Analysis of surface marker expression

Differences in surface marker composition of bone marrow derived MSC, hCh and mnCPC were comparatively examined by flow cytometric analysis (FACS). When reaching 80 % confluence, each cell type was harvested by trypsinization (passage 2). Cells were washed twice with PBS containing 1 % FBS (Biochrom) and 0.02 % sodium azide (Sigma-Aldrich). The cells were incubated for 30 min in ice cold blocking buffer containing specific fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-labelled mouse anti-human monoclonal antibodies. The following antibodies were used for the detection of human surface antigens: CD9, CD45 and CD90 FITC-labelled, CD29, CD31, CD34, CD44, CD49d, CD49e, CD49f (rat IgG2), CD54, CD73, CD105, CD106, CD133/1, CD133/2, CD146 and CD166 PE-labelled. In all experiments, the respective FITC- or PE-labelled non-immune isotype-matched antibodies were used as negative controls.

Surface marker composition was analyzed on a FACScan flow cytometer with dual-laser technology and CELLQuest software V2 (Becton–Dickinson, Franklin Lakes, NJ, USA). For each antibody 1 × 10⁴ cells of each cell type were used. Dead cells were excluded by propidium iodide (Sigma-Aldrich) staining. Cells were gated on forward and side scatter to exclude debris and cell aggregates. FI emitted by the dye-conjugated specific antibody bound to the antigen was determined and normalized to the median FI emitted by cells stained with respective isotype.

CD133/1, CD133/2 were obtained from Miltenyi Biotec, CD34 from Invitrogen (Carlsbad, CA, USA), CD105 from R&D Systems (Minneapolis, MN, USA), CD9, CD31 and CD106 from BioLegend (San Diego, CA, USA). All other antibodies and the isotype controls (FITC mouse IgG1, PE mouse IgG1, PE rat IgG2) were provided by Becton–Dickinson.