title: Isolation of functionally distinct mesenchymal stem cell subsets using antibodies against CD56, CD271, and mesenchymal stem cell antigen-1.
abstract: 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.
  
  
  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 approximately 90-fold enriched in the MSCA-1(+)CD56(-) fraction and approximately 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.
authors.affiliation: University of Tübingen, Department of Internal Medicine II, Medical, Otfried-Müller-Str. 10, 72076, Tübingen, Germany.
authors.forename: Venkata Lokesh
authors.initials: VL
authors.lastname: Battula
authors.forename: Sabrina
authors.initials: S
authors.lastname: Treml
authors.forename: Petra M
authors.initials: PM
authors.lastname: Bareiss
authors.forename: Friederike
authors.initials: F
authors.lastname: Gieseke
authors.forename: Helene
authors.initials: H
authors.lastname: Roelofs
authors.forename: Peter
authors.initials: P
authors.lastname: de Zwart
authors.forename: Ingo
authors.initials: I
authors.lastname: Müller
authors.forename: Bernhard
authors.initials: B
authors.lastname: Schewe
authors.forename: Thomas
authors.initials: T
authors.lastname: Skutella
authors.forename: Willem E
authors.initials: WE
authors.lastname: Fibbe
authors.forename: Lothar
authors.initials: L
authors.lastname: Kanz
authors.forename: Hans-Jörg
authors.initials: HJ
authors.lastname: Bühring
mesh_terms: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis*; D019002:CD56 Antigen / Q000032:analysis; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D017690:Cell Transplantation / Q000379:methods; D019902:Chondrocytes / Q000637:transplantation; D005434:Flow Cytometry; D006801:Humans; D016130:Immunophenotyping; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000276:immunology; D009419:Nerve Tissue Proteins / Q000032:analysis; D017475:Receptors, Nerve Growth Factor / Q000032:analysis
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D019002:CD56 Antigen; C484065:NGFR protein, human; D009419:Nerve Tissue Proteins; D017475:Receptors, Nerve Growth Factor; C540400:W8B2 protein, human