Korean J Physiol Pharmacol Vol 16: 167－174, June, 2012 http://dx.doi.org/10.4196/kjpp.2012.16.3.167 ＋ ＋ Generation of CD2 CD8 in Porcine ＋ NK Cells from c-Kit Bone Marrow Cells Kyu Hee Lim1, Ji-Hui Han1, Yoon Seok Roh2, Bumseok Kim2, Jung-Kee Kwon3, Myoung Jo You4, Ho Jae Han5, Sohail Ejaz6, Chang-W on Kang1, and Jong-Hoon Kim1 1Department of Physiology and Biosafety Research Institute, College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Departments of 2Pathology, 3Laboratroy Animal Medicine, 4Parasitology, College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, 5Department of Veterinary Physiology, College of Veterinary Medicine, Seoul National University, Seoul 151-741, Korea, 6Department of Clinical Neurosciences, Neurology Unit, Addenbrooke’s Hospital, University of Cambridge, Cambridge CB2 0QQ, UK Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. Because bone marrow-derived hematopoietic stem cells (HSCs), lymphoid protenitors, can give rise to NK cells, NK ontogeny has been considered to be exclusively lymphoid. Here, we show that porcine c-kit＋ bone marrow cells (c-kit＋ BM cells) develop into NK cells in vitro in the presence of various cytokines [interleukin (IL)-2, IL-7, IL-15, IL-21, stem cell factor (SCF), and fms-like tyrosine kinase-3 ligand (FLT3L)]. Adding hydrocortisone (HDC) and stromal cells greatly increases the frequency of c-kit＋ BM cells that give rise to CD2＋CD8＋ NK cells. Also, intracellular levels of perforin, granzyme B, and NKG2D were determined by RT-PCR and western blotting analysis. It was found that of perforin, granzyme B, and NKG2D levels significantly were increased in cytokine-stimulated c-kit＋ BM cells than those of controls. And, we compared the ability of the cytotoxicity of CD2＋CD8＋ NK cells differentiated by cytokines from c-kit＋ BM cells against K562 target cells for 28 days. Cytokines-induced NK cells as effector cells were incubated with K562 cells as target in a ratio of 100： 1 for 4 h once a week. In results, CD2＋CD8＋ NK cells induced by cytokines and stromal cells showed a significantly increased cytotoxicity 21 days later. W hereas, our results indicated that c-kit＋ BM cells not pretreated with cytokines have lower levels of cytotoxicity. Taken together, this study suggests that cytokines-induced NK cells from porcine c-kit＋ BM cells may be used as adoptive transfer therapy if the known obstacles to xenografting (e.g. immune and non-immune problems) were overcome in the future. Key Words: Hematopoietic stem cells, Natural killer cells, Differentiation, Cytokines, Cytotoxicity INTRODUCTION The porcine is a useful animal model for biomedical re- search in xeno-transplantation as well as in regenerative medicine. Organ size and physiological similarities between humans and porcine make these animals suitable organ transplantation for humans [1,2]. However, organ trans- plantation across species is prone to vigorous immunologic rejection. Transplantation of whole bone marrow (BM) cells or selected subsets has been a successful strategy to achieve chimerism and induction of tolerance in different xeno- Received February 6, 2012, Revised April 17, 2012, Accepted April 17, 2012 Corresponding to: Jong-Hoon Kim, Department of Physiology, College of Veterinary Medicine, Chonbuk National University, 664-14, Duckjin-dong, Jeonju 561-756, Korea. (Tel) 82-63-270-2563, (Fax) 82-63-270-3780, (E-mail) jhkim1@chonbuk.ac.kr This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 167 transplantation studies [3,4]. Nevertheless, in spite of pro- mising results, this approach remains limited in part be- cause certain BM cell subpopulations may cause graft-ver- sus-host disease (GVHD). BM-derived stem cells are the most primitive cells that establish a fully self-renewing population and retain multi- lineage hematopoietic differentiation potential. Unlike whole BM, stem cell-enriched populations do not contain mature lymphocytes capable of inducing GVHD. In the adult BM, most hematopoietic progenitors express c-kit (CD117), a member of a family of cell-surface receptors with tyrosine kinase activity. Purified c-kit＋ BM cells have been used in mice to induce stable multilineage mixed chimerism and permanent acceptance of donorspecific grafts without any clinical signs of GVHD [3]. Human hematopoietic stem cells (HSCs) with long term engrafting abilities are also almost ABBREVIATIONS: GVHD, graft-versus-host disease; BM, bone marrow; HSCs, hematopoietic stem cells; NK, natural killer; SCF, stem cell factor; IL, interleukin; FLT3L, fms-like tyrosine kinase-3 ligand; HDC, hydrocortisone; FCS, fetal calf serum; DMEM, Dulbecco’s modified eagles medium; FITC, fluorescein isothiocyanate; PE, phycoerythrin; HUVECs, human umbilical vein endothelial cells; SPF, specific pathogen-free.