© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Xenotransplantation 2015: 22: 155–157 doi: 10.1111/xen.12168
XENOTRANSPLANTATION
Literature Update
Xenotransplantation literature update, January–February 2015 Burlak C, Kerns KC. Xenotransplantation literature update, January– February 2015. Xenotransplantation 2015: 22: 155–157. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Christopher Burlak1 and Karl C. Kerns2 1
Schulze Diabetes Institute, Department of Surgery, University of Minnesota School of Medicine, Minneapolis, MN, , 2Division of Animal Sciences, University of Missouri-Columbia, Columbia, MO, USA Key words: coagulation disorder – CRISPR – pig – xenotransplantation Abbreviations: NHP, non-human primate; IS, immune suppression; RIA, radioimmunoassay; ELISA, enzyme-linked immunosorbent assay; aGal, galactose a1,3 galactose. Address reprint requests to Christopher Burlak, Schulze Diabetes Institute, Department of Surgery, University of Minnesota School of Medicine, 420 Delaware St. SE, Minneapolis 55455, MN, USA (E-mail: [email protected]) Received 4 March 2015; Accepted 5 March 2015
Reviews and commentaries
It has become well accepted that porcine-derived islet cells will likely be the source to subside type 1 diabetes; however, there is not a consensus of donor age at which these cells should be isolated. Nagaraju et al. [1] performed a literature review limited to clinically relevant studies of the pig-tonon-human primate (NHP) model. Their review called attention to a lack of comparative studies between adult and neonatal islet xenotransplantation. The variables considered include islet yield, structure, in vitro insulin secretion, pig production costs, ability for cells to proliferate, and time until normoglycemia. The review suggested exact timing of islet isolation during the first month of life is likely not to be critical, but some advantage might be present within the first week of birth. Immunology
Reduction of xenoantigens and expression of complement regulatory proteins have extended the survival time of vascularized grafts. However, in the presence of immune suppression (IS),
incompatibilities between human or NHP and pig coagulation systems can lead to thrombotic microangiopathy or consumptive coagulopathy. Ezzelarab et al. [2] sought to define the unbalanced mechanisms that destabilize the coagulation system in xenograft recipients. Using organ and artery patch models in NHPs, the authors revealed that the expression and deposition of C-reactive protein was elevated in the presence of IS and correlated with increased expression of IL-6 and fibrinogen. Interestingly, when no IS was given, IL-6 levels were lower, suggesting the coagulation deregulation is rooted in either excessive IS or the combination of IS in the NHP model. To support this hypothesis, the authors show that CD14+ monocytes and CD11c+ dendritic cells had significantly elevated CD40 and tissue factor expression after administration of CTLA4Ig or anti-CD154 costimulation blockade. Close examination of the cytokine expression in the presence and absence of IS suggests that inflammation perhaps associated with increased monocytes leads to activation of the coagulation cascade. Le Bas-Bernardet et al. [3] have conducted a multicenter effort to determine the usefulness of 155
Burlak and Kerns galactose a1,3 galactose (aGal )knockout pigs transgenic for complement regulatory proteins in a lifesaving porcine kidney to baboon model of xenotransplantation. Unfortunately, 9–15 days post-transplantation, immunosuppressed baboons exhibited acute humoral rejection with IgM deposition with complement components excluding C4d suggesting activation of the alternative complement pathway. Rejections were accompanied by a predominantly monocyte infiltration almost in the near absence of lymphocytes. The authors conclude that the addition of bortezomib and plasma exchanges had little benefit to graft survival. It may be relevant to consider Ezzelarab et al. and Le Bas-Bernardet et al. together and evaluate the impact of IS protocols on monocyte populations and inflammatory cytokines. Methods
Wang et al. [4] developed a candidate protocol for future solid whole-organ decellularization, using the porcine liver and kidney as an example. The authors compared the use of 1% SDS, 1% Triton X-100, 1% PAA, 1% NaDOC, and phosphatidase with a perfusion method of inserting the feeding tube into the portal vein and renal artery of the liver and kidney, respectively. The fastest visual changes observed by a more translucent appearance occurred with 1% SDS. It should be noted that this treatment led to organ distension; however, the final decellularized organ volume was comparable to the initial. Almost complete removal of cellular components was observed through quantification of residual DNA and histological staining. SDS has been previously criticized as a treatment condition due to a loss of collagen integrity through extracellular matrix damage; however, the protocol described was optimized by the removal of residual SDS with 1% Triton X-100. Further research is undoubtedly needed before application of bioengineered engrafts; however, the procedure outlined should reduce adverse effects on tissue remodeling response and serve as progress toward recellularization and re-endothelialization methods. The commercial radioimmunoassay (RIA) used to measure porcine C-peptide levels from islet culture or after transplantation was discontinued from production in 2013. To determine the level of agreement between the RIA and enzyme-linked immunosorbent assay (ELISA) that is available as a replacement, Graham et al. [5] evaluated the serum C-peptide concentrations in 60 landrace pigs at fasting and glucose challenge. The authors concluded that comparison of RIA and ELISA mea156
surements of the same sample was not equivalent but within a linear range, the assays may correlate. Researchers comparing previously collected RIA data with newly gathered ELISA data should do so with caution. Genetically engineered pigs
The time needed to create precise, desired modifications to the genome has recently decreased substantially and became much more efficient in just a few years. The necessity of such biotechnology is vital for clinical application of xenotransplantation. Li et al. [6] describe the use of CRISPR/Cas9, a novel targeting system for gene disruption by inducing a precise double-strand break, paired with magnetic bead-based cell sorting to efficiently target multiple genetic modifications in the pig. The authors targeted GGTA1, CMAH, and putative iGb3S genes. IB4 lectin/magnetic beads were used to negatively select cells in the absence of aGal. Somatic cell nuclear transfer was then performed with these cells, and 32-day-old fetuses and piglets were analyzed. The process involving magnetic beads yielded a 1.9-fold co-enrichment of Neu5Gc-deficient cells, the gene product of CMAH, and lacked aGal. All three genes had confirmed modification via DNA sequencing and the results were replicable. The work shown by these authors provides an option of cell selection without antibiotic-resistant markers, thus positively reducing one potential barrier to acceptance for clinic xenotransplantation. In the future, this opens the doors for alternative cell-sorting methods, utilizing technologies such as microrafts or florescentactivated cell sorting, to make multiple genetic modification selections and will serve as a model going forward for efficient generation of additional genetic lines. Organ-specific expression of human transgenes in pigs may allow the creation of a source pig that overcomes immunologic hurdles unique to that organ. Wijkstrom et al. [7] have inserted up to three genes under control of the rat insulin II promoter with the mouse PDX-1 gene distal enhancer. Islets isolated from seven pigs were positive for human tissue factor pathway inhibitor, CTLA4Ig, and/or human CD39 under the rat insulin promoter when examined by confocal microscopy. The authors wanted to know whether introducing another insulin promoter would burden the islets to an extent that glucose metabolism would be impaired. The pigs were normoglycemic and produced C-peptide and glucagon in response to glucose and arginine challenge. However, when compared to wild-type and GTKO pigs, pigs with
Xenotransplantation literature update the insulin promoter-driven genes had increased body weight, fasting blood glucose and glucagon, and significantly lower fasting insulin. The authors suggest that differences in the age of pigs and variation among genetic modifications may account for the variability.
4.
5.
References 1. NAGARAJU S, BOTTINO R, WIJKSTROM M, TRUCCO M, COOPER DKC. Islet xenotransplantation: what is the optimal age of the islet-source pig? Xenotransplantation 2015; 22: 7–19. 2. EZZELARAB MB, EKSER B, AZIMZADEH A et al. Systemic inflammation in xenograft recipients precedes activation of coagulation. Xenotransplantation 2015; 22: 32–47. 3. Le BAS-BERNARDET S, TILLOU X, BRANCHEREAU J et al. Bortezomib, C1-inhibitor and plasma exchange do not
6.
7.
prolong the survival of multi-transgenic GalT-KO pig kidney xenografts in baboons. Am J Transplant 2015; 15: 358–370. WANG Y, BAO J, WU Q et al. Method for perfusion decellularization of porcine whole liver and kidney for use as a scaffold for clinical-scale bioengineering engrafts. Xenotransplantation 2015; 22: 48–61. GRAHAM ML, GRESCH SC, HARDY SK et al. Evaluation of commercial ELISA and RIA for measuring porcine C-peptide: implications for research. Xenotransplantation 2015; 22: 62–69. LI P, ESTRADA JL, BURLAK C et al. Efficient generation of genetically distinct pigs in a single pregnancy using multiplexed single-guide RNA and carbohydrate selection. Xenotransplantation 2015; 22: 20–31. WIJKSTROM M, BOTTINO R, IWASE H et al. Glucose metabolism in pigs expressing human genes under an insulin promoter. Xenotransplantation 2015; 22: 70–79.
157
Xenotransplantation 2015: 22: 155–157 doi: 10.1111/xen.12168
XENOTRANSPLANTATION
Literature Update
Xenotransplantation literature update, January–February 2015 Burlak C, Kerns KC. Xenotransplantation literature update, January– February 2015. Xenotransplantation 2015: 22: 155–157. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Christopher Burlak1 and Karl C. Kerns2 1
Schulze Diabetes Institute, Department of Surgery, University of Minnesota School of Medicine, Minneapolis, MN, , 2Division of Animal Sciences, University of Missouri-Columbia, Columbia, MO, USA Key words: coagulation disorder – CRISPR – pig – xenotransplantation Abbreviations: NHP, non-human primate; IS, immune suppression; RIA, radioimmunoassay; ELISA, enzyme-linked immunosorbent assay; aGal, galactose a1,3 galactose. Address reprint requests to Christopher Burlak, Schulze Diabetes Institute, Department of Surgery, University of Minnesota School of Medicine, 420 Delaware St. SE, Minneapolis 55455, MN, USA (E-mail: [email protected]) Received 4 March 2015; Accepted 5 March 2015
Reviews and commentaries
It has become well accepted that porcine-derived islet cells will likely be the source to subside type 1 diabetes; however, there is not a consensus of donor age at which these cells should be isolated. Nagaraju et al. [1] performed a literature review limited to clinically relevant studies of the pig-tonon-human primate (NHP) model. Their review called attention to a lack of comparative studies between adult and neonatal islet xenotransplantation. The variables considered include islet yield, structure, in vitro insulin secretion, pig production costs, ability for cells to proliferate, and time until normoglycemia. The review suggested exact timing of islet isolation during the first month of life is likely not to be critical, but some advantage might be present within the first week of birth. Immunology
Reduction of xenoantigens and expression of complement regulatory proteins have extended the survival time of vascularized grafts. However, in the presence of immune suppression (IS),
incompatibilities between human or NHP and pig coagulation systems can lead to thrombotic microangiopathy or consumptive coagulopathy. Ezzelarab et al. [2] sought to define the unbalanced mechanisms that destabilize the coagulation system in xenograft recipients. Using organ and artery patch models in NHPs, the authors revealed that the expression and deposition of C-reactive protein was elevated in the presence of IS and correlated with increased expression of IL-6 and fibrinogen. Interestingly, when no IS was given, IL-6 levels were lower, suggesting the coagulation deregulation is rooted in either excessive IS or the combination of IS in the NHP model. To support this hypothesis, the authors show that CD14+ monocytes and CD11c+ dendritic cells had significantly elevated CD40 and tissue factor expression after administration of CTLA4Ig or anti-CD154 costimulation blockade. Close examination of the cytokine expression in the presence and absence of IS suggests that inflammation perhaps associated with increased monocytes leads to activation of the coagulation cascade. Le Bas-Bernardet et al. [3] have conducted a multicenter effort to determine the usefulness of 155
Burlak and Kerns galactose a1,3 galactose (aGal )knockout pigs transgenic for complement regulatory proteins in a lifesaving porcine kidney to baboon model of xenotransplantation. Unfortunately, 9–15 days post-transplantation, immunosuppressed baboons exhibited acute humoral rejection with IgM deposition with complement components excluding C4d suggesting activation of the alternative complement pathway. Rejections were accompanied by a predominantly monocyte infiltration almost in the near absence of lymphocytes. The authors conclude that the addition of bortezomib and plasma exchanges had little benefit to graft survival. It may be relevant to consider Ezzelarab et al. and Le Bas-Bernardet et al. together and evaluate the impact of IS protocols on monocyte populations and inflammatory cytokines. Methods
Wang et al. [4] developed a candidate protocol for future solid whole-organ decellularization, using the porcine liver and kidney as an example. The authors compared the use of 1% SDS, 1% Triton X-100, 1% PAA, 1% NaDOC, and phosphatidase with a perfusion method of inserting the feeding tube into the portal vein and renal artery of the liver and kidney, respectively. The fastest visual changes observed by a more translucent appearance occurred with 1% SDS. It should be noted that this treatment led to organ distension; however, the final decellularized organ volume was comparable to the initial. Almost complete removal of cellular components was observed through quantification of residual DNA and histological staining. SDS has been previously criticized as a treatment condition due to a loss of collagen integrity through extracellular matrix damage; however, the protocol described was optimized by the removal of residual SDS with 1% Triton X-100. Further research is undoubtedly needed before application of bioengineered engrafts; however, the procedure outlined should reduce adverse effects on tissue remodeling response and serve as progress toward recellularization and re-endothelialization methods. The commercial radioimmunoassay (RIA) used to measure porcine C-peptide levels from islet culture or after transplantation was discontinued from production in 2013. To determine the level of agreement between the RIA and enzyme-linked immunosorbent assay (ELISA) that is available as a replacement, Graham et al. [5] evaluated the serum C-peptide concentrations in 60 landrace pigs at fasting and glucose challenge. The authors concluded that comparison of RIA and ELISA mea156
surements of the same sample was not equivalent but within a linear range, the assays may correlate. Researchers comparing previously collected RIA data with newly gathered ELISA data should do so with caution. Genetically engineered pigs
The time needed to create precise, desired modifications to the genome has recently decreased substantially and became much more efficient in just a few years. The necessity of such biotechnology is vital for clinical application of xenotransplantation. Li et al. [6] describe the use of CRISPR/Cas9, a novel targeting system for gene disruption by inducing a precise double-strand break, paired with magnetic bead-based cell sorting to efficiently target multiple genetic modifications in the pig. The authors targeted GGTA1, CMAH, and putative iGb3S genes. IB4 lectin/magnetic beads were used to negatively select cells in the absence of aGal. Somatic cell nuclear transfer was then performed with these cells, and 32-day-old fetuses and piglets were analyzed. The process involving magnetic beads yielded a 1.9-fold co-enrichment of Neu5Gc-deficient cells, the gene product of CMAH, and lacked aGal. All three genes had confirmed modification via DNA sequencing and the results were replicable. The work shown by these authors provides an option of cell selection without antibiotic-resistant markers, thus positively reducing one potential barrier to acceptance for clinic xenotransplantation. In the future, this opens the doors for alternative cell-sorting methods, utilizing technologies such as microrafts or florescentactivated cell sorting, to make multiple genetic modification selections and will serve as a model going forward for efficient generation of additional genetic lines. Organ-specific expression of human transgenes in pigs may allow the creation of a source pig that overcomes immunologic hurdles unique to that organ. Wijkstrom et al. [7] have inserted up to three genes under control of the rat insulin II promoter with the mouse PDX-1 gene distal enhancer. Islets isolated from seven pigs were positive for human tissue factor pathway inhibitor, CTLA4Ig, and/or human CD39 under the rat insulin promoter when examined by confocal microscopy. The authors wanted to know whether introducing another insulin promoter would burden the islets to an extent that glucose metabolism would be impaired. The pigs were normoglycemic and produced C-peptide and glucagon in response to glucose and arginine challenge. However, when compared to wild-type and GTKO pigs, pigs with
Xenotransplantation literature update the insulin promoter-driven genes had increased body weight, fasting blood glucose and glucagon, and significantly lower fasting insulin. The authors suggest that differences in the age of pigs and variation among genetic modifications may account for the variability.
4.
5.
References 1. NAGARAJU S, BOTTINO R, WIJKSTROM M, TRUCCO M, COOPER DKC. Islet xenotransplantation: what is the optimal age of the islet-source pig? Xenotransplantation 2015; 22: 7–19. 2. EZZELARAB MB, EKSER B, AZIMZADEH A et al. Systemic inflammation in xenograft recipients precedes activation of coagulation. Xenotransplantation 2015; 22: 32–47. 3. Le BAS-BERNARDET S, TILLOU X, BRANCHEREAU J et al. Bortezomib, C1-inhibitor and plasma exchange do not
6.
7.
prolong the survival of multi-transgenic GalT-KO pig kidney xenografts in baboons. Am J Transplant 2015; 15: 358–370. WANG Y, BAO J, WU Q et al. Method for perfusion decellularization of porcine whole liver and kidney for use as a scaffold for clinical-scale bioengineering engrafts. Xenotransplantation 2015; 22: 48–61. GRAHAM ML, GRESCH SC, HARDY SK et al. Evaluation of commercial ELISA and RIA for measuring porcine C-peptide: implications for research. Xenotransplantation 2015; 22: 62–69. LI P, ESTRADA JL, BURLAK C et al. Efficient generation of genetically distinct pigs in a single pregnancy using multiplexed single-guide RNA and carbohydrate selection. Xenotransplantation 2015; 22: 20–31. WIJKSTROM M, BOTTINO R, IWASE H et al. Glucose metabolism in pigs expressing human genes under an insulin promoter. Xenotransplantation 2015; 22: 70–79.
157
