Isolation of bovine CD34+ bone marrow stem and progenitor cells using a monoclonal antibody against CD34 protein
Résumé
Background: There is currently no commercially available bovine anti-CD34 monoclonal antibody. Bovine Hematopoietic Stem and Progenitor Cells (BHSPC) isolation in vitro has had mixed results.
Methods: CD34 protein sequences from multiple species were compared in silico to the bovine sequence. A suitable antibody was selected and coupled to R-PhycoErythrin (R-PE). Bone marrow samples from 7 Prim’Holstein calves were collected in EDTA and mononuclear cells were isolated using a density gradient. Antibody binding to BHSPC was monitored by flow cytometry. Labeled BHSPC were separated with anti-PE magnetic beads. Enrichment was evaluated by flow cytometry.
Results and discussion: A monoclonal antibody against ovine CD34 was selected based on high homology between bovine and ovine CD34. CD34+ cells accounted for 4.4 to 27.6 % of BHSPC. Variation in CD34+ cells proportion may relate to an individual variation of positive cells in the marrow; however, polymorphism in the coding region of the protein is possible (previously described). Magnetic beads increased CD34+ cells concentration by a 1.96-fold (n = 10, σ = 0.95), but with a weak recovery rate of 1.54 % (n = 8).
Conclusions: This experiment describes a new potential commercial anti-CD34 monoclonal antibody for isolation or identification of BHSPC. Although the enrichment may appear low, the increase in cell purity is sufficient for culture and immortalization.