Abstract
Transplantation of xenogenic porcine chondrocytes could represent a future strategy for the treatment of human articular cartilage defects. Major obstacles are humoral and cellular rejection processes triggered by xenogenic epitopes like α-1,3-Gal and Neu5Gc. Besides knockout (KO) of genes responsible for the biosynthesis of respective epitopes (GGTA1 and CMAH), transgenic expression of human complement inhibitors and anti-apoptotic as well as anti-inflammatory factors (CD46, CD55, CD59, TNFAIP3 and HMOX1) could synergistically prevent hyperacute xenograft rejection. Therefore, chondrocytes from different strains of single- or multi-genetically modified pigs were characterized concerning their protection from xenogeneic complement activation. Articular chondrocytes were isolated from the knee joints of WT, GalTKO, GalT/CMAH-KO, human CD59/CD55//CD46/TNFAIP3/HMOX1-transgenic (TG), GalTKO/TG and GalT/CMAHKO/TG pigs. The tissue-specific effectiveness of the genetic modifications was tested on gene, protein and epitope expression level or by functional assays. After exposure to 20% and 40% normal human serum (NHS), deposition of C3b/iC3b/C3c and formation of the terminal complement complex (TCC, C5b-9) was quantified by specific cell ELISAs, and generation of the anaphylatoxin C5a by ELISA. Chondrocyte lysis was analyzed by Trypan Blue Exclusion Assay. In all respective KO variants, the absence of α -1,3-Gal and Neu5Gc epitope was verified by FACS analysis. In chondrocytes derived from TG animals, expression of CD55 and CD59 could be confirmed on gene and protein level, TNFAIP3 on gene expression level as well as by functional assays and CD46 only on gene expression level whereas transgenic HMOX1 expression was not evident. Complement activation in the presence of NHS indicated mainly effective although incomplete protection against C3b/iC3b/C3c deposition, C5a-generation and C5b-9 formation being lowest in single GalTKO. Chondrocyte viability under exposure to NHS was significantly improved even by single GalTKO and completely preserved by all other variants including TG chondrocytes without KO of xenoepitopes.
Highlights
The treatment of articular cartilage injuries remains a major clinical and experimental challenge
Chondrocytes were detached by trypsin/EDTA treatment and a minimum of 1 × 105 cells were incubated with GS Isolectin B4 (IB4)-FITC for 30 min or immuno-cytologically stained for 20 min in dark with one of the following antibodies or corresponding isotype controls: mouse anti-human CD46 FITC (#315304, 1:5), anti-human CD55 APC (#311312, 1:20), anti-human CD59 PE (#304708, 1:50), IgG1 FITC (#400108, 1:5), IgG1 APC (#400120, 1:20) and IgG2a PE (#400212, 1:50)
Deposition was detected on any variant, but it was significantly reduced on TG, GalTKO/TG, GalT/CMAHKO and GalT/CMAHKO/TG chondrocytes compared to WT
Summary
The treatment of articular cartilage injuries remains a major clinical and experimental challenge. Xenotransplantation approaches have been developed and continuously optimized for a wide range of organ/cell types in order to solve the persistent shortage of available cell/tissue transplants [5] From those studies, it is well known that human natural antibodies, reactive with the α-1,3-Gal epitope, initiate hyperacute rejection which is mainly driven by activation of the hosts’ classical complement pathway. It is well known that human natural antibodies, reactive with the α-1,3-Gal epitope, initiate hyperacute rejection which is mainly driven by activation of the hosts’ classical complement pathway This first stage of rejection can be significantly reduced by inactivation of the GGTA1 gene, encoding α-1,3-galactosyl transferase that synthesizes the α-1,3-Gal epitopes on the cell surface [6]. The comparative study of five variants of genetically modified pigs allowed us to thoroughly assess their relative impact on complement-mediated xenorejection processes in vitro
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