Development and characterization of a novel CD34 monoclonal antibody that identifies sheep hematopoietic stem/progenitor cells

Department of Animal Biotechnology, School of Veterinary Medicine, University of Nevada, Reno, NV 89557-0104, USA.
Experimental Hematology (Impact Factor: 2.48). 01/2009; 36(12):1739-49. DOI: 10.1016/j.exphem.2008.09.003
Source: PubMed


We and many others have long used sheep as a predictive model system in which to explore stem cell transplantation. Unfortunately, while numerous markers are available to identify and isolate human hematopoietic stem cells (HSC), no reagents exist that allow HSC/progenitors from sheep to be identified or purified, greatly impeding the application of this well-established large animal model to the study of autologous or allogeneic HSC transplantation. The current studies were undertaken to create a monoclonal antibody to sheep CD34 that would enable isolation and study of sheep HSC/progenitors.
A partial cDNA to the extracellular domain of the sheep CD34 antigen was polymerase chain reaction cloned, characterized, and used to genetically immunize mice and create hybridomas.
The resultant monoclonal antibody to sheep CD34 allows flow cytometric detection of sheep HSC/progenitors present within bone marrow, cord blood, and mobilized peripheral blood. Moreover, this antibody can be used to enrich for HSC/progenitors with enhanced in vitro colony-forming potential, and also identifies endothelial cells in situ within paraffin-embedded tissue sections, similarly to antibodies to human CD34.
The availability of this monoclonal antibody recognizing the stem cell antigen CD34 in sheep will greatly facilitate the study of autologous and allogeneic HSC transplantation using this clinically relevant large animal model.

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Available from: Duygu Dee Harrison-Findik, Oct 07, 2015
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    • "We used a novel sheep CD34 + antibody that allows flow cytometric detection of sheep HSC/progenitors present within BM, cord blood, and mobilized peripheral blood. This antibody also enriches for HSC/progenitors with enhanced in vitro colonyforming potential (Porada et al., 2008). Sheep CD34 + AF or adult BM cells were selected and transduced overnight with an HIV lentivirus vector containing eGFP. "
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    • "Pluripotent progenitors are defined by their ability to maintain and differentiate upon exposure to specific stimuli. To further characterize the ovine SP-Cpos/CCSPpos cells, we first performed positive selection for CD34 expression, a surface protein widely described on various stem cells and present on ovine hematopoietic stem/progenitor cells [23]. A small population of cells (few thousand) was isolated and characterized as SP-Cpos/CCSPpos cells. "
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    BMC Veterinary Research 11/2013; 9(1):224. DOI:10.1186/1746-6148-9-224 · 1.78 Impact Factor
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    • "Fetal whole blood was stored in liquid nitrogen in freezing medium containing Iscove's modified Dulbecco's medium (IMDM) medium with 10% heat-inactivated fetal calf serum and 10% dimethylsulfoxide (DMSO). To study the mobilization of hematopoietic stem cells, neutrophils and T-cells on experimental days 0, 1 and 3, fetal whole blood samples were stained for the detection of lymphocytes (mouse anti sheep CD45-biotin; AbDSerotec, Düsseldorf, Germany/streptavidin-Horizon V450; BD Biosciences, Bleiswijk, the Netherlands), hematopoietic stem cells (mouse anti sheep CD34; a kind gift by Dr. Porada, University of Nevada, Reno, NV, USA (Porada et al., 2008), with secondary rat anti mouse-R-phycoerythrin (−PE); BD Biosciences), neutrophils (mouse anti-bovine CD11b-Fluorescein isothiocyanate (− FITC); AbDSerotec), T-helper cells (mouse anti sheep CD4-AlexaFluor® 647 (− A647); AbDSerotec), cytotoxic T-cells (mouse anti sheep CD8-R-phycoerythrin (− PE); AbDSerotec) and a viability marker (7-aminoactinomycin D (7-AAD); BD Biosciences) according to the manufacturer's protocol. Stained cells were acquired on a FACS Canto II flow cytometer (BD Biosciences) equipped with FACS Diva software (BD Biosciences). "
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    ABSTRACT: Hypoxic-ischemic encephalopathy (HIE) is common in preterm infants, but currently no curative therapy is available. Cell-based therapy has a great potential in the treatment of hypoxic-ischemic preterm brain injury. Granulocyte-colony stimulating factor (G-CSF) is known to mobilize endogenous hematopoietic stem cells (HSC) and promotes proliferation of endogenous neural stem cells. On these grounds, we hypothesized that systemic G-CSF would be neuroprotective in a large translational animal model of hypoxic-ischemic injury in the preterm brain.
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