Published online 9 December 2008
Haematologica, Vol 94, Issue 2, 173-184 doi:10.3324/haematol.13740
Copyright © 2009 by Ferrata Storti Foundation
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Mesenchymal Stem Cells

Isolation of functionally distinct mesenchymal stem cell subsets using antibodies against CD56, CD271, and mesenchymal stem cell antigen-1

Venkata Lokesh Battula1,*, Sabrina Treml1, Petra M. Bareiss2, Friederike Gieseke3, Helene Roelofs4, Peter de Zwart5, Ingo Müller3, Bernhard Schewe5, Thomas Skutella2, Willem E. Fibbe4, Lothar Kanz3, Hans-Jörg Bühring1

1 University Clinic of Tübingen, Department of Internal Medicine II, Division of Hematology, Oncology, and Immunology, Tübingen
2 University of Tübingen, Institute of Anatomy, Department of Experimental Embryology, Division of Tissue Engineering, Tübingen
3 University Clinic of Tübingen, Children’s Hospital, Department of General Pediatrics, Division of Hematology and Oncology, Tübingen
4 Leiden University Medical Center, Department of Immunohematology and Blood Transfusion, Center for Stem Cell Therapy, Leiden, The Netherlands and
5 Hospital for Workers Compensation Tübingen, Department of Orthopedic Surgery, Tübingen, Germany

Correspondence: Hans-Jörg Bühring, Ph.D., University of Tübingen, Department of Internal Medicine II, Medical, Otfried-Müller-Str. 10, 72076, Tübingen, Germany., E-mail:hans-joerg.buehring{at}uni-tuebingen.de

Background: Conventionally, mesenchymal stem cells are functionally isolated from primary tissue based on their capacity to adhere to a plastic surface. This isolation procedure is hampered by the unpredictable influence of co-cultured hematopoietic and/or other unrelated cells and/or by the elimination of a late adhering mesenchymal stem cells subset during removal of undesired cells. To circumvent these limitations, several antibodies have been developed to facilitate the prospective isolation of mesenchymal stem cells. Recently, we described a panel of monoclonal antibodies with superior selectivity for mesenchymal stem cells, including the monoclonal antibodies W8B2 against human mesenchymal stem cell antigen-1 (MSCA-1) and 39D5 against a CD56 epitope, which is not expressed on natural killer cells.

Design and Methods: Bone marrow derived mesenchymal stem cells from healthy donors were analyzed and isolated by flow cytometry using a large panel of antibodies against surface antigens including CD271, MSCA-1, and CD56. The growth of mesenchymal stem cells was monitored by colony formation unit fibroblast (CFU-F) assays. The differentiation of mesenchymal stem cells into defined lineages was induced by culture in appropriate media and verified by immunostaining.

Results: Multicolor cell sorting and CFU-F assays showed that mesenchymal stem cells were ~90-fold enriched in the MSCA-1+CD56– fraction and ~180-fold in the MSCA-1+CD56+ fraction. Phenotype analysis revealed that the expression of CD10, CD26, CD106, and CD146 was restricted to the MSCA-1+CD56– mesenchymal stem cells subset and CD166 to MSCA-1+CD56± mesenchymal stem cells. Further differentiation of these subsets showed that chondrocytes and pancreatic-like islets were predominantly derived from MSCA-1+CD56± cells whereas adipocytes emerged exclusively from MSCA-1+CD56– cells. The culture of single sorted MSCA-1+CD56+ cells resulted in the appearance of phenotypically heterogeneous clones with distinct proliferation and differentiation capacities.

Conclusions: Novel mesenchymal stem cells subsets with distinct phenotypic and functional properties were identified. Our data suggest that the MSCA-1+CD56+ subset is an attractive starting population for autologous chondrocyte transplantation.

Key words: mesenchymal stem cells, CD56, MSCA-1.