Author’s address (for correspondence): Hiroshi Nagashima, Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama, Kawasaki 214-8571, Japan. E-mail: firstname.lastname@example.org
Regenerative medicine is expected to make a significant contribution by development of novel therapeutic treatments for intractable diseases and for improving the quality of life of patients. Many advances in regenerative medicine, including basic and translational research, have been developed and tested in experimental animals; pigs have played an important role in various aspects of this work. The value of pigs as a model species is being enhanced by the generation of specially designed animals through cloning and genetic modifications, enabling more sophisticated research to be performed and thus accelerating the clinical application of regenerative medicine. This article reviews the significant aspects of the creation and application of cloned and genetically modified pigs in regenerative medicine research and considers the possible future directions of the technology. We also discuss the importance of reproductive biology as an interface between basic science and clinical medicine.
Application of Cloned Pigs to Research into Kidney Regeneration
The creation of functionally and structurally complete organs from pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) (Evans and Kaufman 1981) and induced PSCs (iPSCs) (Takahashi and Yamanaka 2006), is one of the ultimate goals of regenerative medicine. However, there are considerable obstacles to the generation of organs and tissues in vitro because of their complex functions and structures. An alternative approach has been proposed in which organs from human PSCs are generated using the in vivo milieu of developing animal foetuses (Yokoo et al. 2005). To test this concept, Yokoo et al. (2005) injected human mesenchymal stem cells (MSCs) into rat foetuses (gestational age 11.5 days) and, after culturing the foetuses for 2 days in vitro, collected their renal anlagen (metanephroi). They cultured metanephroi in vitro for a further 6 days or implanted in the omentum fat tissue of immune-suppressed adult rats. They then analysed the subsequent growth and tissue development in the metanephroi. They confirmed that the injected human MSCs were incorporated into the kidney tissue that developed from the transplanted metanephroi. In other words, kidney tissue derived from human PSCs was generated using the organ development mechanism of xenogeneic foetuses, suggesting possibility of generating humanized kidney using animal metanephroi as a scaffold. To apply this concept to humans, the size of the generated organ needs to be increased using larger host animals. At the same time, it is necessary to determine whether the renal anlagen of the host animal can function as a scaffold as efficiently as those of rats. Pigs are considered the most suitable of the possible larger animal models, because of their physiological and anatomical similarities to humans, the feasibility of zoonosis control, their long history as domesticated livestock, and the availability of advanced technologies such as genetic modification and embryo manipulation (Niemann and Kues 2003; Lunney 2007).
The strategy that underlies humanized kidney generation assumes the transplantation of xenogenic foetal anlagen to adult patients. To validate this strategy, it is first necessary to show that pig foetal metanephroi can be grown in the body of adult pigs and to determine what structural and functional changes the metanephroi undergo after engraftment. This analysis will provide fundamental knowledge on the potential of pig metanephroi as a scaffold for humanized kidney generation. The use of cloned pigs would be beneficial for this type of transplantation study because metanephroi from a cloned pig foetus could be transplanted into a syngeneic adult cloned pig. Thus, engraftment and growth of the graft could be evaluated without being affected by immune rejection. A significant advantage of somatic cell cloning technology is that clones of different ages can be successively created from cells of the same origin; therefore, the foetal anlagen of the clones can be transplanted to adult animals with syngeneic background. An investigation of the development of transplanted metanephroi reported that they could develop in the fat tissue of the omentum of adult cloned pigs to forming kidney tissue with a urine production capability (Matsunari et al. 2011b). Thus, syngeneic transplantation system using cloned pigs in which foetal anlagen are transplanted into adult animals will provide valuable information for organ regeneration research. Worldwide, there are currently an estimated 1.4 million dialysis patients and the number is rapidly increasing. Thus, the possibility of kidney regeneration has important implications, not only for improving the quality of life of dialysis patients, but also for reducing the medical burden on patients, medical institutions and national governments.
Yokoo et al. (2009) attempted to treat cats suffering from renal disease by transplantation of xenogenic metanephroi. Metanephroi from days 28–30 pig foetuses were transplanted into the omentum of unilaterally nephrectomized adult cats. The kidney tissue that developed from the grafts was found to produce erythropoietin (EPO) derived from the host cats. Administration of exogenous EPO is a standard treatment for end-stage renal failure. Many old pet cats suffer and die from chronic renal failure, which is a major clinical issue for veterinary medicine. Cats can be an irreplaceable companion animal for humans. If a treatment method can be established in which pig metanephroi are transplanted into patient cats, thereby stimulating EPO production by the host cells, this would offer a valuable contribution to treatment for renal failure in cats. In this way, kidney regeneration using pig metanephroi as a scaffold has potential not only in human medicine but also for companion animals.
Application of Cloned Pigs to Pancreas Regeneration Research
Diabetes, a disease with 346 million sufferers worldwide, is a significant health/welfare problem that the modern society faces. Transplantation of a pancreas or of pancreatic islets may offer an important therapy for treating type 1 diabetes. At present, however, transplantation therapy has the problem of an acute shortage of donor organs/tissues. An innovative study has recently been conducted showing that it may be possible to induce pancreatic regeneration. Kobayashi et al. (2010) reported that pancreatic tissue derived from rat PSCs can be generated in the body of mice with a knockout of the pancreatic and duodenal homeobox-1 gene (Pdx1−/−). The study showed that it is possible to generate a xenogeneic organ in the body of pancreatogenesis-disabled mice; this was achieved by injecting rat PSCs into mouse Pdx1−/− blastocysts (interspecific blastocyst complementation; Fig. 1). In other words, a proof of principle was established for induced organogenesis from xenogeneic PSCs using the empty developmental niche of organogenesis-disabled embryos.
The concept of blastocyst complementation was originally developed by Chen et al. (1993). They showed that deficiency of T and B lymphocyte lineage cells in Rag2-deficient (Rag2−/−) mice could be complemented by injecting normal mouse ESCs into Rag2−/− blastocysts. Kobayashi et al. (2010) found that blastocyst complementation was effective not only for the formation of haematopoietic cells, but also for the generation of solid organs such as the pancreas.
Clearly, in vivo xenogeneic organ generation in humans would require a substantial increase in the size of the regenerated organ. Recently, we have conducted experiments to determine whether the blastocyst complementation strategy used in small rodents (Kobayashi et al. 2010) can also be applied to pigs. As developmental patterns differ greatly between small rodents and pigs, it is not feasible to directly apply the methodology used for xenogenic organ regeneration in mice/rats to pigs.
Kobayashi et al. (2010) created pancreatogenesis-disabled mice using embryos homozygous for a knockout of Pdx1, which is the master regulator of pancreatic formation. Because the pancreatogenesis-disabled phenotype is a neonatal lethal, Pdx1−/− adult animals cannot be obtained. However, for research purposes, a realistic strategy is to mate male and female heterozygous knockout mice to obtain homozygous embryos (Fig. 1). Thus, they collected Pdx−/− blastocysts and used these as hosts to create chimeric blastocysts by injection of rat iPSCs. This approach is not possible for research with large animals because of the considerations of effort, time and cost. However, a somatic cell nuclear transfer (SCNT) technology has been developed in pigs and used to establish a genetic modification system based on SCNT (Park et al. 2001; Lai et al. 2002). Cloned embryos and cloned pigs can be created by SCNT from genetically modified primary culture cells as the nuclear donor. Chimeric pigs can also be created by embryo manipulation. Against such a background, we have been attempting to establish an experimental system to create cloned, genetically modified pigs with a pancreatogenesis-disabled phenotype and, through complementation of embryos from these pigs, generate xenogeneic pancreas formation (Fig. 2).
The ‘rescue chimera technology’, in which survival of embryos with poor developmental potential is aided by chimerization with cells from ‘healthy’ embryos, has already been used for preservation of important genetic resources and for propagation of rare livestock and wild animals. The knowledge and expertise needed to create chimeric animals, which have been accumulated in studies in reproductive biology over many years, are now sufficiently advanced to be exploited for organ regeneration in humans.
In regenerative medicine, the generation of a structurally and functionally complete pancreas is not the only goal in terms of overcoming pancreatic disorders. Transplantation of pancreatic islets has already been used to treat type 1 diabetes. Because only pancreatic islets need to be isolated for transplantation then, with respect to pancreatic regeneration for clinical use, we can focus simply on obtaining functional pancreatic islets. Additionally, making use of isolated islets rather than a whole pancreas would minimize the incorporation of xenogenic components, which is a concern when generating human organs in the body of another species.
Just as research into kidney regeneration has potential applications to pet cats, so pancreatic regeneration using interspecific blastocyst complementation has potential for use in companion animals. For example, the generation of dog pancreatic tissue from iPSCs by complementation of pancreatogenesis-disabled pig blastocysts holds promise for future applications (Fig. 2).
Research into blastocyst complementation between pigs and monkeys would serve as an important bridgehead towards human applications (Fig. 2). Our understanding of the possibility of creating such interspecific chimeras is limited because, to date, such research has been largely confined to the rescue of rare or endangered animals. It will be important to conduct interspecific chimerism research in pigs and a range of other mammalian species in the future.
The regeneration of organs using PSCs is aimed at providing medical treatments tailored to needs of the individual patient. Such an aim does not diminish the importance of generic treatments that can be applied to every patient, for example organ transplant. The shortage of donor organs for the latter is the single most important factor hindering progress in this medical field and is also one of the factors stimulating the development of alternative approaches such as regeneration of organs and xenotransplantation.
The shortage of donor organs for organ transplantation is a worldwide problem. Xenotransplantation, the use of organs of non-human origin for transplantation into humans, has been proposed as a potential solution to this shortage, and considerable research is going into making this a realistic goal. As mentioned earlier in relation to organ regeneration research, pigs are considered as the most suitable species to act as xenogenic organ donors (Ibrahim et al. 2006; Yang and Sykes 2007).
Successful application of xenotransplantation between pigs and humans requires that the problem of organ rejection is overcome. There are multiple types of rejection: hyperacute rejection, delayed xenograft rejection and cellular rejection. Clearly, sophisticated and complex (multilayered) genetic modifications will be necessary to overcome all of these aspects of rejection (Cozzi et al. 1994; Fodor et al. 1994; Miyagawa et al. 1994; McCurry et al. 1995). In other words, an efficient strategy is required in which multiple genetic modifications of pigs can be made to counter with the complex mechanism involved in the rejection of xenogenic organs.
Crosses between existing transgenic pig strains is one way to obtain animals with multiple genetic modifications; however, this approach would require enormous time and effort in pigs, which are large animals with a long generation cycle. Another problem is that pigs with desirable genetic modifications may have been developed in different parts of the world, raising difficulties in terms of accessibility and quarantine. One possible solution may be to use an SCNT-based method that will allow the complex genetic engineering required for the introduction or deletion of genes into or out of cultured cells derived from an existing genetically modified pig (Dai et al. 2002; Lai et al. 2002). For example, cloned pigs with genetic modifications have already been created by knocking out the α1,3-galactosyltransferase (GalT) gene from the cells of a polytransgenic pig that had integration of multiple transgenes (Ramsoondar et al. 2003; Takahagi et al. 2005). Somatic cell nuclear transfer also has the advantage of allowing investigation of the phenotypic characteristics of the derived genetically modified pigs using only one or a few of the resulting clones. This analysis can be used to decide on which further genetic modifications should be added in subsequent generations (Fig. 3). Gene knockout (KO) in pig primary culture cells was initially a very inefficient method; however, with the advent of methods that use zinc finger nuclease, it has developed almost to the point of being routine (Watanabe et al. 2010; Hauschild et al. 2011).
The SCNT approach has been used to generate pigs with several different transgenes in addition to GalT-KO, but, to date, they have not been used in clinical xenotransplantation studies (Takahagi et al. 2005; Yamada et al. 2005; Cooper et al. 2007). Undoubtedly, future research will be directed at improving existing stocks of genetically modified pigs by introducing additional genes by SCNT. The generation of pigs with such a multiplicity of genetic modifications is obviously based on the repeated application of somatic cell cloning (Fig. 3). There is evidence from mice that SCNT does indeed have the necessary repeatability as six generations of cloned mice have been successfully created by SCNT (Wakayama et al. 2000) and, recently, it has been reported that further clonal generations can also be created (T Wakayama, personal communication).
Currently, it is unclear whether there is any limit to the number of times cloning can be repeated in pigs; Matsunari et al. (2010) reported that they have successfully created six generations of cloned pigs. Importantly, Kurome et al. (2008) showed that telomere lengths in cloned pigs did not change after three repetitions of somatic cell cloning.
The accumulation of epigenetic modifications and the reduction in telomere length have been major concerns with regard to repeated cloning of animals (Wakayama et al. 2000; Kubota et al. 2004). However, the possibility of resetting epigenetic modifications has been demonstrated using histone deacetylase inhibitors (HDACi) (Kishigami et al. 2006; Zhao et al. 2010a); in combination with advances in nuclear transfer technology in recent years, this suggests that repeated somatic cell cloning may be more feasible than previously predicted (Matsunari et al. 2010). Improvements in the production efficiency and health of somatic cell cloned pigs will be an important topic for future research.
Disease Model Pigs
Disease model animals are useful for deepening our understanding of disease pathogenesis and for obtaining information of value for developing clinical therapies. Many animal models of disease have been developed and used in numerous studies (Dauer and Przedborski 2003; Rees and Alcolado 2005; Carver and Pandolfi 2006; Guilbault et al. 2007; Recchia and Lionetti 2007). However, in many instances, genetic mutations that cause disease do not show the same range of effects in humans and in the animal model, particularly in rodents (Grubb and Boucher 1999). Translational research in regenerative medicine requires that an animal disease model produces findings that can be immediately extrapolated to humans. This is one of the important advantages of creating and using disease model pigs as they often show similar symptoms and effects as humans.
The anatomical and physiological similarities of pigs and humans are wide-ranging, covering the cardiovascular system, digestive system, central nervous system, skeleton and feeding habit (omnivorous). Given these similarities, it is unsurprising that pigs and humans also share pathological conditions (Niemann and Kues 2003; Lunney 2007; Pearce et al. 2007). Another advantage of pigs as an experimental animal is that because of the similarity in body and organ sizes, the surgical and endoscopic procedures developed in humans can be applied to pigs.
Despite the many advantages, only a few practical disease model pigs have been established to date. Additionally, the techniques for working with pig ESCs and iPSCs are still under development, and unlike mice, cell lines capable of germ line transmission are not yet available. For these reasons, creation of disease model pigs using gene KO technology is still limited. The progress to date on this aspect of research has recently been reviewed by Aigner et al. (2010) and Whyte and Prather (2011). Here, we focus on maintenance and exploitation of the currently available disease model pigs. Although disease model pigs have obvious value to research, the expression of severe disease can affect growth and reproduction. Thus, there is a conundrum that whereas disease model pigs have high value and utility, their use may be limited by problems of reproduction.
The nature of this problem and a possible means of resolving it are illustrated by some recent work on diabetic transgenic-cloned pigs. Umeyama et al. (2009) created diabetes model pigs by inducing a dominant negative mutation of the hepatocyte nuclear factor 1α (HNF1α), the causal gene for maturity onset diabetes of the young (MODY) type 3. The pigs exhibited constant high blood glucose levels before weaning and, if left untreated, died as pre-adults during the growth period. However, it was shown that these disease model pigs could be efficiently reproduced using somatic cell cloning (Umeyama et al. 2009). These results indicate that where a genetic modification is detrimental to the growth of newborns, then proliferation by means of somatic cell cloning is a feasible option. However, use of somatic cell cloning for propagating disease model pigs raises the concern of possible differences in pathological phenotypes among the derived individuals because of differences in epigenetic modifications (Zhao et al. 2010b). Thus, for example, Umeyama et al. (2009) reported marked differences in non-fasting blood glucose levels and longevities among clones of their diabetes model pig, which might reflect variations in the epigenetic status among the cloned siblings. The developmental potential of cloned embryos has been reported to improve dramatically by treatment with HDACi (Zhao et al. 2009), and we have confirmed this effect (Matsunari et al. 2012). However, we also found that the phenotypes of cloned pigs developed from SCNT embryos treated with HDACi showed as wide variation as non-treated clones (H Matsunari and H Nagashima, unpublished data). The induction and control of diseases by epigenetic modification will be an important future issue for future research.
Cryopreservation of Cloned Embryos
The production efficiency of somatic cell cloned pigs (the number of cloned piglets/the number of cloned embryos transferred) is currently no more than 1–5% (Kurome et al. 2008; Matsunari et al. 2008; Zhao et al. 2009). Therefore, several dozen cloned embryos must be created to perform an embryo transfer to a single recipient female. A solution to this difficulty would be to cryopreserve somatic cell cloned pig embryos; as pig embryos are notoriously sensitive to damage during cryopreservation, improvements in the methodology are required to increase the production efficiency of cloned pigs.
Nakano et al. (2011b) reported that cloned piglets could be produced from cloned embryos that had been cryopreserved using a vitrification method, with a production efficiency equivalent to that for non-vitrified embryos. In this study, the cloned embryos were vitrified at the morula stage following the removal of cytoplasmic lipid droplets (delipation) (Nagashima et al. 1995). The effectiveness of delipation for the survival of the cryosensitive porcine embryos was originally reported by Nagashima et al. (1995) and has since been confirmed by other researchers (Li et al. 2006, 2009; Du et al. 2007; Nakano et al. 2011b).
Recently, we have developed a new embryo cryopreservation method called the hollow fibre vitrification (HFV) method (Matsunari et al. 2011a). This new method is highly efficient at producing piglets from embryos and does not involve use of delipation (Nakano et al. 2011a). We have also shown that the HFV method is effective for vitrification of porcine embryos derived from in vitro matured/fertilized oocytes (Maehara et al. 2011, 2011). The advantages of the HFV method should stimulate greater use of cryopreserved porcine cloned embryos.
The creation and use of cloned pigs have made a significant contribution to various fields in basic and applied research for regenerative medicine such as treatment for intractable diseases, stem cell therapy and organ/tissue transplantation. It is important to verify the findings obtained from the in vitro studies in a complex system of individual animals. The role of research that uses cloned pigs as a platform is significant in terms of producing findings truly useful for clinical application.
This work was supported by JST, ERATO, Nakauchi Stem Cell and Organ Regeneration Project, Tokyo.
Conflicts of interest
None of the authors have any conflicts of interest to declare.