Fig 1: Anti-BCAM/Lu antibodies do not inhibit CD34 + HSPC attachment to LM-511 and LM-521 nor do enhance CD34 + HSPC proliferation in presence of LM-511 and LM-521. (A) Cell-matrix adhesion assays were performed with MACS-sorted CD34 + HSPC with LM-411 and LM-511, laminins which were immobilized on the plastic dish. Cell adhesion could only be observed to LM-511, but not to LM-411. (B) CD34 + HSPC were allowed to adhere to LM-511, LM-521 in the presence of anti-BCAM/Lu antibodies and without antibodies (control). Representative image of three independent experiments. (C) Cell-matrix adhesion assays were performed with MACS-sorted CD34 + HSPC with LM-511 and LM-521, in presence of vehicle and recombinant BCAM/Lu protein. Cell adhesion to LM-511 and LM-521 could be inhibited by recombinant BCAM/Lu protein. (D) MACS-sorted CD34+ cells from three donors incubated in serum-free expansion medium with 10 μg/ml LM-511 and LM-521 or without (control) were cultured for three consecutive days. Cells were harvested each day, and the proliferation rate was determined. LM 511 and LM 521 drastically diminished CD34+ cell proliferation. (E) Antibodies against BCAM/Lu did not interfere with CD34 + cell proliferation which were cultured for 3 days in serum-free expansion medium in the presence of 10 μg/ml anti-BCAM/Lu antibodies and LM-511 or LM-521. The control without laminin isoforms is not shown in these diagrams. *p < 0.05; **p < 0.005.
Fig 2: Human HSPC and bone marrow niche cells express BCAM/Lu. (A) RT-PCR analysis of CD34 + HSPC, human umbilical vein endothelial cells (HUVEC), bone marrow mesenchymal stromal cells (MSC), and primary osteoblasts (pOB) showing the expression of human BCAM/Lu on all cell types. (B) qRT-PCR analysis showing the quantification of expression of BCAM/Lu, and the fold change in expression relative to HUVEC was determined. (C) Immunoblotting using a monoclonal antibody against the extracellular domain of BCAM/Lu revealed the expression of BCAM/Lu on HUVEC, CD34 + HSPC, MSC, and pOB, but not on K562 or KG1a cells.
Fig 3: BCAM/Lu regulates transmigration of CD34 + HSPC through endothelial barrier and regulates differentiation. (A) MACS-sorted CD34+ cells from three donors were allowed to migrate through an 8 μm polycarbonate membrane pre-coated with a confluent layer of HUVEC. The adherent endothelial cell layer was either cultured without antibodies or treated with 10 μg/ml of BCAM/Lu or W6/32.HK antibodies added to the endothelial cells. The lower chamber was filled with SFEM with or without 100 ng/ml SDF-1α. The lower chamber without the cytokine was the negative control, whereas that containing SDF-1α acted as positive control. The CD34+ cells were allowed to migrate through the membrane for 16 h. The number of migrated cells in the lower chamber was determined measuring the DNA content with the CyQuant. BCAM/Lu antibodies strongly inhibited the SDF-1α induced transmigration in all three donors, whereas the control antibody W6/32.HK showed no inhibitory effect. (B) MACS-sorted CD34+ cells from three donors were pre-treated with antibodies against BCAM/Lu for 3 days, and 1000 cells were then plated on methylcellulose. The colonies were scored at day 14. The treatment with antibodies against BCAM/Lu induced lower number of colony formation. *p < 0.05; **p < 0.005; ***p < 0.0005.
Fig 4: BCAM/Lu expression on CD34+ cells, HUVEC, MSC, and pOB. (A) Left: Lineage-negative CBMNC were double-stained with mouse-anti-BCAM/Lu and rabbit-anti-CD34 antibodies followed by Cy3- and Alexa 488-conjugated secondary antibodies, respectively. Cell nuclei of all lineage- cells were counterstained with DAPI (blue signal). The majority of the CD34 + HSPC express BCAM/Lu as seen in the merged immunofluorescence micrograph. Scale bar: 50 μm. Right: FACS blots representing the expression of BCAM/Lu on 50% of the CD34 + HSPCs. (B) Left panel: The adherent cells – endothelial, mesenchymal stromal or osteoblastic cells – were labeled with the BCAM/Lu antibody and Cy3-conjugated secondary antibody. Cell nuclei were counterstained with DAPI. Strong immunofluorescence signals were seen on HUVEC and MSC, weaker signals were found on pOB. Scale bar: 50 μm. Right panel: FACS blots representing the expression of BCAM/Lu on HUVEC, MSC, and pOB (top to bottom). Representative plots of three independent experiments.
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