Immortalization And Immumodulation Of Bone Marrow Derived Mesenchymal Stem Cells From An Inbred Porcine Breeding

PURPOSESThe nature immunomodulatory properties of MSCs, their ability to support hematopoiesis and be genetically modified stably, their capacity of mutipotential differentiation and participating in the regeneration of damaged tissues, suggest that MSCs can be utilized in many novel applications in clinical stem cell therapy. It was reported that allogenic mesenchymal stem cells (MSCs) derived from bone marrow could survive in both human and animal transplantation models. Cotransplantation with autologous or allologous MSCs could prolong skin graft survival or promote hematopoietic stem cell (HSC) engraftment. Namely, allogenic MSCs were believed to be low immunogenicity as well as immunomodulator. Recently, it was found that the possible reason is allogenic MSCs posses ability to inhibit T cell proliferation.Banna Minipig Inbred Line (BMI), a highly inbred porcine, was showed to be hopeful candidate for xenotransplantation. MSCs derived from bone marrow of this highly inbred porcine breeding were especially valuable in basic and clinic research of stem cell therapy. But it is far from clinic use for understanding of its basic biology was limited by short life span. Therefore, it is of great importance to establish a reliable and stable BMI-MSCs sourceand explore its biological characteristics. It has been reported that expression of simian virus 40 (SV40) large T antigen gene is sufficient for some somatic as well as stem cells escaping senescence.Therefore, the aims of this study are to (1) investigating whether or not MSCs from BMI could be immortalized by introducing SV40 gene into the cells; (2) evaluating the suppression effects on human peripheral blood lymphocytes (hPBLs) proliferation and explore the possible mechanism underlying by using the immortalized BMI-MSCs. MATERIALS AND METHODSBMI-MSCsMSCs were isolated from bone marrow of BMI (the 18th generation of subline JS) and transfected with pSV3neo (generously supplied by R. Weinberg), a plasmid coding for SV40 large T antigen and a neomycin-resistance gene at population doubling (PD)2. Proliferation, expression of stem cell markers, potential of differentiation, senescence and tumorgenicity were investigated by classical methods.Mixed lymphocyte reaction(MLR)To determine whether BMI-MSCs could stimulate a proliferative response, 7X 104 hPBLs were cultured with the same amount of BMI-MSCs, BMI-PBLs, human endothelial cells (ECV-304) and l0 g/ml ConA in 96-well microtitre plate. To evaluate suppressive effect on hPBLs proliferation by BMI-MSCs, 7X104 stimulated hPBLs were cultured with different amount of BMI-MSCs. To identify soluble factors responsible for the inhibitory effect, antibodies against FasL, TGF1 and IL-10 were added in system. The proliferation of hPBLs were measured by means of MTS assay..Statistical analysisThe stimulation index (SI) was calculated using the following formula: proliferation of stimulated lymphocyte with or without MSCs/proliferation ofunstimulated lymphocyte alone. The data were statistically analyzed by SPSS 10.0 software and the student t test was used to evaluate the significant differences between samples.RESULTSNormal BMI-MSCss would undergo growth arrest with strong activity of senescence-associated galactosidase (SA-P-Gal) while its PD number exceeded 20. However, SV40-transfected cells were still proliferative at PD180 and only small amount of cells were SA-P-Gal positive. As same as that of normal BMI-MSCss, transfected MSCss were positive for stem cell marker including SH2, SH3, SH4, SB 10, SB20, SB21. Bone formation was initiated in the pores of HA/TCP implants loaded immortalized BMI-MSCs 7 weeks postimplantation. There was no clone formed by these cells in soft-agarose culture experiment and no tumorgenises in nude mice.When 7 X 104 BMI-MSCs were used to stimulate the same amount of hPBLs, no obvious proliferation response was detected. However, hPBLs showed strong proliferative response to allogenic endothelial cells, xenogenic PBLs and nonspecific mitogenic stimuli ConA. It was indicated that BMI-MSCs pos...