170 KH Lim, et al heat-inactivated fetal bovine serum (FBS) (Sigma, St Louis, USA), penicillin (Sigma, St Louis, USA) (100 U/ml) and streptomycin (Sigma, St Louis, USA) (100 mg/ml) in a 5% CO2 humidified incubator (standard culture conditions). Washed in complete medium, the K562 target cells (5×106) were labeled with 100 μCi of Na251CrO4 (Radio Chemical Center, Otwock-Świerk, Poland) at 37oC for 1 h. The labeled target cells were then washed five times using RPMI con- taining 2% FBS and adjusted to 1×105/ml in complete me- dium The targets cells (2×104) were cultured in round-bot- tomed microwells plates (Nun, Roskilde, Denmark) with various concentrations of effector cells (effector: target cells ratios: 100：1 in a total volume of 200 μl) in triplicate, un- der standard culture conditions for 4 h. Spontaneous 51Cr release wells had target cells plus 100 μl of complete me- dium and the maximum release wells contained target cells plus 100 μl of complete medium with 5% Triton X-100 (Sigma, St Louis, USA). The assay was terminated at the 4th h by centrifuging the plates (500×g, 5 min). Then 100 μl of supernatant was removed from each well. An isotope count and the percentage of specific lysis (specific 51Cr re- lease) were performed with a gamma counter (Baker System 9120, Allentown, USA) as follows: Specific 51Cr release= Experimental 51Cr release－spontaneous 51Cr release Maximum 51Cr release－spontaneous 51Cr release ×100 Statistical analysis All data were expressed as the mean±SD. Data were ana- lyzed for statistical significance using one-way (ANOVA), followed by Tukey’s test as a post-hoc test with SPSS soft- ware (SPSS for Windows, Version 10). A probability value of p＜0.05 was considered to indicate statistical signi- ficance, and all experiments were performed at least three times. RESULTS Phenotype analysis of porcine c-kit＋ BM cells We examined the expression levels of porcine c-kit, Fig. 1. Phenotyping of porcine bone marrow derived c-kit＋ cells. Porcine c-kit＋ BM cells were isolated from bone marrow using a ficoll and mag- netic activated cell sorter (MACS) and confirmed by fluorescence cell sorting (A). Histogram of isotype unstained control is shown (A: the part shown in dotted line area). Total number of CD117＋ (c-kit＋) BM cells is shown (A: the part shown in black area). Panel 1; Respective isotype control in unstained c-kit＋ BM cells gate is shown. Panel 2; Frequency of CD117＋ cells after staining with monoclonal antibodies CD117-FITC is shown in Q4 region: (Q4=98.9%). Porcine c-kit＋ cell colony obtained from porcine BM cells was photographed in stem cell medium (B, Bar: 50 μm). Fig. 2. Phenotypic analysis of CD2＋CD8＋ cells subpopulations in length of time for 28 days. There was a significant difference in expression of CD2 and CD8 molecules three weeks later from the treatment of cytokines compared to control (*p＜0.01). CD117. The fluorescent intensity of Allexa Fluor 488-CD117 was used to measure the expression of c-kit. As shown in Fig. 1A, porcine BM cells were stained with more than 98.9±3.7% compare to isotype control. After 16 h, BM cells were aggregated each other to clump (Fig. 1B). Cytokines-induced NK cells identified by CD2＋ CD8＋ cells Surface marker CD2 and CD8 molecules are highly spe- cific for porcine NK cells [31]. Cytokines-activated cells were isolated from co-culture plate with HUVEC. Then, CD2＋CD8＋ cells were determined using a FACS analysis. Only primary cultured cells were used, because they have the ability to become various types of cells under specific culture conditions. Therefore, cytokines-activated c-kit＋ BM cells for 7th, 14th, 21th and 28th days were confirmed by FACS analysis using porcine NK cells marker-specific antibodies (Fig. 2, *p＜0.01). The FACS results demon- strate that 1.2±1.7, 5.5±3.4, 9.7±7.3, 35.5±6.8 and 34.7±8.7% of the cytokines-induced NK cells expressed CD2 and CD8 molecules compared to c-kit＋ BM cells not pretreated with cytokines (2.5±1.8, 2.7±2.4, 2.8±2.2, 2.2±2.3 and 2.3±1.9%, respectively) in length of time (Fig. 2, *p＜0.01). Whereas, all c-kit＋ BM cells did not differentiate to CD2＋CD8＋ NK