Development of novel monoclonal antibodies that define differentiation stages of human stromal (mesenchymal) stem cells

Ditte Caroline Andersen, Angela Kortesidis, Andrew C W Zannettino, Irina Kratchmarova, Li Chen, Ole Nørregaard Jensen, Børge Teisner, Stan Gronthos, Charlotte H Jensen, Moustapha Kassem

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Resumé

Human mesenchymal stem cells (hMSC) are currently being introduced for cell therapy, yet, antibodies specific for native and differentiated MSCs are required for their identification prior to clinical use. Herein, high quality antibodies against MSC surface proteins were developed by immunizing mice with hMSC, and by using a panel of subsequent screening methods. Flow cytometry analysis revealed that 83.5, 1.1, and 8.5% of primary cultures of hMSC were double positive for STRO-1 and either of DJ 3, 9, and 18, respectively. However, none of the three DJ antibodies allowed enrichment of clonogenic hMSC from BMMNCs as single reagents. Using mass-spectrometric analysis, we identified the antigen recognised by DJ3 as CD44, whereas DJ9 and DJ18 recognized HLA-DRB1 and Collagen VI, respectively. The identified proteins were highly expressed throughout in vitro osteogenic- and adipogenic differentiation. Interestingly, undifferentiated cells revealed a sole cytoplasmic distribution pattern of Collagen VI, which however changed to an extracellular matrix appearance upon osteogenic- and adipogenic differentiation. In relation to this, we found that STRO-1(+/-)/Collagen VI(-) sorted hMSC contained fewer differentiated alkaline phosphatase(+) cells compared to STRO-1(+/-)/Collagen VI(+) hMSC, suggesting that Collagen VI on the cell membrane exclusively defines differentiated MSCs. In conclusion, we have generated a panel of high quality antibodies to be used for characterization of MSCs, and in addition our results may suggest that the DJ18 generated antibody against Collagen VI can be used for negative selection of cultured undifferentiated MSCs.
OriginalsprogEngelsk
TidsskriftMolecules and Cells
Vol/bind32
Udgave nummer2
Sider (fra-til)133-142
Antal sider10
ISSN1016-8478
DOI
StatusUdgivet - 2011

Fingeraftryk

Mesenchymal Stromal Cells
HLA-DRB1 Chains
Alkaline Phosphatase
Flow Cytometry
Membrane Proteins
Cell Membrane
Proteins

Citer dette

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title = "Development of novel monoclonal antibodies that define differentiation stages of human stromal (mesenchymal) stem cells",
abstract = "Human mesenchymal stem cells (hMSC) are currently being introduced for cell therapy, yet, antibodies specific for native and differentiated MSCs are required for their identification prior to clinical use. Herein, high quality antibodies against MSC surface proteins were developed by immunizing mice with hMSC, and by using a panel of subsequent screening methods. Flow cytometry analysis revealed that 83.5, 1.1, and 8.5{\%} of primary cultures of hMSC were double positive for STRO-1 and either of DJ 3, 9, and 18, respectively. However, none of the three DJ antibodies allowed enrichment of clonogenic hMSC from BMMNCs as single reagents. Using mass-spectrometric analysis, we identified the antigen recognised by DJ3 as CD44, whereas DJ9 and DJ18 recognized HLA-DRB1 and Collagen VI, respectively. The identified proteins were highly expressed throughout in vitro osteogenic- and adipogenic differentiation. Interestingly, undifferentiated cells revealed a sole cytoplasmic distribution pattern of Collagen VI, which however changed to an extracellular matrix appearance upon osteogenic- and adipogenic differentiation. In relation to this, we found that STRO-1(+/-)/Collagen VI(-) sorted hMSC contained fewer differentiated alkaline phosphatase(+) cells compared to STRO-1(+/-)/Collagen VI(+) hMSC, suggesting that Collagen VI on the cell membrane exclusively defines differentiated MSCs. In conclusion, we have generated a panel of high quality antibodies to be used for characterization of MSCs, and in addition our results may suggest that the DJ18 generated antibody against Collagen VI can be used for negative selection of cultured undifferentiated MSCs.",
author = "Andersen, {Ditte Caroline} and Angela Kortesidis and Zannettino, {Andrew C W} and Irina Kratchmarova and Li Chen and Jensen, {Ole N{\o}rregaard} and B{\o}rge Teisner and Stan Gronthos and Jensen, {Charlotte H} and Moustapha Kassem",
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Development of novel monoclonal antibodies that define differentiation stages of human stromal (mesenchymal) stem cells. / Andersen, Ditte Caroline; Kortesidis, Angela; Zannettino, Andrew C W; Kratchmarova, Irina; Chen, Li; Jensen, Ole Nørregaard; Teisner, Børge; Gronthos, Stan; Jensen, Charlotte H; Kassem, Moustapha.

I: Molecules and Cells, Bind 32, Nr. 2, 2011, s. 133-142.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Development of novel monoclonal antibodies that define differentiation stages of human stromal (mesenchymal) stem cells

AU - Andersen, Ditte Caroline

AU - Kortesidis, Angela

AU - Zannettino, Andrew C W

AU - Kratchmarova, Irina

AU - Chen, Li

AU - Jensen, Ole Nørregaard

AU - Teisner, Børge

AU - Gronthos, Stan

AU - Jensen, Charlotte H

AU - Kassem, Moustapha

PY - 2011

Y1 - 2011

N2 - Human mesenchymal stem cells (hMSC) are currently being introduced for cell therapy, yet, antibodies specific for native and differentiated MSCs are required for their identification prior to clinical use. Herein, high quality antibodies against MSC surface proteins were developed by immunizing mice with hMSC, and by using a panel of subsequent screening methods. Flow cytometry analysis revealed that 83.5, 1.1, and 8.5% of primary cultures of hMSC were double positive for STRO-1 and either of DJ 3, 9, and 18, respectively. However, none of the three DJ antibodies allowed enrichment of clonogenic hMSC from BMMNCs as single reagents. Using mass-spectrometric analysis, we identified the antigen recognised by DJ3 as CD44, whereas DJ9 and DJ18 recognized HLA-DRB1 and Collagen VI, respectively. The identified proteins were highly expressed throughout in vitro osteogenic- and adipogenic differentiation. Interestingly, undifferentiated cells revealed a sole cytoplasmic distribution pattern of Collagen VI, which however changed to an extracellular matrix appearance upon osteogenic- and adipogenic differentiation. In relation to this, we found that STRO-1(+/-)/Collagen VI(-) sorted hMSC contained fewer differentiated alkaline phosphatase(+) cells compared to STRO-1(+/-)/Collagen VI(+) hMSC, suggesting that Collagen VI on the cell membrane exclusively defines differentiated MSCs. In conclusion, we have generated a panel of high quality antibodies to be used for characterization of MSCs, and in addition our results may suggest that the DJ18 generated antibody against Collagen VI can be used for negative selection of cultured undifferentiated MSCs.

AB - Human mesenchymal stem cells (hMSC) are currently being introduced for cell therapy, yet, antibodies specific for native and differentiated MSCs are required for their identification prior to clinical use. Herein, high quality antibodies against MSC surface proteins were developed by immunizing mice with hMSC, and by using a panel of subsequent screening methods. Flow cytometry analysis revealed that 83.5, 1.1, and 8.5% of primary cultures of hMSC were double positive for STRO-1 and either of DJ 3, 9, and 18, respectively. However, none of the three DJ antibodies allowed enrichment of clonogenic hMSC from BMMNCs as single reagents. Using mass-spectrometric analysis, we identified the antigen recognised by DJ3 as CD44, whereas DJ9 and DJ18 recognized HLA-DRB1 and Collagen VI, respectively. The identified proteins were highly expressed throughout in vitro osteogenic- and adipogenic differentiation. Interestingly, undifferentiated cells revealed a sole cytoplasmic distribution pattern of Collagen VI, which however changed to an extracellular matrix appearance upon osteogenic- and adipogenic differentiation. In relation to this, we found that STRO-1(+/-)/Collagen VI(-) sorted hMSC contained fewer differentiated alkaline phosphatase(+) cells compared to STRO-1(+/-)/Collagen VI(+) hMSC, suggesting that Collagen VI on the cell membrane exclusively defines differentiated MSCs. In conclusion, we have generated a panel of high quality antibodies to be used for characterization of MSCs, and in addition our results may suggest that the DJ18 generated antibody against Collagen VI can be used for negative selection of cultured undifferentiated MSCs.

U2 - 10.1007/s10059-011-2277-7

DO - 10.1007/s10059-011-2277-7

M3 - Journal article

C2 - 21614487

VL - 32

SP - 133

EP - 142

JO - Molecules and Cells

JF - Molecules and Cells

SN - 1016-8478

IS - 2

ER -