In vitro generation of mature neutrophils from canine LinÀ bone marrow cells Leticia G. Leo ´n a , Luciana K. Ostronoff a , Marı ´a Luisa Fermı ´n b , Cristina Fragı ´o b , Elisabeth Kremmer c , Hans-Jochem Kolb d , Concepcio ´n Tejero a, * a Departamento de Bioquı ´mica y Biologı ´a Molecular, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain b Departamento de Medicina y Cirugı ´a Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain c Institut fu ¨r Immunologie, GSF-Forschungszentrum fu ¨r Umwelt und Gesundheit, Marchioninstr 25, 81377 Mu ¨nchen, Germany d Institut fu ¨r Haematologie, GSF-Forschungszentrum fu ¨r Umwelt und Gesundheit, Marchioninstr 25, 81377 Mu ¨nchen, Germany Received 27 July 2004; received in revised form 23 December 2004; accepted 14 March 2005 Abstract The major goal of this work was to describe the in vitro generation of mature functional neutrophils derived from a canine enriched haematopoietic progenitor cell population. We have utilised lineage depletion by immunomagnetic selection to isolate a canine haematopoietic progenitor cell population. The physical, immunological, metabolical and morphological methodologies employed in this study have permitted us to isolate and define a cell population enriched in Rh-123 low and CD34+ cells. Irradiated pre-established long-term bone marrow cultures (LTBMC) were utilised to determine the self-renewal ability of lineage negative (LinÀ) cells, as well as their capacity to differentiate into mature functional neutrophils. The authors demonstrate for the first time that canine neutrophils derived from LinÀ cells are able to produce oxyradicals, express a specific neutrophil surface antigen, and contain gelatinase granules. These characteristics enable them to migrate through basement membranes to act as a first line defence mechanism. The fact that these cells are able to differentiate into functional mature cells, and give rise to long-term culture-initiating cells (LTC-IC) after 35 days of culture, allows the authors to assure that the isolated canine enriched haematopoietic cell population exhibit functional characteristics, associated with primitive haematopoietic cells. # 2005 Elsevier B.V. All rights reserved. Keywords: Canine haematopoiesis; LinÀ population; In vitro differentiation; Neutrophil functionality www.elsevier.com/locate/vetimm Veterinary Immunology and Immunopathology 107 (2005) 41–50 Abbreviations: BFU-E, burst forming unit-erythroid; cG-CSF, canine granulocyte-colony-stimulating factor; cSCF, canine stem cell factor; CFU-GM, colony forming unit-granulocyte-macrophage; DHR, dihydrorhodamine 123; FACS, fluorescent-activated cell sorting; hEPO, human erythropoietin; hGM-CSF, human granulocyte/macrophage-colony stimulating factor; LinÀ, lineage negative; Lin+, lineage positive; LTBMC, long-term bone marrow culture; LTC-IC, long-term culture-initiating cells; MMP, metalloproteinase; MWM, molecular weight markers; PE, phycoerythrin; Rh-123, rhodamine 123 * Corresponding author. Tel.: +34 913 943899; fax: +34 913 943909. E-mail address: contejor@vet.ucm.es (C. Tejero). 0165-2427/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2005.03.014