Contents lists available at ScienceDirect Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm Research paper Long-term culture and differentiation of porcine red bone marrow hematopoietic cells co-cultured with immortalized mesenchymal cells Abubakar Garba, Delphine D. Acar, Inge D.M. Roukaerts, Lowiese M.B. Desmarets, Bert Devriendt, Hans J. Nauwynck ⁎ Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium ARTICLE INFO Keywords: Red bone marrow Hematopoietic cells Mesenchymal cells Swine ABSTRACT Mesenchymal cells are multipotent stromal cells with self-renewal, differentiation and immunomodulatory capabilities. We aimed to develop a co-culture model for differentiating hematopoietic cells on top of im- mortalized mesenchymal cells for studying interactions between hematopoietic and mesenchymal cells, useful for adequately exploring the therapeutic potential of mesenchymal cells. In this study, we investigated the survival, proliferation and differentiation of porcine red bone marrow hematopoietic cells co-cultured with immortalized porcine bone marrow mesenchymal cells for a period of five weeks. Directly after collection, primary porcine bone marrow mesenchymal cells adhered firmly to the bottom of the culture plates and showed a fibroblast-like appearance, one week after isolation. Upon immortalization, porcine bone marrow mesench- ymal cells were continuously proliferating. They were positive for simian virus 40 (SV40) large T antigen and the mesenchymal cell markers CD44 and CD55. Isolated red bone marrow cells were added to these immortalized mesenchymal cells. Five weeks post-seeding, 92 ± 6% of the red bone marrow hematopoietic cells were still alive and their number increased 3-fold during five weekly subpassages on top of the immortalized mesenchymal cells. The red bone marrow hematopoietic cells were originally small and round; later, the cells increased in size. Some of them became elongated, while others remained round. Tiny dendrites appeared attaching hematopoietic cells to the underlying immortalized mesenchymal cells. Furthermore, weekly differential-quick staining of the cells indicated the presence of monoblasts, monocytes, macrophages and lymphocytes in the co-cultures. At three weeks of co-culture, flow cytometry analysis showed an increased surface expression of CD172a, CD14, CD163, CD169, CD4 and CD8 up to 37 ± 0.8%, 40 ± 8%, 41 ± 4%, 23 ± 3% and 19 ± 5% of the he- matopoietic cells, respectively. In conclusion, continuous mesenchymal cell cultures were successfully estab- lished and characterized and they supported the proliferation of red bone marrow hematopoietic cells, which finally differentiated into monocytic cells and CD4 + and CD8 + cells. 1. Introduction The red bone marrow serves as the main source of white blood cells in mammals. Red bone marrow consists of two main cell types, the hematopoietic cells and non-hematopoietic stromal cells. The hemato- poietic cells comprise megakaryocytes and erythrocytic, myeloid, and lymphocytic cells. The bone marrow stroma consists of a heterogeneous cell population, which provide mechanical and physiological support to the hematopoietic cells (Summerfield et al., 2001). They include stem cell-like cells that under specific culture conditions can differentiate into osteocytes, chondrocytes and adipocytes. Mesenchymal cells drive the differentiation of hematopoietic cells and are characterized by their ability to adhere to cell culture plates and their expression of specific surface markers such as CD44, CD55, CD73, CD29 and CD90 (Dominici et al., 2006; Wagner et al., 2007; Nesselmann et al., 2008). Leukocyte progenitor cells are used in the treatment of red bone marrow defects either inherited or caused by disease. Red bone marrow defects may also be caused by chemo- or radiotherapy and restored by transplanting auto- or allogenic cells (Zimmermann and Moghaddam, 2010). There are however limitations regarding the availability of red bone marrow hematopoietic cells for fundamental research. To date, human and mouse red bone marrow is widely used as a source of hematopoietic progenitor cells. However, in man only a little amount of bone marrow can be obtained by biopsy under strict ethical conditions, while in mice, red bone marrow is not always accessible in sufficient quantities, re- sulting in euthanasia of large numbers of mice (Ingersoll et al., 2010). This is in conflict with the 3R principle of animal welfare. In contrast, pigs are ideal donors as large amounts of hematopoietic cells can be http://dx.doi.org/10.1016/j.vetimm.2017.08.002 Received 27 February 2017; Received in revised form 24 July 2017; Accepted 3 August 2017 ⁎ Corresponding author. E-mail address: hans.nauwynck@ugent.be (H.J. Nauwynck). Veterinary Immunology and Immunopathology 191 (2017) 44–50 0165-2427/ © 2017 Published by Elsevier B.V. MARK