1. Basic research
Studies of discordant xenografts, such as guinea‐pig to rat and pig to human, were started more than 30 years ago. The first subject to be addressed was the mechanism of discordant xenograft rejection , i.e., hyperacute rejection. After verification of the reaction of the complement system, xeno‐specific glycoantigens in pig‐to‐human xenotransplantation, such as the α‐gal epitope, were then studied [2, 3], followed by the other immune systems.
Therefore, the first gene modification on pigs was focused on issues related to complement regulatory proteins (CRPs) such as Membrane Cofactor Protein (MCP, CD46), Decay Accelerating Factor (DAF, CD55), and CD59 . DAF (CD55)‐transgenic pigs were then first produced in 1994 [5, 6], followed by other CRP‐transgenic pigs [7, 8]. On the other hand, different from the mouse system, pig embryonic stem (ES) cells had not yet been established. Therefore, other methods for reducing the α‐gal epitope, such as the overexpression of α1,2 fucosyltransferase , End‐β‐GalC , and GnT‐III [11, 12], were examined .
Fortunately, the gene targeting technique was combined with (fetus) fibroblasts and the nuclear transfer techniques, resulting in the successful development of α‐gal knockout (KO) pigs in 2002 .
Many kinds of CRP and glycoantigens  are now being nominated for transgenic and knockout, respectively, based on improved genetic engineering (GE) techniques. The next obstacle to xenograft is cellular rejection by the innate immune system, which comprises natural killer (NK) cells and monocytes/macrophages.
Strategies for suppressing NK function on the pig cells have been extensively examined. HLA‐class Ia molecules, such as HLA‐C, but also class Ib, HLA‐G1 [16, 17] and –E [18, 19], has been considered in the case of the transgenic pig. In addition, changing the pattern of glycosylation on the surfaces of pig cells is also a reasonable strategy [20–24].
The issue of how to regulate monocytes/macrophages, it is now known that only CD47  binds to SIRPα on the surface of monocytes/macrophages that contains the immune receptor tyrosine–based inhibition motif (ITIM). Therefore, until quite recently, other routes to the downregulation of monocytes/macrophages have not been not well studied.
However, especially in these past 5 years, additional key molecules for suppressing monocytes/macrophages have clearly been identified. Thus, for example, HLA class Ib, HLA‐G1 , and –E  were identified as having a suppressive function not only for NK cells but also for monocytes/macrophages as well. Monocytes/macrophages actually have common receptors in common with NK cells. In addition, changes in glycoantigens, such as the overexpression of the α2,6‐sialic acids, as well as other methods , also function to downregulate monocytes/macrophages .
In addition, meanwhile, the many strategies for suppressing the movement of T cells have been proposed, such as class II dominant negative (CIIDN) [30, 31], HLA class I‐KO , FasL, and tumor necrosis factor receptor I IgG‐Fc (TNFRI‐Fc) . In addition to immunological studies, studies of coagulation systems, such as thrombomodulin (TM), the tissue factor pathway inhibitor (TFPI), the endothelial cell protein C receptor (EPCR), CD39 and CD73, and anti‐apoptotic and anti‐inflammatory genes, such as heme oxygenase 1 (HO‐1) and A20, have also progressed.
2. Genetic engineering
The most progress during these past 5 years involves gene targeting technology. One involves zinc‐finger nucleases (ZFN)  and is continued by the transcription activator‐like effector nuclease (TALEN)  method, and finally the clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/CAS) . These methods had brought about a revolution in certain aspects of gene‐targeting technology. Therefore, the KO of a special gene became extremely easier than in the past. Not only α‐gal KO but also cytidine monophospho‐
In addition, as a new strategy, attempts are being made to retain the fixed expression of transgenes, because the transgenic expression of each gene was sometimes not stable over generations. Knockin (KI) human genes to the ROSA locus of the pig genome became of interest .
3. Preclinical study
Surprisingly, heterotopic hearts from the GE‐pigs continued to beat for almost 2.5 years, when implanted in the monkey abdomen , and pig life‐supporting kidney could function for nearly 1 year in monkeys .
Concerning islet cells, trials in which islet cells from GE‐pigs are transplanted in monkeys have been reported. Several groups have reported survival periods of more than 1 year, using adult pig islets (APIs) [46, 47]. Generally speaking, results using neonatal porcine islet‐like cell clusters (NPCCs) were worse than those using APIs. It is noteworthy that one group reported a survival of over 600 days using API from wild‐type pigs , suggesting that the combination of API from GE‐pigs and excellent drug therapy may permit islets to survive for more than 2–3 years.
4. Porcine endogenous retrovirus (PERV)
Concerning the problems associated with the porcine endogenous retrovirus (PERV) , new studies have appeared during these past 5 years, trials to knockout all PERV genes from the pig genome were done using the new techniques, ZFN and CRISPR [50, 51]. However, success has not yet been achieved.
However, in spite of hundreds of patients undergoing transplantation of pig organs or tissue, no reports have appeared of suffering . The controversy associated with the risks of PERV has already been minimized.
5. For clinic
In Japan, in 2014, a law related to the pig cell (islets) transplants was passed. In 2016, the guidelines for xenotransplantation were revised. At this moment, clinical detection systems for identifying infectious diseases from pig tissue are being improved. Thus, it has already become possible to start clinical pig islets transplantation. In addition, in the USA, the councilors of the International Xenotransplantation Association (IXA) will be holding meetings with FDA‐staff concerning the start of clinical trials in this September at IXA2017 in Baltimore. We are hoping for positive results from this meeting.
In the near future, possibly within 1 or 2 years, in Japan, the USA, and Europe, some clinical trials involving the use of genetic‐modified pigs or microencapsulation pancreatic islets in xenotransplantation will start.
Miyagawa S, Hirose H, Shirakura R, et al. The mechanism of discordant xenograft rejection. Transplantation. 1988; 46:825-830
Galili U, Swanson K. Gene sequences suggest inactivation of alpha‐1,3‐galactosyltransferase in catarrhines after the divergence of apes from monkeys. Proceedings of the National Academy of Sciences of the United States. 1991; 88:7401-7404
Cooper DKC. Depletion of natural antibodies in non‐human primates – A step towards successful discordant xenografting in humans. Clinical Transplantation. 1992; 6:178-183
Miyagawa S, Yamamoto A, Matsunami K, et al. Complement regulation in the GalT KO era. Xenotransplantation. 2010; 17:11-25
Dalmasso AP, Vercellotti GM, Platt JL, et al. Inhibition of complement‐mediated endothelial cell cytotoxicity by decay‐accelerating factor. Potential for prevention of xenograft hyperacute rejection. Transplantation. 1991; 52:530-533
Cozzi E, White DJG. The generation of transgenic pigs as potential organ donors for humans. Nature Medicine. 1995; 1:964-966
Loveland BE, Milland J, Kyriakou P, et al. Characterization of a CD46 transgenic pig and protection of transgenic kidneys against hyperacute rejection in non‐immunosuppressed baboons. Xenotransplantation. 2004; 11:171-183
Takahagi Y, Fujimura T, Miyagawa S, et al. Production of alpha‐1,3‐galactosyltransferase gene knockout pigs expressing both human decay‐accelerating factor and N‐acetylglucosaminyltransferase III. Molecular Reproduction and Development. 2005; 71:331-338
Sandrin MS, Fodor WL, Mouhtouris E, et al. Enzymatic remodelling of the carbohydrate surface of a xenogenic cell substantially reduces human antibody binding and complement‐mediated cytolysis. Nature Medicine. 1995; 1:1261-1267
Ogawa H, Muramatsu H, Kobayashi T, et al. Molecular cloning of endo‐beta‐galactosidase C and its application in removing alpha‐galactosyl xenoantigen from blood vessels in the pig kidney. Journal of Biological Chemistry. 2000; 275:19368-19374
Tanemura M, Miyagawa S, Ihara Y, et al. Significant downregulation of the major swine xenoantigen by N‐acetylglucosaminyltransferase III gene transfection. Biochemical and Biophysical Research Communications. 1997; 235:359-364
Miyagawa S, Murakami H, Takahagi Y, et al. Remodeling of the major pig xenoantigen by N‐acetylglucosaminyltransferase III in transgenic pig. Journal of Biological Chemistry. 2001; 276:39310-39319
Byrne GW, Du Z, Stalboerger P, et al. Cloning and expression of porcine β1,4 N‐acetylgalactosaminyl transferase encoding a new xenoreactive antigen. Xenotransplantation. 2014; 21:543-554
Dai Y, Vaught TD, Boone J, et al. Targeted disruption of the alpha‐1,3‐galactosyltransferase gene in cloned pigs. Nature Biotechnology. 2002; 20:251-255
Miyagawa S. Xenotransplantation and glycomedicine. In: Kamerling JP, editor. Comprehensive Glycoscience. Elsevier Press; 2007. pp. 533-553.
Sasaki H, Xu XC, Mohanakumar T. HLA‐E and HLA‐G expression on porcine endothelial cells inhibit xenoreactive human NK cells through CD94/NKG2‐dependent and ‐independent pathways. Journal of Immunology. 1999; 163:6301-6305
Matsunami K, Miyagawa S, Nakai R, et al. The possible use of HLA‐G1 and G3 in the inhibition of NK cell‐mediated swine endothelial cell lysis. Clinical & Experimental Immunology. 2001; 126:165-172
Matsunami K, Miyagawa S, Nakai R, et al. Modulation of the leader peptide sequence of the HLA‐E gene up‐regulates its expression and down‐regulates natural killer cell‐mediated swine endothelial cell lysis. Transplantation. 2002; 73:1582-1589
Matsunami K, Kusama T, Okura E, et al. Involvement of position‐147 for HLA‐E expression. Biochemical and Biophysical Research Communications. 2006; 347:692-697
Miyagawa S, Nakai R, Yamada M, et al. Regulation of natural killer cell‐mediated swine endothelial cell lysis through genetic remodeling of a glycoantigen. Journal of Biochemical. 1999; 126:1067-1073
Inverardi L, Clissi B, Stolzer AL, et al. Human natural killer lymphocytes directly recognize evolutionarily conserved oligosaccharide ligands expressed by xenogeneic tissues. Transplantation. 1997; 63:1318-1330
Artrip JH, Kwiatkowski P, Michler RE, et al. Target cell susceptibility to lysis by human natural killer cells is augmented by alpha‐(1,3)‐galactosyltransferase and reduced by alpha‐(1,2)‐fucosyltransferase. Journal of Biological Chemistry. 1999; 274:10717-10722
Baumann BC, Schneider MK, Lilienfeld BG, et al. Endothelial cells derived from pigs lacking Gal alpha‐(1,3)‐gal: No reduction of human leukocyte adhesion and natural killer cell cytotoxicity. Transplantation. 2005; 79:1067-1072
Christiansen D, Mouhtouris E, Milland J, et al. Recognition of a carbohydrate xenoepitope by human NKRP1A (CD161). Xenotransplantation. 2006; 13:440-446
Ide K, Wang H, Tahara H, et al. Role for CD47‐SIRPα signaling in xenograft rejection by macrophages. Proceedings of the National Academy of Sciences of the United States of America. 2007; 104:5062-5066
Esquivel EL, Maeda A, Eguchi H, et al. Suppression of human macrophage‐mediated cytotoxicity by transgenic swine endothelial cell expression of HLA‐G. Transplant Immunology. 2015; 32:109-115
Maeda A, Kawamura T, Ueno T, et al. The suppression of inflammatory macrophage‐mediated cytotoxicity and proinflammatory cytokine production by transgenic expression of HLA‐E. Transplant Immunology. 2013; 29:76-81
Sakai R, Maeda A, Choi T‐V, et al. Human CD200 suppresses macrophage‐mediated xenogeneic cytotoxicity and phagocytosis. Surgery Today. 2017. DOI: 10.1007/s00595-017-1546-2
Maeda A, Kawamura T, Nakahata K, et al. Regulation of macrophage‐mediated xenocytotoxicity by overexpression of alpha‐2,6‐sialyltransferase in swine endothelial cells. Transplantation Proceedings. 2014; 46:1256-1258
Hara H, Witt W, Crossley T, et al. Human dominant‐negative class II transactivator transgenic pigs – effect on the human anti‐pig T‐cell immune response and immune status. Immunology. 2013; 140:39-46
Yun S, Gustafsson K, Fabre JW. Suppression of human anti‐porcine T‐cell immune responses by major histocompati bility complex class II transactivator constructs lacking the amino terminal domain. Transplantation. 1998; 66:103-111
Reyes LM, Estrada JL, Wang ZY, et al. Creating class I MHC‐null pigs using guide RNA and the Cas9 endonuclease. Journal of Immunology. 2014; 193:5751-5757
Park SJ, Cho B, Koo OJ, et al. Production and characterization of soluble human TNFRI‐Fc and human HO‐1(HMOX1) transgenic pigs by using the F2A peptide. Transgenic Research. 2014; 23:407-419
Hauschild J, Petersen B, Santiago Y, et al. Efficient generation of a biallelic knockout in pigs using zinc‐finger nucleases. Proceedings of the National Academy of Sciences of the United States of America. 2011; 108:12013-12017
Miyagawa S, Matsunari H, Watanabe M, et al. Generation of α1,3‐galactosyltransferase and cytidine monophospho‐N‐acetylneuraminic acid hydroxylase gene double‐knockout pigs. Journal of Reproduction and Development. 2015; 61:449-457
Kwon DJ, Kim DH, Hwang IS, et al. Generation of α‐1,3‐galactosyltransferase knocked‐out transgenic cloned pigs with knocked‐in five human genes. Transgenic Research. 2017; 26:153-163
Martens GR, Reyes LM, Butler JR, et al. Humoral reactivity of renal transplant‐waitlisted patients to cells from GGTA1/CMAH/B4GalNT2, and SLA class I knockout pigs. Transplantation. 2017; 101:e86‐e92
Szymczak AL, Workman CJ, Wang Y, et al. Correction of multi‐gene deficiency in vivo using a single ‘self‐cleaving’ 2A peptide‐based retroviral vector. Nature Biotechnology. 2004; 22:589-594
Fisicaro N, Londrigan SL, Brady JL, et al. Versatile co‐expression of graft‐protective proteins using 2A‐linked cassettes. Xenotransplantation. 2011; 18:121-1130
Li S, Flisikowska T, Kurome M, et al. Dual fluorescent reporter pig for Cre recombination: Transgene placement at the ROSA26 locus. PLoS One. 2014; 9:e102455
Cooper DK, Satyananda V, Ekser B, et al. Progress in pig‐to‐non‐human primate transplantation models (1998-2013): A comprehensive review of the literature. Xenotransplantation. 2014; 21:397-419
Cooper DK, Bottino R. Recent advances in understanding xenotransplantation: Implications for the clinic. Expert Review of Clinical Immunology. 2015; 11:1379-1390
Puga Yung GL, Rieben R, Bühler L, et al. Xenotransplantation: Where do we stand in 2016? Swiss Medical Weekly. 2017; 147:w14403
Mohiuddin MM, Singh AK, Corcoran PC, et al. Chimeric 2C10R4 anti‐CD40 antibody therapy is critical for long‐term survival of GTKO.hCD46.hTBM pig‐to‐primate cardiac xenograft. Nature Communications. 2016; 7:11138
Iwase H, Hara H, Ezzelarab M, et al. Immunological and physiological observations in baboons with life‐supporting genetically engineered pig kidney grafts. Xenotransplantation. 2017. Mar; 24(2). DOI: 10.1111/xen.12293. Epub 2017 Mar 17
Bottino R, Wijkstrom M, van der Windt DJ, et al. Pig‐to‐monkey islet xenotransplantation using multi‐transgenic pigs. American Journal of Transplantation. 2014; 14:2275-2287
Hawthorne WJ, Salvaris EJ, Phillips P, et al. Control of IBMIR in neonatal porcine islet xenotransplantation in baboons. American Journal of Transplantation. 2014; 14:1300-1309
Shin JS, Kim JM, Kim JS, et al. Long‐term control of diabetes in immunosuppressed nonhuman primates (NHP) by the transplantation of adult porcine islets. American Journal of Transplantation. 2015; 15:2837-2850
Patience C, Takeuchi Y, Weiss RA. Infection of human cells by an endogenous retrovirus of pigs. Nature Medicine 1997; 3:282-286
Semaan M, Ivanusic D, Denner J, et al. Cytotoxic effects during knock out of multiple porcine endogenous retrovirus (PERV) sequences in the pig genome by zinc finger nucleases (ZFN). PLoS One. 2015; 10:e0122059
Yang L, Güell M, Niu D, et al. Genome‐wide inactivation of porcine endogenous retroviruses (PERVs). Science. 2015; 350:1101-1104
Morozov VA, Wynyard S, Matsumoto S, et al. No PERV transmission during a clinical trial of pig islet cell transplantation. Virus Research. 2017; 227:34-40
Groth CG, Korsgren O, Tibell A, et al. Transplantation of porcine fetal pancreas to diabetic patients. Lancet. 1994; 344:1402-1404
Wang W, Mo Z, Ye B, et al. A clinical trial of xenotransplantation of neonatal pig islets for diabetic patients. J Cent South Univ Med Sci. 2011; 36:1134-1140
Valdés‐González RA, Dorantes LM, Garibay GN, et al. Xenotransplantation of porcine neonatal islets of Langerhans and Sertoli cells: A 4‐year study. European Journal of Endocrinology. 2005; 153:419-427
Elliott RB, Escobar L, Tan PL, et al. Live encapsulated porcine islets from a type 1 diabetic patient 9.5 yr after xenotransplantation. Xenotransplantation. 2007; 14:157-161