To establish the consensus about the conditions for undertaking clinical trials in xenocorneal transplantation in Korea, specific issues regarding the xenocorneal transplantation on ethical and regulatory aspects are addressed, and the guidelines to conduct clinical trial of the xenocorneal transplantation are proposed.

Chapter 1 reviews the key ethical requirements and progress of a Korean regulatory framework for clinical trials of xenocorneal transplantation. Chapters 2–7 provide recommendations on source pigs, quality control of porcine corneal procurement, preclinical efficacy required to justify a clinical trial, strategies to prevent transmission of porcine endogenous retrovirus (PERV), patient selection for clinical trials, and informed consent in xenocorneal transplantation using either cellularized or decellularized porcine graft, which are essentially based on the International Xenotransplantation Association (IXA) islet xenotransplantation consensus statement. The consensus statement of the inclusion criteria for the patients' selection has been made by the executive board members in Korean External Eye Disease Society.

This consensus statement will be a good initiative for Korean Food and Drug Administration to discuss final regulatory guidelines in conducting clinical trials of xenocorneal transplantation in Korea and for International Xenotransplantation Association to develop International Consensus Standards of Xenocorneal Transplantation.

We previously generated humanized chimeric mice by transplanting h-hepatocytes into the livers of the diseased-liver transgenic mouse model with immunodeficient background. These mice with livers mostly replaced by human (h) hepatocytes have been proved to be useful for research on drug metabolism and toxicity and on intrahepatic pathogens such as hepatitis. However, their small body size prohibited collecting sufficient biological samples and made surgical manipulation difficult, which motivated us to produce humanized larger animal(s) bearing h-hepatocytes.

Fischer 344 (F344) rats at 2 weeks of age were administrated with hepatotoxin retrorsine (RS) and then transplanted with syngeneic F344 rat (r)- or h-hepatocytes via the portal vein. The hosts were injected daily with FK506 immunosuppressant. The livers were harvested periodically for determining donor-cell replacement ratios and compared with those of the humanized chimeric mice, and liver-specific mRNA and protein expressions by immunohistochemistry and reverse-transcription PCR.

RS treatment of infant rats inhibited hepatocyte proliferation, resulting in decreased liver weight and megalocytic changes in hepatocytes. R-hepatocytes transplanted into RS-treated rats engrafted into and repopulated the liver at ratios of 16.4 ± 6.7% and 48.3 ± 29.3% at 3 and 6 weeks after transplantation, respectively. H-hepatocytes also engrafted into the rat liver and showed a repopulation ratio of 2.5 ± 1.5% at 3 weeks post-transplantation, which was comparable to the ratio in the humanized chimeric mouse model at least until 3 weeks. Propagated h-hepatocytes in the rat liver expressed hepatocyte-specific mRNA and proteins at least 3 weeks after transplantation.

Xenogeneic hepatocytes were able to engraft rat liver and grow well therein for at least 3 weeks post-transplantation in rats when immunosuppression was combined appropriately with liver injury at comparable levels to the well-characterized humanized chimeric mouse model.

Novel threads of discovery provide the basis for optimism for the development of a stem-cell-based strategy for the treatment of retinal blindness. Accordingly, achievement to suitable cell source with potential-to-long-term survival and appropriate differentiation can be an effective step in this direction.

After derivation of human adipose-derived mesenchymal stem cells (HAD-MSCs), they were stably transfected with a vector containing Turbo-green fluorescent protein (GFP) and JRed to be able to trace them after transplantation. Labeled HAD-MSCs were transplanted into the intact adult rat eye and their survival, integration, and migration during 6 months post-transplantation were assesed.

The transplanted cells were traceable in the rat vitreous humor (VH) up until 90 days after transplantation, with gradual reduction in numbers, their adhesion and expansion capacity after recovery. These cells were also integrated into the ocular tissues. Nonetheless, some of the implanted cells succeeded to cross the blood–retina barrier (BRB) and accumulate in the spleen with time.

The survival of the HAD-MSCs for a period of 90 days in VH and even longer period of up to 6 months in other eye tissues makes them a promising source to be considered in regenerative medicine of eye diseases. However, the potency of crossing the BRB by the implanted cells suggests that use of HAD-MSCs must be handled with extreme caution.

Reactive oxygen species (ROS) and nitric oxide species (NOS) are pivotal after ischemia–reperfusion. However, the role of different cells on the formation of free radical species after xenotransplantation remains elusive. We hypothesized that ROS and NOS formed during hyperacute rejection are dependent on leukocytes, erythrocytes, activated thrombocytes, and Kupffer cells (KCs). To address this issue, we developed a model of xenoperfused rat liver and assessed the relationship between free radical production and graft dysfunction.

Livers from Sprague-Dawley rats were isolated, flushed with cold Ringer solution, and perfused at physically flow rates for 120 min after 1 h of ischemia. The control group was perfused with rat whole blood (n = 9). In the study groups, the livers were perfused with human whole blood, human plasma with erythrocytes, and plasma with erythrocytes and isolated thrombocytes (n = 9/group). In an additional group, gadolinium chloride (GdCl3), a selective Kupffer cell (KC) toxic agent, was applied. Liver damage, hyperacute rejection, and the depletion of KCs were monitored histologically. Liver damage and function were determined by means of liver enzymes, portal pressure, and bile production. Malondialdehyde (MDA), nitric oxide formation, and peroxynitrite concentration, as well as total glutathione (tGSH) level, were measured as indicators for free radical formation and anti-oxidative status.

Significant differences in the MDA, NO, peroxynitrite levels, and GSH levels after reperfusion with various cell populations were observed. Markedly high ROS/RNS production was evident in the KCs and the xenogeneic whole-blood group. The oxidative stress was mainly caused by leukocytes and to lower extent by KCs, but only in combination with leukocytes. Neither erythrocytes, thrombocytes, nor hepatocytes had an effect on the release of ROS and RNS, as we could not observe significant differences in the MDA, peroxynitrite, and NO levels in these groups compared with control. Tissue injury and hyperacute rejection were more evident in the KC and whole-blood livers. No sign of damage was observed for the control, erythrocyte, and thrombocyte group. Removal of leukocytes from the perfusate by filtration had a major protective effect on the liver function and the grade of hyperacute rejection, whereas KC depletion reduced the ROS production, but did not have an impact on the hyperacute rejection and liver damage. In all xenogeneic perfused groups, the activation of the complement was histologically observed by positive C3c and C9b. Neither KC depletion nor the removal of leukocytes or thrombocytes from the perfusate had an effect on the activation of the complement system. Damage of the rat liver by the complement system was only observed in association with leukocytes.

Our data revealed that various cell populations contribute to the formation of free radicals in our model. The production of free radicals was mainly linked to leukocytes and to a minor extent to KCs, but only in combination with leukocytes. Free radicals critically contribute to injury, rejection, and dysfunction of the xenotransplanted liver. Furthermore, hyperacute rejection in the xenogeneic perfused liver is triggered by the complement system only in the presence of leukocytes and free radical formation.

Nuclear transfer (NT) technologies offer a means for producing the genetically modified pigs necessary to develop swine models for mechanistic studies of disease processes as well as to serve as organ donors for xenotransplantation. Most previous studies have used commercial pigs as surrogates.

In this study, we established a cloning technique for miniature pigs by somatic cell nuclear transfer (SCNT) using Nippon Institute for Biological Science (NIBS) miniature pigs as surrogates. Moreover, utilizing this technique, we have successfully produced an α-1, 3-galactosyltransferase knockout (GalT-KO) miniature swine. Fibroblasts procured from a NIBS miniature pig fetus were injected into 1312 enucleated oocytes. The cloned embryos were transferred to 11 surrogates of which five successfully delivered 13 cloned offspring; the production efficiency was 1.0% (13/1312). In a second experiment, lung fibroblasts obtained from neonatal GalT-KO MGH miniature swine were used as donor cells and 1953 cloned embryos were transferred to 12 surrogates. Six cloned offspring were born from five surrogates, a production efficiency of 0.3% (6/1953).

These results demonstrate successful establishment of a miniature pig cloning technique by SCNT using NIBS miniature pigs as surrogates. To our knowledge, this is the first demonstration of successful production of GalT-KO miniature swine using miniature swine surrogates. This technique could help to ensure a stable supply of the cloned pigs through the use of miniature pig surrogates and could expand production in countries with limited space or in facilities with special regulations such as specific pathogen-free or good laboratory practice.

To establish the safety of xenotransplantation when cells, tissues, or organs of pigs are used, an effective screening for potential zoonotic microorganisms has to be performed. In doing so, special attendance has to be paid to porcine endogenous retroviruses (PERVs) that are widely distributed as proviruses in the genome of pigs. PERV-A and PERV-B are present in all pigs, they infect human cells in vitro and therefore represent a direct risk. PERV-C infects only pig cells; however, recombinant PERV-A/C infecting human cells and replicating at a higher rate were found in pigs indicating an indirect risk. To prevent the transmission of PERV, it was suggested to use animals characterized by a low expression of PERV-A and PERV-B that are free of PERV-C and cannot generate recombinants. Göttingen minipigs are used for numerous biomedical investigations and they are well characterized; however, the prevalence and the expression of PERV in these animals were not yet investigated.

The presence and expression of all PERVs including a new variant (nv) of PERV-C and PERV-A/C were analyzed using PCR and real-time PCR methods. Altogether, 15 animals belonging to different families were analyzed. To make a low expression better measurable, peripheral blood mononuclear cells (PBMCs) of the animals were stimulated with phytohaemagglutinin generally increasing the expression of PERV and allowing a better classification into animals with high and low expression. As a major end point, the release of virus particles able to infect susceptible human 293 cells was investigated.

PERV-A, PERV-B, PERV-C, and PERV-Cnv were found in the genome of all investigated Göttingen minipigs, but recombinant PERV-A/Cs were not found. When the expression of PERV was compared with that in previously analyzed pig strains, it was higher than in German landrace and some other pigs, but lower than in Yucatan miniature pigs. Virus particles able to infected human 293 cells were not detected even after mitogen treatment of the PBMCs.

The Göttingen minipigs are well defined concerning their physiologic parameters, their health status, and their genetics, and therefore, they may be considered as donor animals for at least cell xenotransplantation. When the prevalence and the expression of PERVs were analyzed in these animals, it was demonstrated that although PERV-A, -B, and -C proviruses were found in all animals, their expression was low. Additional investigations are required to assess the suitability of Göttingen minipigs and other animals for xenotransplantation in terms of microbiological safety.

The successful isolation, purification, and culture of caprine islets has recently been reported. The present study shows arange of size distribution in caprine islet diameter from 50 to 250 μm, in which 80% of the total islet yield was comprised of small islets.

Caprine islets were isolated and purified. Islets were handpicked and the diameter of the islets was recorded using light microscopy. Viablility of the islets was analyzed by confocal microscopy. Insulin secretion assay was carried out and analyzed by ELISA.

When tested at 48 h after isolation, these small islets were 29.3% more viable compared to the large-sized islets. Large islets showed a high ratio (P < 0.01) of central core necrosis (29.5% ± 1.92) whilst no significant core death was observed in small islets (2.33% ± 0.59). The annexin assay demonstrated 5.21% ± 0.97 and 7.34% ± 0.78 apoptotic death for small and large islets, respectively. During static incubation, small islets released 2.89-fold (1.39 ± 0.2 ng/IE) higher insulin level under low glucose induction (3.3 mm) and simultaneously 2.92-fold (2.95 ± 0.33 ng/IE) more insulin under high glucose condition (16.7 mm) in comparison to large islets at the same islet equivalents (P < 0.05).

The present findings evidenced the superior quality of smaller caprine islets compared to larger ones under an optimized basal maintenance condition. As it is equally important to preserve the quality of larger caprine islets, this work warrants further investigation on special culture conditions to support these islets.

Activation of the clotting cascade is central in acute xenograft rejection (AHXR) that occurs when pig organs are transplanted into primates. The coagulopathy reported in this model is a very complex process that involves simultaneously coagulation factors, platelets and phospholipid-bearing cells (i.e., leukocytes, red blood cells, and endothelial cells). Choosing whole blood for coagulation analysis theoretically appears more favorable compared with plasma. Whole blood rotation thromboelastometry (ROTEM^®) is a point-of-care global coagulation analyzer able to evaluate the characteristics of clot formation and lysis by dynamic monitoring. The aim of this study was to record thromboelastographic profiles, performed by ROTEM^®, in a series of immunosuppressed nephrectomized primates that received a life-supporting kidney.

Of the eight primates, n = 4 received a pig kidney transgenic for human decay-accelerating factor (hDAF/Gal+); n = 2, an α 1,3-galactosyltransferase gene-knockout (GT-KO) pig kidney transgenic for human CD39, CD55, CD59 and fucosyltransferase (HTF); and n = 2, a GT-KO pig kidney transgenic for hDAF. Blood samples were collected before and at least once per week after transplantation till euthanasia. Intrinsic (INTEM) and extrinsic (EXTEM) coagulation pathways and the function of fibrinogen (FIBTEM) were evaluated. Thromboelastographic parameters considered were clotting time (CT, seconds) and clot formation time (CFT, seconds) in INTEM and EXTEM and maximum clot firmness (MCF, mm) in FIBTEM. The correlations between CT in INTEM and activated partial thromboplastin time (aPTT), CT in EXTEM and PT, CFT in INTEM and EXTEM, and platelet counts and MCF in FIBTEM and fibrinogen plasma levels were also considered.

In all animals, thromboelastographic profiles showed progressive prolongation of CT (activation of coagulative cascade) in INTEM. A close correspondence was observed between (i) the prolongation of the CFT values (propagation of clot formation), both in INTEM and EXTEM, and the decrease in platelet counts; (ii) the reduction in MCF values (clot firmness) ​​in FIBTEM and the decrease in fibrinogen plasma levels. No concordance between CT in INTEM and aPTT and between CT in EXTEM and PT was observed.

Our study demonstrated that ROTEM^® analyzer could be a useful and complementary tool to study the consumptive coagulopathy, either “compensated” or “non-compensated,” that takes place when transgenic pig kidneys are transplanted into primates. Larger and prospective studies are needed to confirm our results and to evaluate the role of ROTEM^® to guide the management of consumptive coagulopathy in order to prolong the survival of the transplanted organ.

We investigated whether graft produced anti-human CD2, mediated by adenovirus (Adv) transduction of pig neonatal islet cell clusters (pNICC), would protect xenografts in a humanized mouse model from immune attack and whether such immunosuppression would remain local.

A mouse anti-human CD2 Ab (CD2hb11) previously generated by us was genetically engineered to produce chimeric and humanized versions. The three forms of CD2hb11 were named dilimomab (mouse), diliximab (chimeric) and dilizumab (humanized). All 3 forms of CD2hb11 Ab were tested for their ability to bind CD3⁺ human T cells and to inhibit a human anti-pig xenogeneic mixed lymphocyte reaction (MLR). They were administered systemically in a humanized mouse model in order to test their ability to deplete human CD3⁺ T cells and whether they induced a cytokine storm. An adenoviral vector expressing diliximab was generated for transduction of pNICC. Humanized mice were transplanted with either control-transduced pNICC or diliximab-transduced pNICC and human T cells within grafts and spleens were enumerated by flow cytometry.

Dilimomab and diliximab inhibited a human anti-pig xenogeneic response but dilizumab did not. All 3 forms of CD2hb11 Ab bound human T cells in vitro though dilimomab and diliximab exhibited 300-fold higher avidity than dilizumab. All 3 anti-CD2 Abs could deplete human CD3⁺ T cells in vivo in a humanized mouse model without inducing upregulation of activation markers or significant release of cytokines. Humanized mice transplanted with diliximab-transduced pNICC afforded depletion of CD3⁺ T cells at the graft site leaving the peripheral immune system intact.

Local production of a single Ab against T cells can reduce graft infiltration at the xenograft site and may reduce the need for conventional, systemic immunosuppression.

Stem cell therapy may help restore cardiac function after acute myocardial infarction (AMI), but the optimal therapeutic cell type has not been identified.

We examined the effects of CD34-/CD45- human unrestricted somatic stem cells (USSCs) in pigs (n = 30) with an AMI created by a 90-min occlusion of the left anterior descending coronary artery. Pigs were randomly assigned to receive either USSCs (302 ± 23 × 10⁶ cells) or phosphate-buffered saline via 15 NOGA-guided transendocardial injections 10 days after AMI. Cyclosporine A (10 mg/kg orally, twice a day) was started in all pigs 3 days before control or cell treatment. Cardiac function was assessed by echocardiography before injection and at 4 and 8 weeks after treatment. Serum titers for pig IgG antibodies against USSCs were also measured at these time points and before AMI.

Compared with control pigs, USSC-treated pigs showed no significant differences in any of the functional parameters examined. USSC-treated pigs showed variable increases in anti-USSC IgG antibody titers in the blood and chronic inflammatory infiltrates at the cell injection sites. Immunohistochemical studies of the injection sites using human anti-mitochondrial antibodies failed to detect implanted USSCs.

We conclude that human USSCs did not improve cardiac function in a pig model of AMI. Cell transplantation in a xenogeneic setting may obscure the benefits of stem cell therapy.

Delayed xenograft rejection (DXR) involves type II vascular endothelial cell (VEC) activation including upregulation of pro-inflammatory genes, which contributes to infiltration into the graft and a complex process of cytokine production. Approaches to prevent DXR have shown limited success. In this study, we modified heart donors using siRNA in an attempt to attenuate DXR and to improve xenograft survival in the mouse-to-rat heterotopic heart transplant model.

siRNA technology was used to inhibit NF-kappaB p65 gene expression in vivo in mice. After the donor was transfected with siRNA, the effects of NF-kappaB siRNA on DXR and expression of NF-kappaB and pro-inflammatory genes were evaluated in a concordant mouse-to-rat cardiac xenograft model.

Treatment of NF-kappaB siRNA prolonged median heart graft survival time in the recipient rats from 1.7 days in a PBS control group to 5.4 days in the NF-kappaB siRNA-treated group (P < 0.05). Compared with normal mouse hearts, the NF-kappaB p65 mRNA relative levels following siRNA injection in the donors decreased significantly (approximately 70% reduction) in grafts harvested 12 h after transplantation. The mRNA levels of VCAM-1, ICAM-1, and interleukin-1 displayed a similar reduction. Histological evaluation using light and electron microscopy showed that damage of endothelial cells after NF-kappaB siRNA treament occured at a later time.

Transfection of NF-kappaB p65 siRNA in donor animals can delay the emergence of DXR. This treatment may be used as part of strategies to minimize the complex and multi-faceted rejection responses in vascularized xenografts.