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

Abstract

Objective-We and numerous 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 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 (MoAb) to sheep CD34 that would enable isolation and study of sheep HSC/ progenitors.Methods-A partial cDNA to the extracellular domain of the sheep CD34 antigen was PCR cloned, characterized, and used to genetically immunize mice and create hybridomas.Results-The resultant MoAb 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, in similarity to antibodies to human CD34. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conclusions-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. NIH Public Access

Perivascular Mesenchymal Stem Cells in Sheep: Characterization and Autologous Transplantation in a Model of Articular Cartilage Repair

“…The cross-reactivity of the CD146 antibody was validated further when the cultured pericytes from bone marrow were analysed with flow cytometry ( Figure 3C) and immunocytochemistry ( Figure 3D-E). An ovine specific antibody was required to identify CD34 accurately [31].…”

Fetal Gene Therapy

“…If gene transfer was performed at only 54-57 days of gestation, genemarking levels of 5-6% could be achieved in the peripheral blood, a level that could exert a beneficial effect in at least some genetic diseases. Importantly, these gene-marked hematopoietic cells persisted in these sheep over the course of 5 years of study [81,82], transgene-positive CD34+ cells could be detected in the bone marrow of these animals several years post in utero gene transfer [85], and bone marrow cells isolated from these in utero gene transfer recipients successfully engrafted the hematopoietic system of secondary fetal sheep recipients upon re-transplantation. These three pieces of data provide compelling evidence that this approach enabled us to successfully insert genes into the stem cells of the hematopoietic system, suggesting this method could provide lifelong genetic correction.…”

CD34 affinity pheresis attenuates a surge among circulating progenitor cells following vascular injury

“…8D11 antibody reactive to ovine CD34 was characterized 21 and kindly provided by Dr Christopher Porada (University of Nevada, Reno, Nev). This antibody was manufactured by Aldevron (Freiburg, Germany).…”

“…As in the human condition, the marker CD34 has been previously defined as a marker of hematopoietic progenitor cells with colony-forming activity in sheep. 21 A second objective of this study was to evaluate a short-term affinity pheresis approach to mediate depletion of circulating progenitors and the impact if any on mature hematopoietic cells. Despite the apparent technical simplicity of progenitor depletion, selective affinity depletion of progenitor cells from the circulation has not been described previously in the literature.…”