© 2013 John Wiley & Sons A/S
Xenotransplantation 2013: 20: 193–196 Printed in Singapore. All rights reserved doi: 10.1111/xen.12040
XENOTRANSPLANTATION
Literature Update
Xenotransplantation literature update, March–April 2013 Schneider MKJ, Seebach JD. Xenotransplantation literature update, March–April 2013. Xenotransplantation 2013: 20: 193–196. © 2013 John Wiley & Sons A/S.
M arten K. J. Schneider1 and J€org D. Seebach2 1
Division of Internal Medicine, Laboratory of Vascular Immunology, University Hospital Zurich, Zurich, 2Department of Internal Medicine, Service of Immunology and Allergology, University Hospital and Medical Faculty, Geneva, Switzerland Key words: galactose-a1,3-galactose – islet cells – pig – xenotransplantation – xenozoonosis Abbreviations: Ab, antibodies; BM, bone marrow; DXR, delayed xenograft rejection; EXTEM, extrinsic coagulation pathways; FIBTEM, function of fibrinogen; GSIS, glucose-stimulated insulin secretion; GT, a1,3-galactosyltransferase; GT-KO, a1,3-galactosyltransferase knockout; hDAF, human decayaccelerating factor; HO-1, Heme oxygenase-1; INS-1E, rat insulinoma cells; INTEM, intrinsic coagulation pathways; LDC, liver-derived cells; mCD47INS-1E, INS-1E cells transfected with mouse CD47; MLR, mixed-lymphocyte reaction; NHP, non-human primate; pEC, porcine endothelial cells; piPSC, pig induced pluripotent stem cells; pNICC, pig neonatal islet cell clusters; ROTEM, whole blood rotation thromboelastometry; siRNA, small interfering RNA; SIRP, signal regulatory protein a; SCNT, somatic cell nuclear transfer; ZFN, zinc finger nucleases Address reprint requests to M arten K. J. Schneider, PhD, Division of Internal Medicine, Laboratory of Vascular Immunology, University Hospital Zurich, Raemistrasse 100, LAB C 7, CH-8091 Zurich, Switzerland (E-mail: [email protected]) Received 17 April 2013; Accepted 17 April 2013
Commentary
In light of the progress in experimental pig islet xenotransplantation and the prospects for clinical trials, Cooper et al. [1] discussed whether revisions to the guidelines proposed by the International Xenotransplantation Association in 2009 are warranted. The authors suggest five major topics of discussion: (i) is there a need for additional experimental work, (ii) which preliminary clinical studies need to have been carried out, (iii) the need for microbiological safety testing of pigs and pig islets, (iv) patient selection for the initial clinical
trial, and finally, (v) on what basis would a trial be considered successful? The authors conclude by stating that the human suffering associated with diabetes is so significant that pig islet xenotransplantation, with its considerable potential as a clinical therapy, should not be regulated to the point that no advances can be made or will be delayed unnecessarily. Preclinical
Coagulopathy in pig-to-primate organ transplantation is a complex process involving coagulation 193
Schneider and Seebach factors, platelets, and phospholipid-bearing cells. In this context, Spiezia et al. [2] examined thromboelastographic profiles by performing whole blood rotation thromboelastometry (ROTEM) in immunosuppressed nephrectomized primates following renal transplantation. Cynomolgus monkeys received lifesupporting kidneys from pigs transgenic for (i) human decay-accelerating factor (hDAF), (ii) human CD39, CD55, CD59, and fucosyltransferase on a a1,3-galactosyltransferase knock-out (GTKO) background, or (iii) hDAF and GT-KO. Three standard ROTEM assays were performed analyzing intrinsic (INTEM) and extrinsic (EXTEM) coagulation pathways and the function of fibrinogen (FIBTEM). All recipients showed progressive prolongation of clotting time in INTEM. Prolonged clot formation times in both INTEM and EXTEM corresponded with decreased platelet counts, whereas a reduction in maximum clot firmness in FIBTEM corresponded with decreased fibrinogen plasma levels. No concordance was seen between clotting time in INTEM and activated partial thromboplastin time or between clotting time in EXTEM and prothrombin time. The authors conclude that the ROTEM analyzer could be a useful and complementary tool to study consumptive coagulopathy in pig-to-primate xenotransplantation studies. Pre-treatment with donor hematopoietic cells may induce transplantation tolerance via mixed chimerism. Jie et al. [3] studied the survival of porcine corneal xenografts in rhesus monkeys pretreated with either cyclophosphamide alone or with cyclophosphamide followed by pig bone marrow (BM) cell transplantation. The mean graft survival time was 18 days in control recipients treated with cyclophosphamide alone. Pretreatment with donor BM increased the mean graft survival to 36 days with minimal inflammatory cell infiltration and a mean chimerism of around 5% after 1 week and less than 1% at 3 weeks. BMtreated recipients also showed less immune reactivity to donor spleen cells in mixed-lymphocyte reactions (MLR) and lower serum immunoglobulin and complement levels as compared to control recipients. In conclusion, corneal xenograft survival may be prolonged by prior BM transplantation. Islets
The suitability and the predictive value of preclinical pig-to-non-human primate (NHP) islet xenotransplantation remain critical issues because of species differences, including the insulin secretory characteristics of islets. Mueller et al. [4] analyzed glucose-stimulated insulin secretion in vitro of islets 194
isolated from humans, NHP, and adult and juvenile pigs. Insulin secretion was measured after perifusion of pancreata with medium at basal glucose followed by high glucose concentrations. The total glucose-stimulated insulin secretion (GSIS) of NHP islets was 3 times higher than that of adult pig islets. Furthermore, the GSIS of human islets was 3 and 30 times higher than that of adult and juvenile pig islets, respectively. The insulin content was similar in human, NHP, and adult pig islets, but much smaller in juvenile pig islets. Thus, although human, NHP, and adult pig islets contain similar amounts of insulin, the significantly higher GSIS of human and NHP islets compared to pig islets suggests the need for increased dosing of islets in pig-to-primate xenotransplantation. Vakhshiteh et al. [5] examined the viability and insulin secretion of caprine islets in vitro. Caprine islet had a diameter between 50 and 250 lm, in which 80% of the total islet yield was defined as small islets with a diameter of 150 lm. At 48 h after isolation, the small islets were more viable than large islets, the latter showing a higher rate of central core necrosis and apoptosis. Small islets secreted almost three times more insulin under both low and high glucose incubation. Thus, smaller caprine islets show a superior quality compared to larger islets under an optimized basal maintenance condition. Immunobiology
Interspecies incompatibility between the signal regulatory protein a (SIRPa) and CD47 results in phagocytosis of xenogeneic cells by host macrophages. Teraoka et al. [6] investigated whether transgenic expression of mouse CD47 in rat insulinoma cells (INS-1E) could inhibit macrophagemediated xenograft rejection. INS-1E cells transfected with mouse CD47 (mCD47-INS-1E) induced SIRPa-tyrosine phosphorylation in mouse macrophages in vitro, whereas control vector cells did not. When injected into streptozotocin-induced diabetic Rag2 / c / mice (T, B, and NK cell deficient), the expression of mouse CD47 on the INS1E cells reduced the susceptibility to phagocytosis by macrophages. Furthermore, injection of mCD47-INS-1E cells induced normoglycemia, which could be prevented by blocking antibodies (Ab) against SIRPa. In conclusion, these results further suggest that the expression of recipient CD47 on xenogeneic donor cells may prevent macrophage-mediated xenograft rejection. CD2 is a costimulation and adhesion molecule expressed on lymphocytes, including T cells. Brady et al. [7] investigated whether secretion of
Xenotransplantation literature update anti-human CD2 Ab by pig neonatal islet cell clusters (pNICC) would induce local immune protection. For this, three forms of a mouse anti-human CD2 Ab, dilimomab (mouse), diliximab (chimeric), and dilizumab (humanized) were utilized. All 3 forms of Ab bound human T cells in vitro, although dilimomab and diliximab exhibited a 300-fold higher avidity than dilizumab. Dilimomab and diliximab, but not dilizumab, inhibited a human anti-pig xenogeneic MLR. When administered systemically in humanized mice, all 3 antiCD2 Ab were able to deplete human CD3+ T cells in vivo without inducing an upregulation of activation markers or a significant cytokine release. Finally, when humanized mice were transplanted with diliximab-transduced pNICC, a depletion of CD3+ T cells at the graft site was observed, leaving the peripheral immune system intact. The authors conclude that a local production of a single Ab against T cells can reduce graft infiltration at the xenograft site, which may reduce the need for conventional, systemic immunosuppression. Delayed xenograft rejection (DXR) involves type II vascular endothelial cell activation, including upregulation of proinflammatory genes. In an attempt to attenuate DXR and to improve the survival of mouse-to-rat heterotopic heart xenografts, Shen et al. [8] used small interfering RNA (siRNA) technology to inhibit NF-jB p65 gene expression in vivo in donor mice. Donor treatment with NFjB siRNA 1 day before transplantation prolonged the median heart graft survival time to 5.4 days compared to 1.7 days for hearts from PBS-treated control donors. Pre-treatment with NF-jB siRNA caused a significant decrease in mRNA levels of NF-jB p65, VCAM-1, ICAM-1, and interleukin-1 at 12 h post-transplantation and a delay of endothelial cell damage. In summary, NF-jB p65 siRNA treatment of donor animals has the potential to delay the emergence of DXR. Heme oxygenase-1 (HO-1) expression has been reported as essential to ensure accommodation in concordant xenotransplantation models. In an approach to establish an accommodation model in vitro, Zhang et al. [9] investigated whether overexpression of HO-1 would protect porcine endothelial cells (pEC) against lysis mediated by human xenoreactive Ab and complement. Overexpression of HO-1 in aortic pEC was achieved by incubation with cobalt-protoporphyrin IX. However, HO-1 overexpression did not protect pEC against lysis mediated by human sera. Therefore, the authors conclude that the induction of HO-1 overexpression alone is not enough to protect pEC from human antibody plus complement-mediated humoral injury.
Donor animals
Engineered zinc finger nucleases (ZFN) have recently been used to generate genetically modified pigs. Li et al. [10] introduced a ZFN pair targeted to exon 8 of the a1,3-galactosyltransferase (GT) gene into adult pig liver-derived cells (LDC) by electroporation. A ZFN activity of 6.5% was detected in LDC, indicating 6.5% mutated alleles in the pooled cell population. GT-KO cells were selected and used as nuclear donors for somatic cell nuclear transfer (SCNT). A total of six fetuses and 13 piglets were produced by SCNT. All fetuses and piglets had biallelic mutations in the ZFN targeted region and were negative for the galactose-a1,3-galactose epitope. In conclusion, biallelic GT gene disruption in LDC was efficiently generated by ZFN. With their long life span and robust growth rate, LDC has the potential to endure multiple sequential genetic modifications in vitro prior to using SCNT, which may accelerate the production of genetically modified pigs for xenotransplantation. The development of highly proliferative porcine induced pluripotent stem cells (piPSC) from GT-KO tissue provides a valuable cell source for complex genetic manipulations. Liu et al. [11] generated GT-KO piPSC by transducing porcine GT-KO fibroblasts with six human reprogramming pluripotency genes (POU5F1, SOX2, NANOG, LIN28, KLF-4, and C-MYC). The piPSC showed classical stem cell morphology and characteristics, expressing the integrated reprogramming genes in addition to endogenous pluripotent markers. The piPSC were highly proliferative and possessed doubling times and telomerase activity similar to human embryonic stem cells. In conclusion, GT-KO piPSC may provide an excellent immortal cell source for the generation of pigs with complex genetic modifications for xenotransplantation, somatic cell nuclear transfer, and chimera formation. References 1. COOPER DK, BOTTINO R, SATYANANDA V, WIJKSTROM M, TRUCCO M. Toward clinical islet xenotransplantation - are revisions to the IXA guidelines warranted? Xenotransplantation 2013; 20: 68–74. 2. SPIEZIA L, BOLDRIN M, RADU C et al. Thromboelastographic evaluation of coagulative profiles in pig-to-monkey kidney xenotransplantation. Xenotransplantation 2013; 20: 89–99. 3. JIE Y, LIU L, PAN Z, WANG L. Survival of pig-to-rhesus corneal xenografts prolonged by prior donor bone marrow transplantation. Mol Med Rep 2013; 7: 869–874. 4. MUELLER KR, BALAMURUGAN AN, CLINE GW et al. Differences in glucose-stimulated insulin secretion in vitro of
195
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5.
6.
7.
8.
196
islets from human, nonhuman primate, and porcine origin. Xenotransplantation 2013; 20: 75–81. VAKHSHITEH F, ALLAUDIN ZN, MOHD LILA MA, HANI H. Size-related assessment on viability and insulin secretion of caprine islets in vitro. Xenotransplantation 2013; 20: 82–88. TERAOKA Y, IDE K, MORIMOTO H, TAHARA H, OHDAN H. Expression of recipient CD47 on rat insulinoma cell xenografts prevents macrophage-mediated rejection through SIRPalpha inhibitory signaling in mice. PLoS ONE 2013; 8: e58359. BRADY JL, SUTHERLAND RM, HANCOCK M et al. Anti-CD2 producing pig xenografts effect localized depletion of human T cells in a huSCID model. Xenotransplantation 2013; 20: 100–109. SHEN Z, YE W, TEN X. Suppression of NF-kappaB p65 expression attenuates delayed xenograft rejection. Xenotransplantation 2013; 20: 123–130.
9. ZHANG C, WANG L, ZHONG S et al. Over-expression of heme oxygenase-1 does not protect porcine endothelial cells from human xenoantibodies and complement-mediated lysis. J Huazhong Univ Sci Technolog Med Sci 2013; 33: 102–106. 10. LI P, ESTRADA JL, BURLAK C, TECTOR AJ. Biallelic knockout of the alpha-1,3 galactosyltransferase gene in porcine liver-derived cells using zinc finger nucleases. J Surg Res 2013; 181: e39–e45. 11. LIU Y, YANG JY, LU Y et al. Alpha-1,3-galactosyltransferase knockout pig induced pluripotent stem cells: a cell source for the production of xenotransplant pigs. Cell Reprogram 2013; 15: 107–116.
Xenotransplantation 2013: 20: 193–196 Printed in Singapore. All rights reserved doi: 10.1111/xen.12040
XENOTRANSPLANTATION
Literature Update
Xenotransplantation literature update, March–April 2013 Schneider MKJ, Seebach JD. Xenotransplantation literature update, March–April 2013. Xenotransplantation 2013: 20: 193–196. © 2013 John Wiley & Sons A/S.
M arten K. J. Schneider1 and J€org D. Seebach2 1
Division of Internal Medicine, Laboratory of Vascular Immunology, University Hospital Zurich, Zurich, 2Department of Internal Medicine, Service of Immunology and Allergology, University Hospital and Medical Faculty, Geneva, Switzerland Key words: galactose-a1,3-galactose – islet cells – pig – xenotransplantation – xenozoonosis Abbreviations: Ab, antibodies; BM, bone marrow; DXR, delayed xenograft rejection; EXTEM, extrinsic coagulation pathways; FIBTEM, function of fibrinogen; GSIS, glucose-stimulated insulin secretion; GT, a1,3-galactosyltransferase; GT-KO, a1,3-galactosyltransferase knockout; hDAF, human decayaccelerating factor; HO-1, Heme oxygenase-1; INS-1E, rat insulinoma cells; INTEM, intrinsic coagulation pathways; LDC, liver-derived cells; mCD47INS-1E, INS-1E cells transfected with mouse CD47; MLR, mixed-lymphocyte reaction; NHP, non-human primate; pEC, porcine endothelial cells; piPSC, pig induced pluripotent stem cells; pNICC, pig neonatal islet cell clusters; ROTEM, whole blood rotation thromboelastometry; siRNA, small interfering RNA; SIRP, signal regulatory protein a; SCNT, somatic cell nuclear transfer; ZFN, zinc finger nucleases Address reprint requests to M arten K. J. Schneider, PhD, Division of Internal Medicine, Laboratory of Vascular Immunology, University Hospital Zurich, Raemistrasse 100, LAB C 7, CH-8091 Zurich, Switzerland (E-mail: [email protected]) Received 17 April 2013; Accepted 17 April 2013
Commentary
In light of the progress in experimental pig islet xenotransplantation and the prospects for clinical trials, Cooper et al. [1] discussed whether revisions to the guidelines proposed by the International Xenotransplantation Association in 2009 are warranted. The authors suggest five major topics of discussion: (i) is there a need for additional experimental work, (ii) which preliminary clinical studies need to have been carried out, (iii) the need for microbiological safety testing of pigs and pig islets, (iv) patient selection for the initial clinical
trial, and finally, (v) on what basis would a trial be considered successful? The authors conclude by stating that the human suffering associated with diabetes is so significant that pig islet xenotransplantation, with its considerable potential as a clinical therapy, should not be regulated to the point that no advances can be made or will be delayed unnecessarily. Preclinical
Coagulopathy in pig-to-primate organ transplantation is a complex process involving coagulation 193
Schneider and Seebach factors, platelets, and phospholipid-bearing cells. In this context, Spiezia et al. [2] examined thromboelastographic profiles by performing whole blood rotation thromboelastometry (ROTEM) in immunosuppressed nephrectomized primates following renal transplantation. Cynomolgus monkeys received lifesupporting kidneys from pigs transgenic for (i) human decay-accelerating factor (hDAF), (ii) human CD39, CD55, CD59, and fucosyltransferase on a a1,3-galactosyltransferase knock-out (GTKO) background, or (iii) hDAF and GT-KO. Three standard ROTEM assays were performed analyzing intrinsic (INTEM) and extrinsic (EXTEM) coagulation pathways and the function of fibrinogen (FIBTEM). All recipients showed progressive prolongation of clotting time in INTEM. Prolonged clot formation times in both INTEM and EXTEM corresponded with decreased platelet counts, whereas a reduction in maximum clot firmness in FIBTEM corresponded with decreased fibrinogen plasma levels. No concordance was seen between clotting time in INTEM and activated partial thromboplastin time or between clotting time in EXTEM and prothrombin time. The authors conclude that the ROTEM analyzer could be a useful and complementary tool to study consumptive coagulopathy in pig-to-primate xenotransplantation studies. Pre-treatment with donor hematopoietic cells may induce transplantation tolerance via mixed chimerism. Jie et al. [3] studied the survival of porcine corneal xenografts in rhesus monkeys pretreated with either cyclophosphamide alone or with cyclophosphamide followed by pig bone marrow (BM) cell transplantation. The mean graft survival time was 18 days in control recipients treated with cyclophosphamide alone. Pretreatment with donor BM increased the mean graft survival to 36 days with minimal inflammatory cell infiltration and a mean chimerism of around 5% after 1 week and less than 1% at 3 weeks. BMtreated recipients also showed less immune reactivity to donor spleen cells in mixed-lymphocyte reactions (MLR) and lower serum immunoglobulin and complement levels as compared to control recipients. In conclusion, corneal xenograft survival may be prolonged by prior BM transplantation. Islets
The suitability and the predictive value of preclinical pig-to-non-human primate (NHP) islet xenotransplantation remain critical issues because of species differences, including the insulin secretory characteristics of islets. Mueller et al. [4] analyzed glucose-stimulated insulin secretion in vitro of islets 194
isolated from humans, NHP, and adult and juvenile pigs. Insulin secretion was measured after perifusion of pancreata with medium at basal glucose followed by high glucose concentrations. The total glucose-stimulated insulin secretion (GSIS) of NHP islets was 3 times higher than that of adult pig islets. Furthermore, the GSIS of human islets was 3 and 30 times higher than that of adult and juvenile pig islets, respectively. The insulin content was similar in human, NHP, and adult pig islets, but much smaller in juvenile pig islets. Thus, although human, NHP, and adult pig islets contain similar amounts of insulin, the significantly higher GSIS of human and NHP islets compared to pig islets suggests the need for increased dosing of islets in pig-to-primate xenotransplantation. Vakhshiteh et al. [5] examined the viability and insulin secretion of caprine islets in vitro. Caprine islet had a diameter between 50 and 250 lm, in which 80% of the total islet yield was defined as small islets with a diameter of 150 lm. At 48 h after isolation, the small islets were more viable than large islets, the latter showing a higher rate of central core necrosis and apoptosis. Small islets secreted almost three times more insulin under both low and high glucose incubation. Thus, smaller caprine islets show a superior quality compared to larger islets under an optimized basal maintenance condition. Immunobiology
Interspecies incompatibility between the signal regulatory protein a (SIRPa) and CD47 results in phagocytosis of xenogeneic cells by host macrophages. Teraoka et al. [6] investigated whether transgenic expression of mouse CD47 in rat insulinoma cells (INS-1E) could inhibit macrophagemediated xenograft rejection. INS-1E cells transfected with mouse CD47 (mCD47-INS-1E) induced SIRPa-tyrosine phosphorylation in mouse macrophages in vitro, whereas control vector cells did not. When injected into streptozotocin-induced diabetic Rag2 / c / mice (T, B, and NK cell deficient), the expression of mouse CD47 on the INS1E cells reduced the susceptibility to phagocytosis by macrophages. Furthermore, injection of mCD47-INS-1E cells induced normoglycemia, which could be prevented by blocking antibodies (Ab) against SIRPa. In conclusion, these results further suggest that the expression of recipient CD47 on xenogeneic donor cells may prevent macrophage-mediated xenograft rejection. CD2 is a costimulation and adhesion molecule expressed on lymphocytes, including T cells. Brady et al. [7] investigated whether secretion of
Xenotransplantation literature update anti-human CD2 Ab by pig neonatal islet cell clusters (pNICC) would induce local immune protection. For this, three forms of a mouse anti-human CD2 Ab, dilimomab (mouse), diliximab (chimeric), and dilizumab (humanized) were utilized. All 3 forms of Ab bound human T cells in vitro, although dilimomab and diliximab exhibited a 300-fold higher avidity than dilizumab. Dilimomab and diliximab, but not dilizumab, inhibited a human anti-pig xenogeneic MLR. When administered systemically in humanized mice, all 3 antiCD2 Ab were able to deplete human CD3+ T cells in vivo without inducing an upregulation of activation markers or a significant cytokine release. Finally, when humanized mice were transplanted with diliximab-transduced pNICC, a depletion of CD3+ T cells at the graft site was observed, leaving the peripheral immune system intact. The authors conclude that a local production of a single Ab against T cells can reduce graft infiltration at the xenograft site, which may reduce the need for conventional, systemic immunosuppression. Delayed xenograft rejection (DXR) involves type II vascular endothelial cell activation, including upregulation of proinflammatory genes. In an attempt to attenuate DXR and to improve the survival of mouse-to-rat heterotopic heart xenografts, Shen et al. [8] used small interfering RNA (siRNA) technology to inhibit NF-jB p65 gene expression in vivo in donor mice. Donor treatment with NFjB siRNA 1 day before transplantation prolonged the median heart graft survival time to 5.4 days compared to 1.7 days for hearts from PBS-treated control donors. Pre-treatment with NF-jB siRNA caused a significant decrease in mRNA levels of NF-jB p65, VCAM-1, ICAM-1, and interleukin-1 at 12 h post-transplantation and a delay of endothelial cell damage. In summary, NF-jB p65 siRNA treatment of donor animals has the potential to delay the emergence of DXR. Heme oxygenase-1 (HO-1) expression has been reported as essential to ensure accommodation in concordant xenotransplantation models. In an approach to establish an accommodation model in vitro, Zhang et al. [9] investigated whether overexpression of HO-1 would protect porcine endothelial cells (pEC) against lysis mediated by human xenoreactive Ab and complement. Overexpression of HO-1 in aortic pEC was achieved by incubation with cobalt-protoporphyrin IX. However, HO-1 overexpression did not protect pEC against lysis mediated by human sera. Therefore, the authors conclude that the induction of HO-1 overexpression alone is not enough to protect pEC from human antibody plus complement-mediated humoral injury.
Donor animals
Engineered zinc finger nucleases (ZFN) have recently been used to generate genetically modified pigs. Li et al. [10] introduced a ZFN pair targeted to exon 8 of the a1,3-galactosyltransferase (GT) gene into adult pig liver-derived cells (LDC) by electroporation. A ZFN activity of 6.5% was detected in LDC, indicating 6.5% mutated alleles in the pooled cell population. GT-KO cells were selected and used as nuclear donors for somatic cell nuclear transfer (SCNT). A total of six fetuses and 13 piglets were produced by SCNT. All fetuses and piglets had biallelic mutations in the ZFN targeted region and were negative for the galactose-a1,3-galactose epitope. In conclusion, biallelic GT gene disruption in LDC was efficiently generated by ZFN. With their long life span and robust growth rate, LDC has the potential to endure multiple sequential genetic modifications in vitro prior to using SCNT, which may accelerate the production of genetically modified pigs for xenotransplantation. The development of highly proliferative porcine induced pluripotent stem cells (piPSC) from GT-KO tissue provides a valuable cell source for complex genetic manipulations. Liu et al. [11] generated GT-KO piPSC by transducing porcine GT-KO fibroblasts with six human reprogramming pluripotency genes (POU5F1, SOX2, NANOG, LIN28, KLF-4, and C-MYC). The piPSC showed classical stem cell morphology and characteristics, expressing the integrated reprogramming genes in addition to endogenous pluripotent markers. The piPSC were highly proliferative and possessed doubling times and telomerase activity similar to human embryonic stem cells. In conclusion, GT-KO piPSC may provide an excellent immortal cell source for the generation of pigs with complex genetic modifications for xenotransplantation, somatic cell nuclear transfer, and chimera formation. References 1. COOPER DK, BOTTINO R, SATYANANDA V, WIJKSTROM M, TRUCCO M. Toward clinical islet xenotransplantation - are revisions to the IXA guidelines warranted? Xenotransplantation 2013; 20: 68–74. 2. SPIEZIA L, BOLDRIN M, RADU C et al. Thromboelastographic evaluation of coagulative profiles in pig-to-monkey kidney xenotransplantation. Xenotransplantation 2013; 20: 89–99. 3. JIE Y, LIU L, PAN Z, WANG L. Survival of pig-to-rhesus corneal xenografts prolonged by prior donor bone marrow transplantation. Mol Med Rep 2013; 7: 869–874. 4. MUELLER KR, BALAMURUGAN AN, CLINE GW et al. Differences in glucose-stimulated insulin secretion in vitro of
195
Schneider and Seebach
5.
6.
7.
8.
196
islets from human, nonhuman primate, and porcine origin. Xenotransplantation 2013; 20: 75–81. VAKHSHITEH F, ALLAUDIN ZN, MOHD LILA MA, HANI H. Size-related assessment on viability and insulin secretion of caprine islets in vitro. Xenotransplantation 2013; 20: 82–88. TERAOKA Y, IDE K, MORIMOTO H, TAHARA H, OHDAN H. Expression of recipient CD47 on rat insulinoma cell xenografts prevents macrophage-mediated rejection through SIRPalpha inhibitory signaling in mice. PLoS ONE 2013; 8: e58359. BRADY JL, SUTHERLAND RM, HANCOCK M et al. Anti-CD2 producing pig xenografts effect localized depletion of human T cells in a huSCID model. Xenotransplantation 2013; 20: 100–109. SHEN Z, YE W, TEN X. Suppression of NF-kappaB p65 expression attenuates delayed xenograft rejection. Xenotransplantation 2013; 20: 123–130.
9. ZHANG C, WANG L, ZHONG S et al. Over-expression of heme oxygenase-1 does not protect porcine endothelial cells from human xenoantibodies and complement-mediated lysis. J Huazhong Univ Sci Technolog Med Sci 2013; 33: 102–106. 10. LI P, ESTRADA JL, BURLAK C, TECTOR AJ. Biallelic knockout of the alpha-1,3 galactosyltransferase gene in porcine liver-derived cells using zinc finger nucleases. J Surg Res 2013; 181: e39–e45. 11. LIU Y, YANG JY, LU Y et al. Alpha-1,3-galactosyltransferase knockout pig induced pluripotent stem cells: a cell source for the production of xenotransplant pigs. Cell Reprogram 2013; 15: 107–116.
