Ditte C. Andersen *" /> Angela Kortesidis " /> " /> " /> " /> " /> " /> " /> " /> and Moustapha Kassem" /> Ditte C. Andersen*, Angela Kortesidis, Andrew C.W. Zannettino, Irina Kratchmarova, Li Chen, Ole N. Jensen, Børge Teisner, Stan Gronthos, Charlotte H. Jensen, and Moustapha Kassem" /> Ditte C. Andersen*, Angela Kortesidis, Andrew C.W. Zannettino, Irina Kratchmarova, Li Chen, Ole N. Jensen, Børge Teisner, Stan Gronthos, Charlotte H. Jensen, and Moustapha Kassem. Mol. Cells 2011;32:133-42. https://doi.org/10.1007/s10059-011-2277-7">
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Mol. Cells 2011; 32(2): 133-142

Published online August 31, 2011

https://doi.org/10.1007/s10059-011-2277-7

© The Korean Society for Molecular and Cellular Biology

Development of Novel Monoclonal Antibodies that Define Differentiation Stages of Human Stromal (Mesenchymal) Stem Cells

Ditte C. Andersen1,7,*, Angela Kortesidis2, Andrew C.W. Zannettino3, Irina Kratchmarova4, Li Chen5, Ole N. Jensen4, Børge Teisner1, Stan Gronthos2, Charlotte H. Jensen1,7, and Moustapha Kassem5,6

1Department of Cancer and Inflammation Research, University of Southern Denmark, Denmark, 2Mesenchymal Stem Cell Group, Department of Haematology, Institute of Medical and Veterinary Science/Hanson Institute, Adelaide, South Australia, Australia, 3Myeloma Research Laboratory, Department of Haematology, Institute of Medical and Veterinary Science/Hanson Institute, Adelaide, South Australia, Australia, 4Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark, 5Department of Endocrinology and Metabolism, Odense University Hospital, Odense, Denmark, 6Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud Univeristy, KSA, 7Present address: Laboratory of Molecular and Cellular Cardiology, Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark

Correspondence to : *Correspondence: dandersen@health.sdu.dk

Received: November 8, 2011; Revised: April 28, 2011; Accepted: May 4, 2011

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 phospha-tase+ 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.

Keywords mesenchymal stem cells (MSC), monoclonal antibodies, MSC differentiation, MSC markers, osteogenesis

Article

Research Article

Mol. Cells 2011; 32(2): 133-142

Published online August 31, 2011 https://doi.org/10.1007/s10059-011-2277-7

Copyright © The Korean Society for Molecular and Cellular Biology.

Development of Novel Monoclonal Antibodies that Define Differentiation Stages of Human Stromal (Mesenchymal) Stem Cells

Ditte C. Andersen1,7,*, Angela Kortesidis2, Andrew C.W. Zannettino3, Irina Kratchmarova4, Li Chen5, Ole N. Jensen4, Børge Teisner1, Stan Gronthos2, Charlotte H. Jensen1,7, and Moustapha Kassem5,6

1Department of Cancer and Inflammation Research, University of Southern Denmark, Denmark, 2Mesenchymal Stem Cell Group, Department of Haematology, Institute of Medical and Veterinary Science/Hanson Institute, Adelaide, South Australia, Australia, 3Myeloma Research Laboratory, Department of Haematology, Institute of Medical and Veterinary Science/Hanson Institute, Adelaide, South Australia, Australia, 4Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark, 5Department of Endocrinology and Metabolism, Odense University Hospital, Odense, Denmark, 6Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud Univeristy, KSA, 7Present address: Laboratory of Molecular and Cellular Cardiology, Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark

Correspondence to:*Correspondence: dandersen@health.sdu.dk

Received: November 8, 2011; Revised: April 28, 2011; Accepted: May 4, 2011

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 phospha-tase+ 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.

Keywords: mesenchymal stem cells (MSC), monoclonal antibodies, MSC differentiation, MSC markers, osteogenesis

Mol. Cells
Aug 31, 2022 Vol.45 No.8, pp. 513~602
COVER PICTURE
Cryo-EM structure of human porphyrin transporter ABCB6 (main figure) shows that binding of hemin (inset, magenta) in concert with two glutathione molecules (cyan) primes ABCB6 for high ATP turnover (Kim et al., pp. 575-587).

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