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The cell surface proteome of human mesenchymal stromal cells.

Niehage C, Steenblock C, Pursche T, Bornhäuser M, Corbeil D, Hoflack B - PLoS ONE (2011)

Bottom Line: The CD markers are distributed homogenously within plastic-adherent hMSC populations and their expression is modulated during the process of adipogenesis or osteogenesis.Moreover, our in silico analysis revealed a significant difference between the cell surface proteome of hMSCs and that of human embryonic stem cells reported previously.Collectively, our analytical methods not only provide a basis for further studies of mechanisms maintaining the multipotency of hMSCs within their niches and triggering their differentiation after signaling, but also a toolbox for a refined antibody-based identification of hMSC populations from different tissues and their isolation for therapeutic intervention.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Center, Dresden University of Technology, Dresden, Germany.

ABSTRACT

Background: Multipotent human mesenchymal stromal cells (hMSCs) are considered as promising biological tools for regenerative medicine. Their antibody-based isolation relies on the identification of reliable cell surface markers.

Methodology/principal findings: To obtain a comprehensive view of the cell surface proteome of bone marrow-derived hMSCs, we have developed an analytical pipeline relying on cell surface biotinylation of intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin to enrich the plasma membrane proteins and mass spectrometry for identification with extremely high confidence. Among the 888 proteins identified, we found ≈200 bona fide plasma membrane proteins including 33 cell adhesion molecules and 26 signaling receptors. In total 41 CD markers including 5 novel ones (CD97, CD112, CD239, CD276, and CD316) were identified. The CD markers are distributed homogenously within plastic-adherent hMSC populations and their expression is modulated during the process of adipogenesis or osteogenesis. Moreover, our in silico analysis revealed a significant difference between the cell surface proteome of hMSCs and that of human embryonic stem cells reported previously.

Conclusions/significance: Collectively, our analytical methods not only provide a basis for further studies of mechanisms maintaining the multipotency of hMSCs within their niches and triggering their differentiation after signaling, but also a toolbox for a refined antibody-based identification of hMSC populations from different tissues and their isolation for therapeutic intervention.

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Flow cytometric analysis of hMSC surface CD markers.Cells were harvested and labeled with fluorescence-conjugated antibodies recognizing cell surface markers. Black: isotype control; blue: Ab against surface markers. At least three independent experiments were performed. Shown are representative flow cytometry histograms.
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pone-0020399-g002: Flow cytometric analysis of hMSC surface CD markers.Cells were harvested and labeled with fluorescence-conjugated antibodies recognizing cell surface markers. Black: isotype control; blue: Ab against surface markers. At least three independent experiments were performed. Shown are representative flow cytometry histograms.

Mentions: The MS-based proteomic identifications were confirmed by flow cytometry using a panel of specific antibodies recognizing some of the CD markers identified by MS. Our analysis demonstrated that the expanded, plastic-adherent hMSCs were positive for a number of surface markers (Figure 2). During all cell passages, the hMSCs were positive for the hMSC-associated CD markers CD29, CD44, CD73, CD90, CD105, CD146, and CD166, and negative for the hematopoietic markers CD14, CD34, and CD45 as well as CD133 as previously reported [33]. In addition, the hMSCs were positive for a panel of other surface markers like CD54, CD56, CD61, CD63, CD71, CD97, CD98, CD99, CD106, CD112, CD155, CD276 and CD304 in agreement with the MS data (except for CD56) (Figure 2, Table 1, and Table S2). Furthermore, CD325 expression was confirmed by western blot analysis using total cell extracts from hMSCs (data not shown).


The cell surface proteome of human mesenchymal stromal cells.

Niehage C, Steenblock C, Pursche T, Bornhäuser M, Corbeil D, Hoflack B - PLoS ONE (2011)

Flow cytometric analysis of hMSC surface CD markers.Cells were harvested and labeled with fluorescence-conjugated antibodies recognizing cell surface markers. Black: isotype control; blue: Ab against surface markers. At least three independent experiments were performed. Shown are representative flow cytometry histograms.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102717&req=5

pone-0020399-g002: Flow cytometric analysis of hMSC surface CD markers.Cells were harvested and labeled with fluorescence-conjugated antibodies recognizing cell surface markers. Black: isotype control; blue: Ab against surface markers. At least three independent experiments were performed. Shown are representative flow cytometry histograms.
Mentions: The MS-based proteomic identifications were confirmed by flow cytometry using a panel of specific antibodies recognizing some of the CD markers identified by MS. Our analysis demonstrated that the expanded, plastic-adherent hMSCs were positive for a number of surface markers (Figure 2). During all cell passages, the hMSCs were positive for the hMSC-associated CD markers CD29, CD44, CD73, CD90, CD105, CD146, and CD166, and negative for the hematopoietic markers CD14, CD34, and CD45 as well as CD133 as previously reported [33]. In addition, the hMSCs were positive for a panel of other surface markers like CD54, CD56, CD61, CD63, CD71, CD97, CD98, CD99, CD106, CD112, CD155, CD276 and CD304 in agreement with the MS data (except for CD56) (Figure 2, Table 1, and Table S2). Furthermore, CD325 expression was confirmed by western blot analysis using total cell extracts from hMSCs (data not shown).

Bottom Line: The CD markers are distributed homogenously within plastic-adherent hMSC populations and their expression is modulated during the process of adipogenesis or osteogenesis.Moreover, our in silico analysis revealed a significant difference between the cell surface proteome of hMSCs and that of human embryonic stem cells reported previously.Collectively, our analytical methods not only provide a basis for further studies of mechanisms maintaining the multipotency of hMSCs within their niches and triggering their differentiation after signaling, but also a toolbox for a refined antibody-based identification of hMSC populations from different tissues and their isolation for therapeutic intervention.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Center, Dresden University of Technology, Dresden, Germany.

ABSTRACT

Background: Multipotent human mesenchymal stromal cells (hMSCs) are considered as promising biological tools for regenerative medicine. Their antibody-based isolation relies on the identification of reliable cell surface markers.

Methodology/principal findings: To obtain a comprehensive view of the cell surface proteome of bone marrow-derived hMSCs, we have developed an analytical pipeline relying on cell surface biotinylation of intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin to enrich the plasma membrane proteins and mass spectrometry for identification with extremely high confidence. Among the 888 proteins identified, we found ≈200 bona fide plasma membrane proteins including 33 cell adhesion molecules and 26 signaling receptors. In total 41 CD markers including 5 novel ones (CD97, CD112, CD239, CD276, and CD316) were identified. The CD markers are distributed homogenously within plastic-adherent hMSC populations and their expression is modulated during the process of adipogenesis or osteogenesis. Moreover, our in silico analysis revealed a significant difference between the cell surface proteome of hMSCs and that of human embryonic stem cells reported previously.

Conclusions/significance: Collectively, our analytical methods not only provide a basis for further studies of mechanisms maintaining the multipotency of hMSCs within their niches and triggering their differentiation after signaling, but also a toolbox for a refined antibody-based identification of hMSC populations from different tissues and their isolation for therapeutic intervention.

Show MeSH
Related in: MedlinePlus