Optimal pig donor selection in islet xenotransplantation: current status and future perspectives

Optimal pig donor selection in islet xenotransplantation: current status and future perspectives 异种胰岛移植中供体猪的筛选：现状与进展 first-author †E-mail:zht0915@163.combobwang75@yeah.net †E-mail:zht0915@163.combobwang75@yeah.net zht0915@163.com corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zht0915@163.combobwang75@yeah.net †E-mail:zht0915@163.combobwang75@yeah.net zht0915@163.com Department of Hepatobiliary Surgery, the First Affiliated Hospital, Medical College, Xi’an Jiaotong University, Xi’an 710061, China Department of Hepatobiliary Surgery, the First Affiliated Hospital, Medical College, Xi’an Jiaotong University, Xi’an 710061, China Islet transplantation is an attractive treatment of type 1 diabetes mellitus. Xenotransplantation, using the pig as a donor, offers the possibility of an unlimited supply of islet grafts. Published studies demonstrated that pig islets could function in diabetic primates for a long time (>6 months). However, pig-islet xenotransplantation must overcome the selection of an optimal pig donor to obtain an adequate supply of islets with high-quality, to reduce xeno-antigenicity of islet and prolong xenograft survival, and to translate experimental findings into clinical application. This review discusses the suitable pig donor for islet xenotransplantation in terms of pig age, strain, structure/function of islet, and genetically modified pig. 猪胰岛细胞异种移植是一种极具潜力治疗糖尿病的方法。其临床应用主要面临两大挑战：高质量的活性胰岛短缺与异种免疫排斥反应。合适的猪供体筛选（年龄、性别、品系、基因表型）是获取充足的高性能胰岛移植物的重要前提，并可有效地减轻胰岛异种抗原性及体内异种排斥反应，进而延长移植物的功能存活时间。在文中，我们总结了异种移植中猪胰岛的相关研究进展，为供体猪的筛选提供了依据；同时表明了无特殊病原体的成年转基因猪可能是目前最佳的异种胰岛供体，有望在未来的临床研究中大规模应用。 Islet Pig Xenotransplantation Donor 胰岛 猪 异种移植 供体 Introduction 1 Pancreatic islet transplantation is emerging as a viable and prospective treatment option for patients with type 1 diabetes mellitus (T1DM), but shortage of donors (islet supply crisis) greatly restricts the development of clinical islet transplantation (Shapiro et al., 2000 2000 ; van der Windt et al., 2012 2012 ). The inability of human donor programs to meet present demands stimulates exploration of alternative sources of islet grafts. Xenotransplantation, using the pig as the source of islet cells, is a potential and promising solution to this dilemma (Cardona et al., 2007 2007 ; Wang et al., 2011 2011 ; Cooper et al., 2013 2013 ; Lee et al., 2013 2013 ; Hu et al., 2014 2014 ). The pig is considered as the most suitable candidate for xenologous islet grafts owing to several advantages including (1) structural similarity of insulin between pig and human, just only one amino acid difference; (2) availability in sufficient quantities (Gray et al., 2004 2004 ; Yonekawa et al., 2005 2005 ; Zhu et al., 2012 2012 ); (3) feasibility in genetic immunomodulation (Klymiuk et al., 2012 2012 ; Yeom et al., 2012 2012 ; Nagaraju et al., 2013 2013 ); (4) lack of amyloid formation (Potter et al., 2010 2010 ); (5) resistance to recurrent autoimmunity (Koulmanda et al., 2003 2003 ); and, (6) few ethical problems associated with slaughter pigs. Nevertheless, initiation of clinical application of pig islets still requires additional progress to overcome several major hurdles: inadequate supply of islet cells with high-quality, xenorejection, and xenosis (Rood et al., 2006 2006 ; Elliott, 2011 2011 ; van der Windt et al., 2012 2012 ; Hu et al., 2014 2014 ). Donor pig age (islet type) contributes much to islet yield and functionality, as well as immunogenicity. Strain and breed of donor pigs are the important parameters influencing the outcome of islet isolation. The production of pathogen-free pigs can partially solve the problem of xenosis. In addition, xenogeneic rejection may be overcome by the current production of genetically modified pigs (Klymiuk et al., 2012 2012 ; Yeom et al., 2012 2012 ). Therefore, in order to form a basis for a breeding project and effectively push islet xenotransplantation into routine clinical practice, a suitable candidate pig must be determined and selected; and a summary of current knowledge about pig donor selection is necessary, which is the purpose of this review. Optimum age of donor pig 1 A significant debate has taken place about whether the most optimal islet grafts for preclinical/clinical xenotransplantation should be from embryonic, fetal, neonatal, young, or adult pigs (Dufrane and Gianello, 2008 2008 ; van der Windt et al., 2012 2012 ) (Table 1 1 ). At present, only islet xenografts isolated from neonatal (2–3 d) and adult pigs ( > 6 months) display the ability to reverse diabetes in non-human primates (NHPs) or humans (Hering et al., 2006 2006 ; Elliott et al., 2007 2007 ; Dufrane et al., 2010 2010 ; Thompson et al., 2012 2012 ). Donor pig age Benefit Drawback  Embryonic Sterile procurement; no isolation procedure; proliferation and maturation in vivo ; relatively reduced immunogenicity; low risk of pathogen transmission Delayed in vivo functionality ( > 3 months); tumorigenicity  Fetal Sterile procurement; easy islet isolation and no purification process; resistance against ischemic injury; proliferation in vivo ; low risk of pathogen transmission No response to glucose in vitro ; delayed in vivo functionality ( > 2 months); ethical problem (need for many donors)  Neonatal Sterile procurement; easy islet isolation and no purification process; proliferation and differentiation in vivo ; resistance against ischemic injury; more responsive to glucose vs. fetal Delayed in vivo functionality ( > 4 weeks); lower islet yields vs. adult  Adult Consistent and high islet yields (possible one pig required per patient); direct islet function and diabetes correction Lack of sterile environment (slaughterhouse); difficult isolation and purification process; high costs Embryonic pig islet tissue 2 Pig embryos can be easily obtained from pregnant sows at precisely-defined stages of their pregnancy. Embryonic pancreatic tissues are then extracted under a light microscope and maintained in culture medium at 4 °C pending transplantation (Eventov-Friedman et al., 2006 2006 ). Usually, only islet tissues harvested at embryonic gestational age 42 d (E42) are regarded as the best choice for xenotransplantation in diabetic rodents or monkeys (with immunosuppression) with several advantages including reduced immunogenicity, long-term proliferative potential, revascularization by host endothelium, and favorable endocrine/exocrine ratio (primarily composed of endocrine tissue with minimal exocrine activity) (Eventov-Friedman et al., 2006 2006 ; Hecht et al., 2009 2009 ; Tchorsh-Yutsis et al., 2009 2009 ; 2011 2011 ). However, the embryonic implants exhibit a delayed insulin response to glucose in vivo , and a period of > 3 months of growth is required to effectively restore normoglycemia (Table 1 1 ) (Eventov-Friedman et al., 2006 2006 ; Hecht et al., 2009 2009 ; Tchorsh-Yutsis et al., 2009 2009 ; 2011 2011 ). In addition, further fine tuning of immunosuppressive strategy, tissue dose, risk of tumorigenesis, and implantation techniques are still to be addressed. Fetal pig islet 2 Fetal pig islet-like cell clusters (ICCs) and neonatal pig islets (NPIs) are immature cells, which can be easily obtained by enzymatic digestion and simple pre-transplantation culture (4–9 d) on account of relative lack of exocrine tissues and concomitant relative abundance of endocrine tissues (Korbutt et al., 1996 1996 ; Sӧderlund et al., 1999 1999 ; Rajotte, 2008 2008 ). In general, fetuses at 66–86 d of gestation are used for islet graft preparation since these ICCs possess better survival potential (Groth et al., 1994 1994 ; Georges et al., 2002 2002 ). Both ICCs and NPIs are also resistant to ischemic and inflammatory damages during the isolation process, which makes islet recovery more efficient. However, the main problem with ICCs is the need for long-term maturation (2–3 months) to achieve in vivo functionality (Hardikar et al., 2002 2002 ). A second disadvantage is the relatively high expression of galactose-α-1,3-galactose (Gal) on the surface of ICCs, rendering wild-type (WT) pig ICCs more susceptible to xenorejection than adult pig islets (APIs), which express negligible amounts of Gal epitope (only 5% of total cells) (Bennet et al., 2000 2000 ; Rayat et al., 2003 2003 ; Dor et al., 2004 2004 ; Komoda et al., 2004 2004 ). In non-diabetic cynomolgus monkeys, pig ICCs transplanted under kidney capsules were almost completely destroyed by immune rejection within 12 d post-transplantation (Sӧderlund et al., 1999 1999 ). Although a previous study demonstrated that the transplanted pig ICCs could survive in the human body (Groth et al., 1994 1994 ), up until now, the sustained long-term function of ICCs has not been documented in diabetic primates. Furthermore, an increasing improvement in achieving good outcomes in API isolation is stimulating a move away from ICCs in preclinical studies. Neonatal pig islet 2 Generally, newborn pigs (1–5 d old) are selected for neonatal islet cell preparation. After in vitro culture (usually 4–9 d), tissue from one neonatal pancreas can yield about 50 000 NPI aggregates, consisting of fully differentiated pancreatic endocrine cells (approximately 35%) and endocrine precursor cells (approximately 57%) (Korbutt et al., 1996 1996 ; Rajotte, 2008 2008 ; Dufrane and Gianello, 2009 2009 ). NPIs are able to functionally correct hyperglycemia in diabetic recipients, which is mainly due to β cell proliferation and/or striking differentiation of precursor cells into β cells (Korbutt et al., 1996 1996 ; Yoon et al., 1999 1999 ; Trivedi et al., 2001 2001 ; Nielsen et al., 2003 2003 ). Though insulin secretion is also delayed after transplantation in vivo , the maturation time of NPIs ( > 4 weeks) is significantly shorter than that of ICCs ( > 2 months) (Table 1 1 ). The origin of xeno-antigenicity of pig NPIs is mainly N -linked sugars including sialic acid and α-Gal antigens (up to 11%–19%) (Rayat et al., 2003 2003 ; Omori et al., 2006 2006 ); however, the transplanted NPIs induce a lower T-cell response than APIs in patients with insulin-dependent diabetes mellitus (Bloch et al., 1999 1999 ). Recently, the growing demonstrations of prolonged diabetes reversal after transplantation of WT NPIs in immunosuppressed primates make neonatal pig an alternative source of islet grafts for future clinical application (Cardona et al., 2006 2006 ; Elliott et al., 2007 2007 ; Thompson et al., 2011a 2011a ; Wang et al., 2011 2011 ). Adult and young pig islets 2 Adult pig is regarded as the primary donor source of islet xenografts, which can supply an adequate amount of viable islet cells and start functioning immediately after implantation (O'Neil et al., 2001 2001 ; Dufrane et al., 2006 2006 ). More than 200 000 islet cells with high purity (80%–95%) can be isolated from an adult pig donor (Ricordi et al., 1990 1990 ; Inoue et al., 1992 1992 ). Normally, insulin independence in diabetic NHPs can be achieved when a sufficient islet cell mass (25 000–100 000 islet equivalents (IEQ)/kg recipient body weight) is implanted (Hering et al., 2006 2006 ; Casu et al., 2008 2008 ; van der Windt et al., 2009 2009 ; Thompson et al., 2011a 2011a ; 2012 2012 ), which requires pooling of islets from 2–4 adult pig donors or 7–10 neonatal pig donors (Korbutt, 2009 2009 ). Generally, adult pigs aged > 2 years (particularly retired breeders) are preferred as suitable donors, providing a greater number of islets with intact morphology, large size (150–200 μm), and good structure (Dufrane et al., 2005a 2005a ; 2005b 2005b ; Bottino et al., 2007 2007 ). Conversely, pancreata from neonatal or young pigs ( < 6 months) are essentially composed of small islets with a diameter of 50–100 μm (Korbutt et al., 1996 1996 ; Dufrane et al., 2005a 2005a ). In large islets, the centralized structure for both collagen and capillaries can effectively reduce collagenase-induced injury and promote post-transplant revascularization. Although several studies reported poor insulin secretion or low islet recovery from isolated APIs in vitro (Nielsen et al., 2002 2002 ; Yonekawa et al., 2005 2005 ; Rijkelijkhuizen et al., 2006 2006 ), the transplanted APIs still showed strong ability to restore normoglycemia in diabetic recipients in vivo (Nielsen et al., 2002 2002 ; Yonekawa et al., 2005 2005 ; Rijkelijkhuizen et al., 2006 2006 ). Thus, APIs possess a better potential for functional engraftment and prolonged survival in islet xenotransplantation. Selection of pig strain 1 Pig islets are also considered more elusive on account of striking fragility, irregular shape, and quick dissociation into small aggregates or single cells during the isolation process (Bottino et al., 2007 2007 ). Further, several studies have demonstrated great variability of islet capsule, size, and yield from various pig donors (Heiser et al., 1994 1994 ; Kirchhof et al., 1994 1994 ; Ulrichs et al., 1995 1995 ; Meyer et al., 1997 1997 ; Kim et al., 2007 2007 ; Kinasiewicz et al., 2011 2011 ). All these indicate that pig breed plays a vital role in islet quantity and function (Prabhakaran and Hering, 2008 2008 ). Despite several years of investigation, no real consensus exists regarding the most optimal pig strain to provide the substantial amount of viable and functional islet grafts required for preclinical/clinical xenotransplantation. Pig strain selection based on peri-insular matrix 2 An important aspect that may greatly influence islet quality and yield is the amount of extracellular matrix proteins (EMPs) surrounding the islet as a “capsule” which is subject to digestion by the enzyme collagenase (Stendahl et al., 2009 2009 ). Therefore, the peri-insular matrix (“islet capsule”) has a protective effect for the fragile islets. However, compared with humans and rats, the islet capsule in pigs is poorly developed, with very little peri-insular matrix present (van Deijnen et al., 1992 1992 ). Moreover, only 25% of all pig islets are encompassed by the collagen capsule, which covers > 75% of islet surfaces (Ulrichs et al., 1995 1995 ). The major components of the islet capsule are collagen types I, III, IV, laminin, and fibronectin (Meyer et al., 1997 1997 ). While disparity of expression of EMPs among individual pigs in the same strain is minimal, the disparity between different strains is considerable. In Hampshire (HA) and Duroc (DU) breeds, both the perimeter and thickness of islet capsule were significantly lower in comparison with other breeds (e.g., Landrace, Pietrain (PI) pigs) (Kirchhof et al., 1994 1994 ). Among the tested breeds including DU, German Landrace (GL), Deutsches Edelschwein (DE), and hybrid pigs, the overall expression of EMPs appeared to be highest in GL breed and lowest in DU breed; furthermore, only the expression of collagen type I in GL breed was comparable to the human expression (Meyer et al., 1998 1998 ). Of importance, whether in minipig bred or market pigs, older individuals always seemed to display much more EMPs in the islet capsule than younger ones (Meyer et al., 1997 1997 ; 1998 1998 ). However, recent study showed that no significant differences between adult (12–78 months) and juvenile (6–12 months) pigs were detected in islet encapsulation (Hilling et al., 2010 2010 ). Hence, the influence of other matrix elements and collagen subtypes should be further addressed in pig donor selection. In different pig strains, determination of EMP composition and expression level will greatly help to select and develop specific enzyme mixtures to improve islet integrity and yield. Pig strain selection based on islet morphology 2 Morphological screening before isolation is necessary to select eligible donor pigs for acquiring a large number of islets with high-quality, reducing variance of isolation outcomes, and raising economic efficiency. Many studies indicated that a pancreas containing round-shape, well-demarcation, and large-size ( > 100 μm diameter) islets could offer the greatest potential for successful isolation results (Mundwiler et al., 1994 1994 ; Krickhahn et al., 2002 2002 ; Gazda et al., 2005 2005 ). Histomorphometry study conducted by Kirchhof et al. ( 1994 1994 ) showed that GL and German Large White (GLW) pigs were more suitable islet donor breeds than DU, HA, PI, and WT pigs. The GL, GLW, and WT pigs displayed much greater islet numbers per area compared with DU and HA. However, the islet size in situ was smaller in WT boars than in all other breeds. Ulrichs et al. ( 1995 1995 ) further demonstrated that the majority (59.8%–68.9%) of islets were 50–100 μm in diameter in seven domestic races. Nevertheless, GL pigs revealed the greatest number (2%) of large islets (250–300 μm and > 300 μm), and the greatest (up to 3.4%) islet volume density (IVD; percent endocrine tissue of the total pancreas mass) of all pig breeds. By contrast, previous histological analysis of Kinasiewicz et al. ( 2011 2011 ) suggested that PI pigs of 100 kg and market-weight pigs of 200 kg were preferable donors for islet grafts, which exhibited the largest value (range 1.20%–1.44%) of IVD. In addition, the PI pigs showed a greater number of islets/cm 2 than Landrace pigs ((425.45±111.7) islets/cm 2 vs. (315.33±36.59) islets/cm 2 ). Kim et al. ( 2007 2007 ) revealed that pancreata from adult Chicago Medical School (CMS) miniature (CM) pigs contained more islets with a diameter of 150–200 μm (11.2%) than those from adult Prestige World Genetics (PWG) miniature (PM) pigs (2.4%), and more islets with a diameter of 200–250 μm (5.3%) than those from PM (1.6%) or adult market pigs (1.3%; from slaughterhouses); moreover, the mean IVD percentages of CM pigs were dramatically higher (3.71%) than those of PM (1.5%) or market pigs (2.16%). Recently, Wuzhishan (WZS) miniature pigs were considered to be a feasible source of islet grafts with advantageous morphological features of denser islet capsule and larger splenic lobe of pancreas (Jiang et al., 2012 2012 ). More importantly, the anatomical structure of the WZS miniature pig pancreas was more similar to human pancreas than that of market pigs. Pig strain selection based on islet yield 2 Only few studies on pig islet isolation have directly addressed the influence of pig strain on islet yield. Islet yields (pre-purification) from market-weight (100 kg) purebred offspring of Landraces (two specific Landrace boars and Landrace sows) were much higher ((9350±630) IEQ/g and (4800±760) IEQ/g, respectively) than yields from hybrid offspring of the same boars and Yorkshire sows ((6150±780) IEQ/g and (2990±520) IEQ/g, respectively) (Cavanagh et al., 1998 1998 ). In the study by Ulrichs et al. ( 1995 1995 ), GL pigs showed the highest numbers (22 786±3739) of theoretically calculated IEQ/g pancreas; however, only (687±515) IEQ/g could be isolated (just 3.0% average yield), which was much lower than the yields of PI pigs ((2173±301) IEQ/g). Heiser et al. ( 1994 1994 ) further confirmed that purebred PI pigs provided higher islet yields ((2180±317) IEQ/g) than GL pigs ((606±379) IEQ/g), minipigs “Troll” ((691±357) IEQ/g), and adult hybrid pigs (mean 1190 IEQ/g), and the improved islet viability was also observed. By contrast, Sabat et al. ( 2003 2003 ) showed that islet yields in pancreata were less than 1000 IEQ/g from PI and Yokshire strains. The great variability of islet yield in the same breed is possibly related to variations in other factors including pancreas procurement, cold preservation, and isolation procedure/technique. Recently, an extremely high yield ((9589±2823) IEQ/g) of well-functioning islets was obtained from CM pigs. Although the in vitro and in vivo functions of isolated islets showed no difference among experimental groups, the islet yields of adult CM pigs ( > 12 months) were significantly higher than those of adult PM pigs ((1752±874) IEQ/g), adult market pigs ((1931±947) IEQ/g), or young (6–7 months) CM pigs ((3460±1985) IEQ/g). Also, after digestion, islet yields in pancreata from adult inbred WZS miniature pigs (1-year-old, weighing 25–30 kg) were much higher than the yields from market pigs ((6078±1105) IEQ/g vs. (2500±625) IEQ/g); moreover, the isolated islets from WZS pigs could efficiently respond to glucose challenge in vitro and in vivo (Jiang et al., 2012 2012 ). In spite of reproductive history being a preferable donor parameter in miniature and market strain, male pigs usually provided higher islet yields compared with breeder miniature strain pigs (Kim et al., 2009 2009 ). In addition, in comparison with the market-raised breeds (e.g., Landrace) from slaughterhouses, the use of specific pathogen-free (SPF) miniature pigs as islet donors has additional advantages, including axenic and standardized pancreas procurement, short warm/cold ischemia time, and minimal possibility of zoonosis. Taken together, the use of the SPF miniature breed may permit great advances in the future preclinical/clinical application of pig islet xenotransplantation. Genetically engineered pig: an alternative selection of islet graft 1 WT pig (e.g., DU, GLW, Landrace, and miniature) islet xenotransplantation has successfully reversed diabetes in primate models (Hering et al., 2006 2006 ; Dufrane et al., 2010 2010 ; Thompson et al., 2012 2012 ). However, there is a significant loss of the transplanted cells on exposure to fresh recipient blood and heterologous antigen, known as instant blood-mediated inflammatory reaction (IBMIR), as well as subsequent humoral and T-cell response that results in xenorejection of islet grafts (Nilsson, 2008 2008 ; Ekser and Cooper, 2010 2010 ). The immunological rejection, which poses negative impacts on islet engraftment and survival, is still a major obstacle for clinical application of pig islet xenotransplantation. Currently, the genetic engineering of pig seems to play a vital role in overcoming these problems, and the experience with transplantation of islets from these pigs into primates is steadily increasing (Table 2 2 ). The gene-modified pigs offer a number of potential advantages in reducing the IBMIR-related graft loss, protecting the islets from cellular rejection, minimizing the number of required islets, mitigating side effects of systemic immunosuppression, and improving islet activity and survival (Mandel et al., 1997 1997 ; Komoda et al., 2005 2005 ; Tai et al., 2007 2007 ; D'Apice and Cowan, 2008 2008 ; Thompson et al., 2011b 2011b ; Nagaraju et al., 2013 2013 ). Up to now, there are three main strategies to induce genetic modification in pigs, including knocking down/out genes responsible for major antigens targeted by natural anti-pig antibodies, knocking in human complement- and coagulation-regulatory genes, as well as transferring T-cell co-stimulation blocking genes (Table 2 2 ). In diabetic primates, humoral rejection can be fully suppressed by crossbreeding of Gal gene-knockout (GTKO) pigs with transgenic pigs expressing human complement regulators such as CD46, CD59, and human decay-accelerating factor (hDAF, CD55) (Diamond et al., 2001 2001 ; Liu et al., 2007 2007 ; le Bas-Bernardet et al., 2011 2011 ; Chen et al., 2014 2014 ). In a humanized mouse model, islet grafts from cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA4Ig) transgenic pigs display the potential to normalize glucose homeostasis and inhibit cellular rejection (Klymiuk et al., 2012 2012 ). Transgenic expression of human heme oxygenase-1 (HO-1) can protect pig islet cells from ischemia/reperfusion injury, and/or acute rejection mediated by inflammatory cytokines (Yeom et al., 2012 2012 ). Additional pig genetic modification, knocking-out tissue factor (TF) and over-expressing human antithrombotic gene (CD39/thrombomodulin), will certainly prevent the occurrence of IBMIR and coagulation dysfunction (Ekser and Cooper, 2010 2010 ). hDAF (CD55): human decay-accelerating factor; GnT-III: N -acetylglucosaminyltransferase-III; HAR: hyperacute rejection; hCD46: human complement-regulatory protein; GTKO: Gal gene-knockout; hCD59: human CD59; hHT: human α(1,2)fucosyltransferase; Cyno: cynomolgus monkey; Rhesus: rhesus macaques; CsA: cyclosporine A; CP: cyclophosphamide; BQR: brequinar; MMF: mycophenolate mofetil; ATG: antithymocyte globulin; anti-CD154: anti-CD154 monoclonal antibody; CTLA4Ig: cytotoxic T lymphocyte antigen 4-immunoglobulin; anti-LFA-1: anti-lymphocyte function-associated antigen-1 monoclonal antibody Study Genetic engineering and function feature Donor age Diabetic recipient Immunosuppressive therapy Islet xenograft survival Mandel et al., 1997 1997 hDAF (CD55) transgenic pigs; complement regulation Fetal (islet tissues), 70–90 d Cyno Group 1: none; Group 2: CsA+steroids+CP or BQR Group 1: 3–7 d; Group 2: >40 d Komoda et al., 2005 2005 GnT-III transgenic pigs; prevention of HAR Adult, >1 year Cyno ( n =4) None 1–5 d ((3.5±0.87) d) van der Windt et al., 2009 2009 hCD46 transgenic pigs; complement regulation Adult, 2 years Cyno ( n =5) MMF+ATG+anti-CD154 87–396 d Thompson et al., 2011b 2011b GTKO pigs; resistance to complement/preformed xenoantibody mediated injury Neonatal, 1–4 d Rhesus ( n =5) CTLA4Ig+anti-CD154+anti-LFA-1+MMF 50–249 d Chen et al., 2014 2014 GTKO/hCD55/hCD59/hHT transgenic pigs; combinations of genetic modifications Neonatal Baboon ( n =3) ATG+MMF+tacrolimus Significant xenoantibody response took place at 28 d after transplantation Given that genetically modified pig islets provide immunological benefits compared with those from WT, it is essential to determine the functionality of these mutations, particularly in regard to glucose metabolism and insulin secretion. As demonstrated by Casu et al. ( 2010 2010 ), both WT (Landrace) and GTKO pigs had similar basal and stimulated blood glucose levels, as well as analogous reduction in blood glucose during the insulin tolerance test and intravenous glucose tolerance test. The study further demonstrated the good functionality of isolated GTKO islets in vitro , since stimulation indices of insulin secretion were similar and basal insulin secretion was higher than with WT islets. Furthermore, genetic engineering of the pigs (deletion of Gal epitope and/or the addition of human transgenes) also did not cause any abnormalities in hematologic, biochemical, or coagulation indicators, which might impact on animal welfare, and even possibly modified these parameters closer to human values (Ekser et al., 2012 2012 ). Additionally, the risk of microbiological infection is still a possibility in xenotransplantation (Mueller et al., 2011 2011 ). Most microorganisms can be eliminated by rearing the pig donors under qualified and SPF conditions. However, pig endogenous retrovirus (PERV) represents a special concern because it can be integrated into the pig genome (van der Laan et al., 2000 2000 ; Patience et al., 2001 2001 ; Denner, 2008 2008 ). In clinical trials of pig islet xenotransplantation, although no evidence of PERV transmission was found in patients with T1DM after long-term follow-up (Valdes-Gonzalez et al., 2007 2007 ; 2010 2010 ; Wang et al., 2011 2011 ), PERV infection remains a key issue. Previous study recommended domestic GL pig as the preferred islet source, since no evidence for PERV release (either as mRNA, or as protein or viral particles) was observed in the isolated islet cells (Irgang et al., 2008 2008 ). In recent years, the development of RNA interference technology targeting PERV substantially mitigated the possible risk of retrovirus contamination; these transgenic pigs could maintain undetectable levels of PERV expression for a long term ( > 3 years) (Ramsoondar et al., 2009 2009 ; Semaan et al., 2012 2012 ). With the emergence of other pathogens (e.g., pig lymphotropic herpesvirus, pig circovirus, and hepatitis E virus), gene-modified donor pigs will provide one of the best bases for solving bio-safety problems. Conclusions 1 Considering that encouraging achievements in improving islet quality/quantity and extending islet survival have been obtained by pre-isolation donor selection, it is necessary to conduct more comprehensive metabolism, lineage, and genotyping analyses to form a basis for the selection or production of appropriate pig donors for fewer variable, lower microbiological, and more economical and effective islet preparation. 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Progress in isolation and purification of porcine islets . Chin J Reparat Reconstr Surg , 2012 . 26 ( 8 ): 1012 - 1016 . (in Chinese) . Progress in isolation and purification of porcine islets Compliance with ethics guidelines Hai-tao ZHU, Liang YU, Yi LYU, and Bo WANG declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1400120 Project supported by the National Natural Science Foundation of China (No. 30700772) 2014-04-28 2014-07-25 2014-07-21 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2014 2014 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 08 15