New Insights into Xenotransplantation for Cartilage Repair: Porcine Multi-Genetically Modified Chondrocytes as a Promising Cell Source
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 responsib...
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MDPI AG
2021-08-01
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| Online Access: | https://www.mdpi.com/2073-4409/10/8/2152 |
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| author | Hanna Tritschler Konrad Fischer Jochen Seissler Jörg Fiedler Rebecca Halbgebauer Markus Huber-Lang Angelika Schnieke Rolf E. Brenner |
| author_facet | Hanna Tritschler Konrad Fischer Jochen Seissler Jörg Fiedler Rebecca Halbgebauer Markus Huber-Lang Angelika Schnieke Rolf E. Brenner |
| author_sort | Hanna Tritschler |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T08:54:27Z |
| format | Article |
| id | doaj.art-48f44517d73c4437822c6f56ca9ecaaa |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-10T08:54:27Z |
| publishDate | 2021-08-01 |
| publisher | MDPI AG |
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| spelling | doaj.art-48f44517d73c4437822c6f56ca9ecaaa2023-11-22T07:12:29ZengMDPI AGCells2073-44092021-08-01108215210.3390/cells10082152New Insights into Xenotransplantation for Cartilage Repair: Porcine Multi-Genetically Modified Chondrocytes as a Promising Cell SourceHanna Tritschler0Konrad Fischer1Jochen Seissler2Jörg Fiedler3Rebecca Halbgebauer4Markus Huber-Lang5Angelika Schnieke6Rolf E. Brenner7Division for Biochemistry of Joint and Connective Tissue Diseases, Department of Orthopedics, University of Ulm, 89081 Ulm, GermanyChair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technische Universität München, 85354 Freising, GermanyMedizinische Klinik und Poliklinik IV, Diabetes Zentrum—Campus Innenstadt, Klinikum der Ludwig-Maximilians-Universität, 80336 München, GermanyDivision for Biochemistry of Joint and Connective Tissue Diseases, Department of Orthopedics, University of Ulm, 89081 Ulm, GermanyInstitute of Clinical and Experimental Trauma Immunology, University Hospital Ulm, 89081 Ulm, GermanyInstitute of Clinical and Experimental Trauma Immunology, University Hospital Ulm, 89081 Ulm, GermanyChair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technische Universität München, 85354 Freising, GermanyDivision for Biochemistry of Joint and Connective Tissue Diseases, Department of Orthopedics, University of Ulm, 89081 Ulm, GermanyTransplantation 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.https://www.mdpi.com/2073-4409/10/8/2152porcine chondrocytesxenotransplantationcomplementα-1,3-Gal-epitopeNeu5Gc-epitopeCD46 |
| spellingShingle | Hanna Tritschler Konrad Fischer Jochen Seissler Jörg Fiedler Rebecca Halbgebauer Markus Huber-Lang Angelika Schnieke Rolf E. Brenner New Insights into Xenotransplantation for Cartilage Repair: Porcine Multi-Genetically Modified Chondrocytes as a Promising Cell Source Cells porcine chondrocytes xenotransplantation complement α-1,3-Gal-epitope Neu5Gc-epitope CD46 |
| title | New Insights into Xenotransplantation for Cartilage Repair: Porcine Multi-Genetically Modified Chondrocytes as a Promising Cell Source |
| title_full | New Insights into Xenotransplantation for Cartilage Repair: Porcine Multi-Genetically Modified Chondrocytes as a Promising Cell Source |
| title_fullStr | New Insights into Xenotransplantation for Cartilage Repair: Porcine Multi-Genetically Modified Chondrocytes as a Promising Cell Source |
| title_full_unstemmed | New Insights into Xenotransplantation for Cartilage Repair: Porcine Multi-Genetically Modified Chondrocytes as a Promising Cell Source |
| title_short | New Insights into Xenotransplantation for Cartilage Repair: Porcine Multi-Genetically Modified Chondrocytes as a Promising Cell Source |
| title_sort | new insights into xenotransplantation for cartilage repair porcine multi genetically modified chondrocytes as a promising cell source |
| topic | porcine chondrocytes xenotransplantation complement α-1,3-Gal-epitope Neu5Gc-epitope CD46 |
| url | https://www.mdpi.com/2073-4409/10/8/2152 |
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