Fig 1: CXCL12hi cells are identified by EBF3 staining in human adult marrow sections. (A) Immunohistochemical analysis for EBF3 in human adult bone marrow. EBF3+ cells were scattered throughout the bone marrow cavity. (B) Immunohistochemical analysis for EBF3 and CD271 in human adult bone marrow. Membranes of EBF3+ cells were positive for CD271, and EBF3+CD271+ cells displayed long processes that formed a reticular network. (C) Immunohistochemical analysis for EBF3 and CD271 in human adult bone marrow. Almost all DAPI+ nuclei of CD271+ cells (white arrows) were positive for EBF3 in the marrow cavity. (D) Combined in situ hybridization of the CXCL12 and LEPR mRNAs and immunohistochemistry for EBF3 in human adult bone marrow. (E) Combined in situ hybridization of the CXCL12 and LEPR mRNAs and immunohistochemistry for von Willebrand factor (VWF) in human adult bone marrow. Cells expressing CXCL12 (white arrow) in contact with endothelial cells (ECs) of vascular sinuses (S) were shown. Scale bar: (A) 100 µm; (B) 10 µm; (C) 10 µm; (D) 10 µm; (E) 10 µm.
Fig 2: Human adult bone marrow CXCL12hi/LEPR+ cells are adipo-osteogenic progenitors with haematopoiesis-supporting abilities. (A) Expression of CD271, CD140a and CD146 in CXCL12hi/LEPR+ cells and osteoblastic cells from human adult bone marrow, as well as synovial mesenchymal cells, as determined by flow cytometry. (B) The colony-forming unit–fibroblast (CFU–F) frequencies of CXCL12hi/LEPR+ cells (n = 15), LEPR-CD56+ osteoblastic cells (n = 9) and LEPR-CD56- cells (n = 8) within the human adult bone marrow CD45-CD235a-CD71-CD31- population, as well as synovial mesenchymal cells (n = 8). (C) The differentiation potentials of CXCL12hi/LEPR+ cells, LEPR-CD56+ osteoblastic cells and synovial mesenchymal cells towards adipogenic (Oil Red O+), osteogenic (Alizarin Red+) and chondrogenic (Alcian blue+) lineages. Scale bar, 100 µm. (D) Relative expression levels of the mRNAs encoding lineage-associated markers in CXCL12hi/LEPR+ cells cultured in adipogenic, osteogenic, chondrogenic media, as determined by quantitative real-time (qRT)-polymerase chain reaction (PCR; n = 4–6). (E) The percentages of Oil Red O+ (left) or Alizarin Red+ (right) single-CXCL12hi/LEPR+-cell-derived clones cultured in adipogenic (Adipo) or osteogenic (Osteo) medium respectively (n = 3). (F) The numbers of CD34+ haematopoietic cells after one week of co-culture with CXCL12hi/LEPR+ cells (n = 13) or LEPR-CD56+ osteoblastic cells (n = 5) in serum-free media containing stem cell factor (SCF), thrombopoietin (TPO) and FMS-like tyrosine kinase 3 ligand (FLT3L). Data represent mean ± SD (B, D, E and F). Two-tailed Student’s t tests were used to assess statistical significance (B, D, E and F; *, P < 0·05; **, P < 0·01).
Fig 3: p75NTR expression in human brain and platelets. (A) Healthy human cortex (C; 10 µg) and platelets lysates (platelets; 30 µg) were analyzed in denaturing and reducing conditions and blotted with antibodies raised against p75NTR. Left: molecular weight marker in kDa. Right: antibody catalog number. ß-actin was used as a loading control. (B) U251-MG cells and healthy human platelet lysates were either left untreated (untreated) or submitted to 37°C overnight in absence (Sham) or presence of the protein deglycosylation mix II (Deglyco). Membranes were blotted with the HPA004765 antibody against p75NTR ICD. Membranes were then stripped and reblotted for lysosomal-associated membrane protein 1 (LAMP-1) and CD41 as internal controls of enzymatic activity. (C) Human glioblastoma U87-MG cells and healthy human platelets isolated from whole blood were fixed or fixed and permeabilized. Cells were labeled with antibodies directed toward the p75NTR receptor and analyzed by flow cytometry. Results displayed are representative of (A) = 3 independent experiments and = 5 different platelet samples from different donors and (B, C) 3 independent experiments and 3 different donors for platelets samples.
Supplier Page from MilliporeSigma for Anti-NGFR antibody produced in rabbit