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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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Related in: MedlinePlus

Differentiation of hMSCs towards the adipogenic or osteogenic lineage.A+B. Immunocytochemistry after 1 week of differentiation. Nuclei were visualized with DAPI. C+D. Flow cytometry after 2 weeks. The change in the mean fluorescence intensity (MFI) before and after differentiation was calculated taking hMSCs as a reference. At least three independent experiments were performed. Black: hMSCs; red: differentiated cells. Shown are representative stainings.
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pone-0020399-g004: Differentiation of hMSCs towards the adipogenic or osteogenic lineage.A+B. Immunocytochemistry after 1 week of differentiation. Nuclei were visualized with DAPI. C+D. Flow cytometry after 2 weeks. The change in the mean fluorescence intensity (MFI) before and after differentiation was calculated taking hMSCs as a reference. At least three independent experiments were performed. Black: hMSCs; red: differentiated cells. Shown are representative stainings.

Mentions: The hMSCs were differentiated toward the osteogenic or the adipocytic lineages using classical differentiation cocktails. After differentiation, modifications of the cell surface proteome, more precisely the CD markers, were monitored by flow cytometry and immunocytochemistry. These analyses showed that the expression of a panel of markers was changed (Figure 4). CD90, CD97, CD98, CD105, and CD155 were downregulated in both adipocytes and osteoblasts, whereas CD63, CD73. CD112, and CD166 were upregulated in both cell types after 2 weeks of differentiation. We noticed that CD97 expression increased during the first week of differentiation towards osteoblasts but decreased drastically when osteoblasts were mature. The remaining markers, CD9, CD29, CD44, CD54, CD56, CD61, CD71, CD99, CD106, CD146, CD276, and CD304 had different expression patterns during adipogenesis and osteogenesis, respectively, whereas the expression of the negative markers, CD14, CD34, CD45, and CD133, did not change considerably as they were already not detected on hMSCs (Figure 2).


The cell surface proteome of human mesenchymal stromal cells.

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

Differentiation of hMSCs towards the adipogenic or osteogenic lineage.A+B. Immunocytochemistry after 1 week of differentiation. Nuclei were visualized with DAPI. C+D. Flow cytometry after 2 weeks. The change in the mean fluorescence intensity (MFI) before and after differentiation was calculated taking hMSCs as a reference. At least three independent experiments were performed. Black: hMSCs; red: differentiated cells. Shown are representative stainings.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020399-g004: Differentiation of hMSCs towards the adipogenic or osteogenic lineage.A+B. Immunocytochemistry after 1 week of differentiation. Nuclei were visualized with DAPI. C+D. Flow cytometry after 2 weeks. The change in the mean fluorescence intensity (MFI) before and after differentiation was calculated taking hMSCs as a reference. At least three independent experiments were performed. Black: hMSCs; red: differentiated cells. Shown are representative stainings.
Mentions: The hMSCs were differentiated toward the osteogenic or the adipocytic lineages using classical differentiation cocktails. After differentiation, modifications of the cell surface proteome, more precisely the CD markers, were monitored by flow cytometry and immunocytochemistry. These analyses showed that the expression of a panel of markers was changed (Figure 4). CD90, CD97, CD98, CD105, and CD155 were downregulated in both adipocytes and osteoblasts, whereas CD63, CD73. CD112, and CD166 were upregulated in both cell types after 2 weeks of differentiation. We noticed that CD97 expression increased during the first week of differentiation towards osteoblasts but decreased drastically when osteoblasts were mature. The remaining markers, CD9, CD29, CD44, CD54, CD56, CD61, CD71, CD99, CD106, CD146, CD276, and CD304 had different expression patterns during adipogenesis and osteogenesis, respectively, whereas the expression of the negative markers, CD14, CD34, CD45, and CD133, did not change considerably as they were already not detected on hMSCs (Figure 2).

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