of the proteins that were isolated and nanoLC-MS/MS analysis to discover the enriched cell surface proteome.
  • Further, RNA isolation was carried out and quantitative real-time reverse transcriptase PCR analysis was conducted for gene expression analysis.

Results of the Study

  • A total of 1239 proteins were identified through the MS analysis of the enriched MSCs surface protein samples. Out of these, 939 proteins were identified in all six biological samples.
  • 19 of these proteins were appointed to the cluster of differentiation classification system as possible cell surface targets.
  • The patterns of protein and gene expressions were positive for commonly used positive MSCs markers such as CD29, CD44, CD90, CD105, and CD166, and negative for MSCs markers such as CD34, CD45, and CD79a.

Conclusion of the Study

  • The study concludes that enrichment of the MSCs surface proteome by biotinylation followed by MS analysis is a dependable alternative to immunophenotyping of surface markers, particularly when suitable antibodies are not available.
  • The study further emphasized that gene expression analysis can serve as a valuable control analysis for validation of data.

Cite This Article

APA
Bundgaard L, Stensballe A, Elbæk KJ, Berg LC. (2018). Mapping of equine mesenchymal stromal cell surface proteomes for identification of specific markers using proteomics and gene expression analysis: an in vitro cross-sectional study. Stem Cell Res Ther, 9(1), 288. https://doi.org/10.1186/s13287-018-1041-8

Publication

Stem cell research & therapy
ISSN: 1757-6512
NlmUniqueID: 101527581
Country: England
Language: English
Volume: 9
Issue: 1
Pages: 288
PII: 288

Researcher Affiliations

Bundgaard, Louise
  • Department of Veterinary Clinical Sciences, University of Copenhagen, Agrovej 8, DK-2630, Taastrup, Denmark. lb@sund.ku.dk.
Stensballe, Allan
  • Department of Health Science and Technology, Aalborg University, Fredrik Bajers Vej 7, 9220, Aalborg Ø, Denmark.
Elbæk, Kirstine Juul
  • Department of Health Science and Technology, Aalborg University, Fredrik Bajers Vej 7, 9220, Aalborg Ø, Denmark.
Berg, Lise Charlotte
  • Department of Veterinary Clinical Sciences, University of Copenhagen, Agrovej 8, DK-2630, Taastrup, Denmark.

MeSH Terms

  • Animals
  • Biomarkers / metabolism
  • Cell Differentiation
  • Cell Lineage
  • Cell Shape
  • Cross-Sectional Studies
  • Gene Expression Regulation
  • Horses / metabolism
  • Mass Spectrometry
  • Mesenchymal Stem Cells / cytology
  • Proteome / metabolism
  • Proteomics / methods

Conflict of Interest Statement

ETHICS APPROVAL AND CONSENT TO PARTICIPATE: The experimental protocol was approved by the Ethics and Welfare Committee of Department of Veterinary Clinical Sciences, University of Copenhagen, and all procedures were carried out according to the Danish Act on animal experiments. CONSENT FOR PUBLICATION: Not applicable. COMPETING INTERESTS: The authors declare that they have no competing interests. PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Citations

This article has been cited 10 times.
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