Generation of a felinized swine endothelial cell line by expression of feline decay-accelerating factor

Abstract

Embryonic stem cell research has facilitated the generation of many cell types for the production of tissues and organs for both humans and companion animals. Because ≥30% of pet cats suffer from chronic kidney disease (CKD), xenotransplantation between pigs and cats has been studied. For a successful pig to cat xenotransplant, the immune reaction must be overcome, especially hyperacute rejection. In this study, we isolated the gene for feline decay-accelerating factor (fDAF), an inhibitor of complement proteins, and transfected a swine endothelial cell line with fDAF to "felinize" the pig cells. These fDAF-expressing cells were resistant to feline serum containing anti-pig antibodies, suggesting that felinized pig cells were resistant to hyperacute rejection. Our results suggest that a "felinized" pig kidney can be generated for the treatment of CKD in cats in the future.

Fig 1. The whole amino acid sequence of the feline decay-accelerating factor (fDAF) protein aligned with DAFs from other species.

(A) Comparison of the whole amino acid sequence of DAFs. The glycosylphosphatidylinositol (GPI) anchor domains are marked with red letters. Asterisks indicate stop codons. Amino acid residues that are conserved in at least two of the four sequences are shaded, and amino acid residues identical among all four proteins are shown in black. Legend: hDAF, human DAF; mDAF, mouse DAF; sDAF, swine DAF.

Fig 2. Establishment of the feline decay-accelerating factor (fDAF)-expressing swine endothelial cell (sEC) line.

(A) Schematic representation of the fDAF expression vector between the SalI and BamHI sites, containing a cytomegalovirus (CMV) enhancer, a chicken β-actin promoter, fDAF, and the puromycin N-acetyl-transferase gene. (B and C) Immunofluorescent staining of DAF (green) in the MYP30 sEC line, with nuclear counterstaining (blue). The control MYP30 sEC line (B) and fDAF-expressing sEC clone (C) are also shown. Scale bars are 100 m. (D) RT-PCR analysis of endothelial cell makers (CD31 and VE-cadherin) and a house-keeping gene (ß-actin) of both the control MYP30 sEC line (control) and the fDAF-expressing sEC clone (fDAF). DNA maker (DNA 1kbplus ladder, invitrogen) is shown in the far left hand side. (E) Western blot analysis with an anti-DAF antibody, anti-FLAG antibody and anti-β-actin antibody of the mock control and fDAF-FLAG-transfected HEK-293 cells. The most right panel was absorption assay of anti-DAF antibody. The leftmost lane shows protein molecular weight standards in kDa. The red arrow indicates expression of fDAF-FLAG protein and the black arrow indicates endogenous human DAF expression in HEK-293 cell line. (F) Western blot analysis of the DAF protein in the MYP30 sEC line. Both the control sEC line and fDAF-expressing sEC clone were bound to an anti-DAF antibody (left panel) and an anti-β-actin antibody (right panel). The gray arrow indicates fDAF and sDAF expression. The leftmost lane shows protein molecular weight standards in kDa. Other bands indicate endogenous sDAF expression in the MYP30 sEC line.

Fig 3. Analysis of the toxic effects of feline serum on feline DAF (fDAF)-expressing cells.

(A–D) Control swine endothelial cell (sEC) line (fDAF⁻) or fDAF-expressing sEC clone (fDAF⁺) incubated with 80% feline serum. Time zero (0 h) of incubation of the control sEC line (A) and of the fDAF-expressing sEC clone (C). After a 6-h incubation, cells of the control sEC line appear small, round, and detached from the dish (B), whereas cells of the fDAF-expressing sEC clone remain attached to the dish (D). (E and F) A lactate dehydrogenase (LDH) assay of the control cells (fDAF⁻, white box) and the fDAF-expressing sEC clone (fDAF⁺, gray box) incubated with 40% feline serum (E) or 80% feline serum (F). Each data point represents mean ± SEM of five independent experiments (*P < 0.05; **P < 0.01).