TY - JOUR
T1 - New insights into xenotransplantation for cartilage repair
T2 - Porcine multi‐genetically modified chondrocytes as a promising cell source
AU - Tritschler, Hanna
AU - Fischer, Konrad
AU - Seissler, Jochen
AU - Fiedler, Jörg
AU - Halbgebauer, Rebecca
AU - Huber‐lang, Markus
AU - Schnieke, Angelika
AU - Brenner, Rolf E.
N1 - Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2021/8
Y1 - 2021/8
N2 - 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.
AB - 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.
KW - CD46
KW - CD55
KW - CD59
KW - Complement
KW - Heme oxygenase‐1
KW - Neu5Gc-epitope
KW - Porcine chondrocytes
KW - TNFAIP3
KW - Xenotransplantation
KW - α‐1,3‐Gal‐epitope
UR - http://www.scopus.com/inward/record.url?scp=85115144799&partnerID=8YFLogxK
U2 - 10.3390/cells10082152
DO - 10.3390/cells10082152
M3 - Article
C2 - 34440921
AN - SCOPUS:85115144799
SN - 2073-4409
VL - 10
JO - Cells
JF - Cells
IS - 8
M1 - 2152
ER -