title: Isolation of C15: a novel antibody generated by phage display against mesenchymal stem cell-enriched fractions of adult human marrow.
abstract: Adult bone marrow stroma contains a source of mesenchymal stem cells (MSC) that have the capacity to self-renew and differentiate into multiple stromal lineages. These rare cells can be visualised indirectly by the formation of heterogeneous colonies, containing stem cells and their differentiated progeny in long-term culture. If MSC and their associated progenitor and precursor populations are to reach their full therapeutic potential, markers will be required to identify and characterize specific bone marrow stromal subsets. We sought to use phage display to generate antibodies against bone marrow mononuclear cells (BMMNC) enriched for colony forming cells. Initially, we identified our target cell population by comparing the colony forming efficiency (CFE) of CD49a-positive, STRO-1-positive and CD45-negative BMMNC subpopulations with unseparated BMMNC. Selection with anti-CD49a gave the greatest enrichment (19-fold) of colony forming cells and in light of these findings, we generated phage antibodies against CD49a-positive BMMNC by simultaneous positive/negative selection. A dominant clone (C15), generated after 3 rounds of selection, has been isolated and sequenced, then characterized for cell and tissue specificity. Sequence analysis showed that the V(H) and V(L) gene segments of C15 aligned most closely to the VH26/DP-47 and IGLV3S1/DPL16 germline V segments found in the synthetic repertoire. C15 bound to 4% of freshly isolated BMMNC and localized to osteoblastic cells and proximal marrow cells in areas of active bone formation in sections of osteophyte. C15 binding was upregulated in cultured bone marrow stromal cells (BMSC) and was also detected on bone-derived cell lines. This report demonstrates that phage display is a powerful tool for the isolation of antibodies against rare cell populations, and provides a platform for the future application of this technology in the search for antigens on MSC and other rare cell populations.
authors.affiliation: Department of Pharmacy and Pharmacology and Centre for Regenerative Medicine, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom. J.A.Letchford@bath.ac.uk
authors.forename: Julie
authors.initials: J
authors.lastname: Letchford
authors.forename: Angharad M
authors.initials: AM
authors.lastname: Cardwell
authors.forename: Karina
authors.initials: K
authors.lastname: Stewart
authors.forename: Karma K S
authors.initials: KK
authors.lastname: Coogans
authors.forename: Jonathan P L
authors.initials: JP
authors.lastname: Cox
authors.forename: M
authors.initials: M
authors.lastname: Lee
authors.forename: Jon N
authors.initials: JN
authors.lastname: Beresford
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Perry
authors.forename: Melanie J
authors.initials: MJ
authors.lastname: Welham
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D001854:Bone Marrow Cells / Q000276:immunology*; D002460:Cell Line; D002469:Cell Separation; D003114:Colony-Forming Units Assay; D004247:DNA / Q000235:genetics; D005803:Genes, Immunoglobulin; D006801:Humans; D007158:Immunologic Techniques; D039423:Integrin alpha1 / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000276:immunology*; D008969:Molecular Sequence Data; D019151:Peptide Library
chemical_list: D000911:Antibodies, Monoclonal; D039423:Integrin alpha1; D019151:Peptide Library; D004247:DNA