168 KH Lim, et al exclusively in the c-kit＋ population of CD34＋ BM cells [5]. On the other hand, the recent demonstration of the capacity of BM-derived HSCs to differentiate into nonhematopoietic lineages such as hepatocytes, skeletal muscle cells, neurons or epithelial cells of the lung, gastrointestinal tract and skin, has raised hopes as to their therapeutic potential in regenerative medicine [6]. Natural killer (NK) cells are innate immune effector cells. Their derivation from either lymphoid or myeloid lineages was debated early in their discovery [7]. Further research showed that NK cells can be derived from common lym- phoid progenitors and hence have been considered separate from myeloid lineage [8,9]. However, some studies question this and have shown that progenitors expressing myeloid antigens can develop into NK cells [10,11]. In previous study, it is shown that NK cell differentiation from HSCs can be studied in vitro [12,13]. This process depends on cy- tokines, notably interleukin-2 (IL-2) or IL-15, whereas oth- er factors (stem cell factor [SCF], fms-like tyrosine kinase-3 ligand [FLT3L]) induce early HSCs expansion and respon- siveness to IL-2 and IL-15 signaling [14]. To date, the fac- tors regulating NK differentiation from porcine c-kit＋ BM cells are poorly understood. However, it is certain that it involves cell to cell interactions that render NK responsive to soluble factors from the microenvironment, and several factors such as IL-15 are reported to be involved in differen- tiation of NK cells from HSCs [15]. Although the intact BM microenvironment is essential for NK differentiation, the some cytokines might be required by early acting including SCF, FLT3L, and IL-7 [16,17] in porcine as did differentia- tion to NK cells from human HSCs. In the present report, we investigated the role of a various of cytokines in the development of CD2＋CD8＋ NK cells from porcine c-kit＋ BM cells, and analyzed the cytotoxic activities of CD2＋CD8＋ NK cells. Until recently, it is well known that NK cells are effective at clearing tumors of epithelial origin as well as metastases from tumors [18]. Grzywacz et al. showed that human mye- loid precursors, hematopoietic stem cells, can differentiate into NK cells and the NK cells can kill humanerythroleuke- mic cells [19]. Moreover, it is reported that NK cells exhibit spontaneous cytotoxicity against MHC class I-deficient tar- get cells, and they participate in the innate immune re- sponses against transformed cells and tumor metastases in vivo [20]. In addition, it is studied that the porcine NK cells after cytokine stimulation lysis humanerythroleukemic cells [21]. Particularly, Sotiriadis et al. reported that por- cine NK cells can lysis human carcinoma cells [22]. As mentioned above, the concept that NK cells can be derived from the c-kit＋ BM cells advances our understan- ding of the NK ontogeny and relationship to other blood lineages, creating the potential for therapeutic applications. METHODS Animals We used 15 male porcines (10∼12 weeks old, 20∼25 kg) purchased from Samtako (South Korea). An automatically controlled 12 h photoperiod (lights on 07:00 a.m. and off 07:00 p.m.) and thermostatically regulated ambient tempe- rature (20∼21oC) were maintained throughout the experi- ment. The porcines were housed individually in stainless steel crates, fed the standard, completely balanced pelleted dry food twice a day (07:00∼07:30 a.m. and 02:00∼02:30 p.m.), with water available ad libitum. The animals were acclimated to their new environment for about 1 week before the experiments. All efforts were made to minimize animal suffering and to reduce the number of animals used, with all the protocols being reviewed and approved by the Ani- mal Research Ethics Committee of the Chonbuk National University (Jeonju, Korea) for the protection of vertebrate animals used for experimental and other scientific purpose. Purification of porcine c-kit＋ BM cells Porcines were euthanized and processed into a single cell suspension. Porcine bone marrow cells, containing c-kit＋ HSCs, were harvested and isolated by a modification of the technique described previously [23]. Femurs were removed and cleaned of muscle tissue. Femurs were then dissected longitudinally and compressed to excise BM cells. BM cells were collected, washed with Dulbecco’s modified Eagles me- dium (DMEM) containing 1% (v/v) fetal calf serum (FCS) and 5 U/ml heparin, and processed into a single cell sus- pension. Since then, each sample was diluted in 0.9% sterile saline solution (1：1) (v/v). After dilution was completed, the mixture was added to Ficoll-Paque PLUS (1.077 g/ml; #07907, StemCell Technologies, Vancouver, Canada) in an- other sterile tube and centrifuged at 400 g for 30 min at room temperature. Using a sterile Pasteur pipette the plas- ma layer on top was removed and discarded; care being tak- en to prevent any disturbance of the mononuclear cell layer. And, the aspirate of porcine mononuclear cell layer was col- lected and transferred into a sterile 17×100 mm polystyrene tube. Ammonium chloride lysis was performed on the re- maining pellet to remove red blood cells. Once completely lysed, the pellet was three times with 15 ml of McCoy’s 5A culture media, and centrifuged at 400 g for 5 min each time. A cell count was then performed using an automatic cell counter in order to determine the number of nucleated cells in the culture plate. Routine microbiologic monitoring did not reveal any evidence of infection with common patho- gens. Isolation of porcine c-kit＋ BM cells For isolation of c-kit＋ BM cells from mononuclear cells, biotinylated porcine SCF was used to detect and purify por- cine c-kit＋ cells [24]. Briefly, low-density cells separated with Histopaque (Sigma, St. Louis, MO, USA) were incubated for 1 h on ice with biotinylated SCF (320 μg/l×109 BMCs for magnetic bead separation), and then stained for 15 min with streptavidin-coated magnetic microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany). After extensive phos- phate-buffered saline (PBS) washes, c-kit＋ cells were sorted by the MACS system (Miltenyi Biotec). Percentages of c-kit＋ cells fractions used in this study were greater than 95%. Developments of cytokines-induced CD2＋ CD8＋ cells from porcine c-kit＋ BM cells NK The human umbilical vein endothelial cells (HUVECs) was cultured to confluence and was irradiated (30 Gy [3000 rad]) [25]. Porcine c-kit＋ BM cells were cultured with a monolayer of HUVECs plus Dulbecco modified Eagle me- dium (DMEM) (1：2 ratio) with 20% porcine serum, etha- nolamine (50 μM), ascorbic acid (20 mg/l), 5 μg/l sodium selenite (Na2SeO3), β-mercaptoethanol (24 μM), and peni-