title: Optimization of a flow cytometry-based protocol for detection and phenotypic characterization of multipotent mesenchymal stromal cells from human bone marrow.
abstract: BACKGROUND
  To study the biology of rare bone marrow (BM) multipotent mesenchymal stromal cells (MSCs), recognized protocols are needed. Colony-forming unit-fibroblast (CFU-F) assays have historically been used for the enumeration of MSCs. However, the need to isolate and further analyze MSCs requires new strategies based on cell surface markers. The purpose of this work was to verify the phenotype of BM MSCs in vivo and to develop flow cytometry-based methods for their evaluation.
  
  
  METHODS
  Pre-enrichment with D7-FIB-conjugated microbeads, cell sorting for CD45low D7-FIB+ LNGFR+ cells, and CFU-F assay were used to confirm the phenotype of BM MSCs in vivo. Further phenotypic characterization of MSCs was performed using three-color flow cytometry following pre-enrichment or by direct four-color flow cytometry. The sensitivity of direct flow cytometry/rare event analysis for the accurate enumeration of MSCs was validated using 85 samples from patients with neoplastic BM diseases.
  
  
  RESULTS
  In normal BM, a significant correlation was found between the frequencies of CFU-Fs and CD45low D7-FIB+ LNGFR+ cells (n = 19, R = 0.719, P = 0.001). Following cell sorting, 15% of these cells were clonogenic. The same cells were enriched using LNGFR-based positive selection, CD45/Glycophorin A-based depletion, or plastic adherence. CD45low D7-FIB+ LNGFR+ cells expressed classic makers of cultured MSCs CD73/SH3 and CD105/SH2 and markers of stromal reticular cells CD106/VCAM and alkaline phosphatase. Novel markers were identified including leukemia inhibitory factor receptor and gp130. CD45low D7-FIB+ LNGFR+ cells were increased fourfold in the floating fat fraction of normal BM aspirates. Their frequency was decreased in chronic lymphocytic leukemia (threefold, n = 13, P = 0.049) and chronic myelogenous leukemia (ninefold, n = 11, P = 0.001) compared with that in age-matched controls (n = 26 and n = 31, respectively).
  
  
  CONCLUSIONS
  This study demonstrates the usefulness of flow cytometry-based methods for the detection, enumeration and further phenotypic analysis of BM MSCs. These findings have broad applications for the future evaluation of BM MSCs in health and disease.
authors.affiliation: Academic Unit of Musculoskeletal Disease, Leeds Institute for Molecular Medicine,University of Leeds, Leeds, United Kingdom. msjej@leeds.ac.uk
authors.forename: Elena A
authors.initials: EA
authors.lastname: Jones
authors.forename: Anne
authors.initials: A
authors.lastname: English
authors.forename: Sally E
authors.initials: SE
authors.lastname: Kinsey
authors.forename: Liz
authors.initials: L
authors.lastname: Straszynski
authors.forename: Paul
authors.initials: P
authors.lastname: Emery
authors.forename: Frederique
authors.initials: F
authors.lastname: Ponchel
authors.forename: Dennis
authors.initials: D
authors.lastname: McGonagle
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D001854:Bone Marrow Cells / Q000276:immunology*; D002648:Child; D002675:Child, Preschool; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping; D008875:Middle Aged; D039902:Multipotent Stem Cells / Q000166:cytology / Q000276:immunology*; D012680:Sensitivity and Specificity; D017154:Stromal Cells / Q000166:cytology / Q000276:immunology*