Development and Characterization of a Novel CD34 Monoclonal Antibody That Identifies Sheep Hematopoietic Stem/Progenitor Cells.
Authors: Porada Christopher D. , Harrison-Findik Dee , Sanada Chad , Valiente Vincent , Simmons Paul J. , Almeida-Porada Graça , Zanjani Esmail D. 
Published: October 2019

Abstract
To date, the cell surface marker CD34 has been the antigen most widely used for identification and isolation of HSC in humans and numerous other species. CD34 is an integral membrane sialomucin present on roughly 1–3% of BM mononuclear cells (MNC) in a normal adult human, yet its precise function remains largely unknown. The CD34+ population is now known to produce durable reconstitution of all blood lineages in both autologous and allogeneic transplantations, providing evidence that CD34 is expressed on some of the most primitive long-term engrafting HSC. We and others have used the fetal sheep model extensively to study the potential and behavior of human HSC. Unfortunately, no reagents exist that allow sheep HSC to be identified or purified, impeding the development of experimental HSC transplantation strategies in this clinically relevant large animal model. We therefore developed monoclonal antibodies (MoAbs) to the ovine homologue of CD34. We PCR cloned and sequenced an 858bp cDNA corresponding to the extracellular domain of the sheep CD34 antigen, which was found to share 92% homology with the bovine CD34 homologue. When aligned to human CD34, the sheep homologue shared 90% homology from nucleotides 1–84, 78% homology from nucleotides 453–858, and exhibited minimal homology between nucleotides 85 and 452. This cDNA was cloned into an expression vector and used to genetically immunize mice and create monoclonal antibodies. One antibody (8D11) was selected for all subsequent studies. Using flow cytometry, 8D11 identified a small, discrete population of CD45+ cells within the peripheral blood, cord blood, and BM of sheep. This population comprised 0.5–2.5% of the total sheep BM MNC, a proportion in close accord with the previously reported incidence of CD34+ cells in adult human BM. The ability of 8D11 to enrich for sheep hematopoietic progenitors was tested by magnetically sorting 8D11+cells and assessing colony-forming potential (CFU) and the CAFC frequency. Ovine CD34+ cells were 35- to 150-fold enriched for CFU and CAFC as compared with BM mononuclear cells, whereas CD34-negative cells were correspondingly depleted of progenitors. G-CSF mobilization of sheep resulted in a 14-fold increase in the levels of circulating CD34+ cells on day 4, providing further evidence of the utility of 8D11 as a marker of primitive hematopoietic cells in the sheep model. Antibody 8D11 also robustly labeled the lining of blood vessels in both frozen and paraffin-embedded sheep tissues, further extending the utility of this antibody to include analysis of tissue sections. In conclusion, this first successful generation of a monoclonal antibody to sheep CD34 will greatly facilitate the use of the sheep as a clinically relevant large animal model system to study allogeneic HSC transplantation both in utero and in post-natal recipients using bone marrow, mobilized peripheral blood, and cord blood as cell sources.