Immortalized bone‐marrow derived pig endothelial cells

Abstract

Primary cultures of porcine endothelial cells (EC) can only be maintained for a limited number of passages. To facilitate studies of xenogeneic human anti-pig immune responses in vitro, pig microvascular bone-marrow (BM) and macrovascular aortic EC were obtained from our herd of partially inbred miniature swine, homozygous for the major histocompatibility locus, and immortalized with a modified SV40 large T vector. The resulting BM-derived (2A2) and aortic (PEDSV.15) immortalized EC lines showed unlimited growth and EC phenotype as indicated by expression of von Willebrand Factor (vWF) and low density lipoprotein (LDL) receptors as well as by formation of typical cobblestone monolayers. Ultrastructural studies revealed morphological similarities in primary and immortalized EC. Flow cytometry analysis demonstrated constitutive SLA class I expression by all lines whereas SLA class II was only expressed after stimulation with porcine IFNgamma. Furthermore, pig CD34 mRNA was detected by Northern blot analysis in primary and immortalized aortic EC but not in 2A2. Both EC lines expressed a number of myeloid markers, adhesion molecules and xenoantigens, the latter being determined by binding of human natural antibodies. Gene transfer into the porcine EC lines was successfully performed by electroporation or calcium-phosphate transfection, as well as by adenoviral infection. Finally, the functional similarity between primary and immortalized EC was demonstrated in adhesion and cytotoxicity assays. Together, these results suggest that 2A2 and PEDSV. 15 represent valuable tools to study both human cellular and humoral immune responses in vitro against pig EC derived from microvascular and large vessels.

Adhesive Interactions between Human NK Cells and Porcine Endothelial Cells

“…However, adhesion receptor blocking alone is probably not sufficient to protect xenografts from rejection in vivo but it may serve as a h: human; p: porcine; f: freshly isolated; a: activated; r: resting; s: stimulated PBL: peripheral blood lymphocytes or leukocytes; PAEC: primary porcine aortic endothelial cells; PED [29] and IPEC [38]: immortalized porcine EC; *PAEC treated for 5 h with human serum; MS: M.K.J. Schneider, unpublished observations.…”

“…Porcine and human EC share several characteristics, such as the expression of the von Willebrand factor and low density lipoprotein receptors, and are also phenotypically similar [29]. However, the phenotypical evaluation of porcine EC has been hampered by the lack of appropriate monoclonal antibodies (MoAb) directed against porcine cell surface molecules.…”

Porcine UL16-Binding Protein 1 Expressed on the Surface of Endothelial Cells Triggers Human NK Cytotoxicity through NKG2D

“…The SV40-immortalized aortic pEC line PEDSV.15 was established and characterized in our laboratory (34). Porcine aortic endothelial cells (PAEC) were isolated from a normal pig following standard procedures (35) and cultured in DMEM (Invitrogen Life Technologies) supplemented with 10% FCS (PAA Laboratories), 1 mM sodium pyruvate, 2 mM Lglutamine, nonessential amino acids (100ϫ), essential amino acids (50ϫ), and 20 mM HEPES (all Invitrogen Life Technologies).…”

MHC Alloantigens Elicit Secondary, But Not Primary, Indirect In Vitro Proliferative Responses

“…PEDSV.15, an SV40-transformed SLA d PAEC clone, was derived and maintained in culture as previously described (18). Retroviral constructs used in this study included the pPBM14 recombinant genome which contained cDNA sequences for the pig DR␣ and -␤ polypeptide chains of the SLA c allele together with that of the neomycin resistance.…”