Interspecies blastocyst complementation

C H A P T E R

32 Interspecies blastocyst complementation Benjamin S. Freedman Kidney Research Institute, Seattle, WA, USA Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Division of Nephrology, Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA Department of Pathology, University of Washington School of Medicine, Seattle, WA, USA O U T L I N E Introduction

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Allograft considerations

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Basic principles of IBC

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Efficiency of IBC

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Generation of pancreas with IBC

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Breeding schemes

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Transplantability of IBC pancreas

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Gene editing with IBC

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Advantages of IBC

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Suitability of large animal hosts

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Pretransplant immunogenicity

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Ethical concerns

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Posttransplant immunogenicity

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Outlook

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Potential for IBC vasculature

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References

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Autograft tolerance

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Introduction Both the supply of pancreas transplants, as well as their quality, is insufficient for the number of patients who need them. For transplantation purposes, it is important that the graft be human, to avoid provoking a severe rejection response that occurs in response to organs from other species.1,2 Ideally, it would be possible to find a way to produce human organs such as the pancreas on-demand in a fully immunocompatible way, from stem cells. As for other organs, the complexity of the pancreas poses a challenge for stem cell-based bioengineering as a therapeutic strategy in humans. Human pluripotent stem cells (hPSC) can be differentiated into pancreatic islet cells, but the resulting structures (or organoids) are immature, variable in composition, and are furthermore contaminated with non-pancreas cells that could pose Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, Volume 2 https://doi.org/10.1016/B978-0-12-814831-0.00032-4

a risk of tumorigenesis.3–5 In addition, these structures produced in vitro are tiny, avascular, and lack the architectural complexity of the pancreas. Although a more sophisticated tissue could conceivably be bioengineered, bioprinted, or produced as a scaffold, the techniques for accomplishing this remain in their infancy.6–9 The bioengineering and stem cell differentiation fields therefore remain distant from producing a true therapeutic alternative to allograft pancreas transplant. An alternative strategy is to grow a human pancreas in a host species, such as a pig. In theory, such a methodology would enable farming of human organs, which could be harvested as needed for transplantation. An emerging technique to accomplish this feat is interspecies blastocyst complementation (IBC). In IBC, pluripotent stem cells from a donor species are implanted within the embryo of a different host species to fill an organ niche, which consists of a deficiency in the host’s

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© 2020 Elsevier Inc. All rights reserved.

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ability to develop specific cell types. Here, the basic features of IBC and its successes to date will be reviewed, and both technical limitations as well as potential ethical issues arising from this technology will be discussed.

Basic principles of IBC The starting point for blastocyst complementation is an early embryo “host” (typically blastocyst-stage) that is incapable of forming a particular type of embryonic structure. For instance, the host embryo may carry lossof-function mutations in a gene essential for the development of a particular organ, such as Pdx1 in the mouse pancreas. As the host develops, this deficiency would produce a niche (vacuum) that can only be filled by cells from another source.10,11 The supplementation of the host blastocyst embryo with healthy “donor” stem cells (lacking the deficiency) creates a situation in which the donor and host cells combine to make a functional embryo. Blastocyst complementation was first tested between cells and embryos of a single species, Mus musculus. Originally, it was demonstrated that immune cells of the blood lineage (B and T lymphocytes) could be complemented by transferring wild-type mouse pluripotent stem cells into blastocysts deficient in Rag2.11 Decades later, it was determined that a similar strategy could work for the pancreas.10,12 This was followed by successful mouse-to-mouse blastocyst complementation in other solid organs, such as kidney, heart, and eye, as well as vascular endothelium.12–14 In addition, single-species blastocyst complementation has been shown to be possible in larger animal species, such as pigs.15 IBC, introduced in 2010, is a cross-species variation on the classic blastocyst complementation technique, in which the donor stem cells originate from a species different than the host embryo.10 This produces a chimera, containing cells from two different species (Fig.  1). In the absence of an organ niche, chimerism between two species would be very difficult to achieve.16–20 In contrast, in IBC, the incorporation of an organ niche into the experiment produces a developmental pressure that enables interspecies chimerism to succeed, albeit at low rates.

Generation of pancreas with IBC The pancreas was the first solid organ to be generated using IBC and remains the best characterized.10 In rodents, expression of Pdx1 is required to generate pancreas. Mice or rats lacking a functional copy of Pdx1 are unable to form pancreas and do not survive long after birth. Pdx1−/− embryos therefore have a developmental pancreas niche that needs to be filled.

FIG. 1  Schematic representation of human pancreas generation with IBC. A theoretical example of pancreas generation in a pig is shown. Note that a small proportion of pancreas tissue is of pig origin.

Taking advantage of this property, IBC was used to generate a mostly rat pancreas in a Pdx1−/− mouse.10 The pancreatic epithelium in these chimeras appeared to be entirely of rat origin, as assessed by a genetically encoded green fluorescent protein (GFP) tracing label in the donor rat iPS cells (Fig.  2). The pancreas itself was of normal morphology and size for a mouse, contained both exocrine and endocrine tissues by marker analysis, and responded to glucose challenge.10 Although most IBC animals failed to reach adulthood, two did survive, according to the original report.10 The rat-specific GFP label persisted into adulthood in the pancreatic epithelial cells of these animals, and was estimated to be present in ~80% of the cells within the organ. The remaining 20% of cells were of mouse origin, and suggested to be of non-epithelial lineages, although their specific fates were not described in detail. The achievement of IBC pancreas provides general insight into the cell-intrinsic nature of solid organ formation. Although it is known that a Pdx1−/− knockout mouse cannot form pancreas, it is less clear whether Pdx1−/− cells can contribute to a pancreas when mixed with wild-type cells (i.e., benefit from a “neighbor”

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Advantages of IBC

FIG. 2  Generation of IBC rat pancreas in a mouse. Rat iPS cells expressing GFP were implanted inside a mouse blastocyst deficient in Pdx1, and the resultant mouse was sacrificed and imaged for GFP expression. Abdominal organs are labeled with abbreviations.

e­ ffect). The IBC results suggest that the intrinsic Pdx1−/− cells of the embryo remain incapable of contributing to a pancreas when mixed with wild-type neighbors. Rather, the vast majority of cells within the pancreas derive from the implanted IBC donor stem cells. This is an interesting finding that could be further investigated by carefully quantifying the appearance of mouse cells within IBC pancreas tissue, to rule out any contribution to the epithelial lineages independent of Pdx1 for specification.

Transplantability of IBC pancreas The clinical vision for IBC is to utilize it as a method to produce transplantable grafts. To test this as a therapeutic strategy, it is necessary to transplant an IBC graft out of the host into a diseased member of the donor species. Although this could conceivably done by transplanting rat-in-a-mouse pancreas tissue into diabetic rats, it was difficult to generate a sufficient mass of rat IBC pancreas tissue in mice for transplantation, because the rat pancreases grown in mice are mouse-sized and much smaller than rats.10,21 The converse experiment—transplantation of mousein-a-rat pancreatic graft into a diabetic mouse—has been successfully achieved.21 In this study, each single rat, harboring biallelic null alleles of Pdx1, was capable of producing a rat-sized IBC pancreas comprising primarily of mouse cells. Both exocrine and endocrine cells of the pancreas in these rats were primarily of mouse origin. About 200 islets could be harvested from each rat pancreas, which was sufficient to implant two diabetic (streptozotocin-induced) mice beneath the kidney capsule. Transplantation of the IBC islets rapidly and dramatically reduced serum glucose in these animals. Although the sample size was low, this rescue of glucose levels persisted for over 300 days after the transplant, and was rapidly reversible when the kidney containing the islets was removed from the animal by nephrectomy (Fig. 3). Thus, IBC islets were both necessary and sufficient to

sustain the low serum glucose levels in the diabetic mice.21 This work suggests that IBC may be a viable therapeutic strategy, at least for the pancreas. Although the transplantation of IBC islets is undoubtedly a success, it should be noted that no transplantation of whole solid organs has not yet been accomplished using IBC, including pancreas. Thus, the transplantability of IBC pancreas is not yet fully explored.

Advantages of IBC The major advantage of IBC over other methods is its ability to generate fully formed and functional organs of macroscopic size. Other methodologies, such as cadaver islet transplantation, differentiation of pluripotent stem cells in vitro, or bioprinting technologies based on spatially ordering cells in prearranged geometries, cannot produce complete organs. In contrast, the organs generated using IBC are very similar to the natural organs present in the host’s body, because they use the host as an incubator to generate the organ in a way that perfectly matches the organogenesis process. It is somewhat vexing, therefore, that IBC transplantation experiments have to date been limited to islet engraftment, rather than successful transplantation of entire organs. Undoubtedly, whole organ transplant is one of the most attractive potential applications of IBC, but it remains speculative. In addition to the pancreas, many other organs could be theoretically generated with IBC for the purposes of transplantation. IBC could therefore emerge as a competitor for existing allograft solid organ transplantation, which remains the gold standard of treatment in many organ systems. Despite substantial interest in IBC as a source of human pancreas and other organs, its adoption is limited by questions of practicality, technical feasibility, and ethics (Table  1). In the ensuing sections, we will consider some of these issues in detail, in an attempt to define a path forward for IBC in the pancreatic lineage.

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FIG. 3  Functional transplant of IBC islets. Serum glucose levels in two mice (red and blue “X” lines) transplanted beneath the kidney capsule with IBC mouse islets grown in biallelic Pdx1 mutant (Pdx1mu/mu) rat hosts. As a negative control, rat islets were transplanted, or islets were from monoallelic mutant rat hosts (Pdx1+/mu).

TABLE 1  Challenges in the application of IBC in humans Challenge

Possible solution(s)

Low efficiency of chimerism

Carefully match stem cells with embryo host

Failure of human IBC in rodents

Attempt in large domesticated species

Inefficient host breeding

SCNT host; CRISPR IBC

Late-stage rejection in host

Harvest graft early; immunosuppress host

Acute rejection in recipient

Acute immuosuppression with transplant

Hyperacute rejection in recipient

IBC vasculature; xenotransplantoptimized host

HLA mismatch

HLA-edited universal donor cells

Brain or gonadal chimerism ethics Restrict differentiation to target organ

Pretransplant immunogenicity One of the unanswered questions regarding the viability of IBC organs is their vulnerability to immune attack. In general, interspecies grafts provoke severe immune reactions. Thus, immune responses must be considered both pretransplant (during development of the IBC chimera) and posttransplant (after harvest and implantation of the graft).

It is remarkable that interspecies chimeras can exist at all, given the extreme nature of xenotransplant immune responses.2,22 The existence of IBC animals would suggest that immune tolerance is induced by the co-­ development of the chimeric donor cells within the host. However, we do not yet know the extent to which this tolerance induction might factor into the success rate of IBC, which is generally low. Performing IBC in immunodeficient hosts could conceivably improve the efficiency of IBC, if tolerance induction is a major issue in the success of the procedure. Although tolerance does appear to be induced in IBC embryos to some degree, there are concerns that rejection could occur during long-term development. A study in which IBC mouse pancreas was generated in rats noted that the host rats suffered from a form of immunologically mediated juvenile diabetes accompanied by lymphocyte invasion of acinars and islets, and pancreatic deterioration both structurally and functionally.21 This is fascinating as it suggests that the IBC organ may be attacked by the host’s immune system, even though it developed together with the rest of the embryo and clearly enjoys some measure of immune tolerance. It is also noteworthy that most of the IBC animals born to date have not remained viable into adulthood.10,21 Whether this is due to a rejection event, inadequate functionality of the IBC organ, or a consequence of off-target chimerism in other tissues is not yet clear.

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Potential for IBC vasculature

Posttransplant immunogenicity After transplantation, concern shifts to possible rejection of the organ in the donor/recipient species due to the contaminated cells of the IBC host species. We do not yet fully understand how organs like the pancreas form and how many different cell populations may be present. Even a small population of cells, if derived from the non-recipient species, could potentially initiate a rejection event in the recipient. The ensuing destruction of this population, or the rejection event itself, may compromise the function of the IBC organ, or cause systemic problems. For this reason, it is critical to test the safety and longevity of IBC grafts in animal models with functional immune systems, as was done in the IBC mouse-in-rat islet transplantation experiments.21 In those experiments, when IBC islets were transplanted into mice, the graft recipients were dosed with tacrolimus and a set of anti-inflammatory monoclonal antibodies at implantation and for the ensuing 5 days. This appears to have been sufficient to offset any severe immune rejection events that might have endangered the graft. Immunosuppressive therapy was discontinued after 5 days, and the mice survived for many months afterwards with normal glycemic control. As the islets were probably not 100% donor-derived, it does appear that at least some foreign-species cells can be tolerated by the transplant recipient, when immunosuppressed in an acute way following the operation. Given the attractive potential of IBC for whole organ transplantation, it is worth considering why islets were transplanted instead of whole pancreas in the rodent model. While it is true that whole pancreas transplant in the mouse requires substantial technical skill to accomplish the surgery, it can be successfully performed, and would have made a more dramatic demonstration of transformative potential of IBC than islet transplantation. Size mismatch between organs of rat and mouse, although considerable, may also have been a surmountable challenge, just as children can be transplanted with organs taken from adults. Why, then, was whole IBC pancreas transplant not performed? A possible answer to this question may lie in the observation that IBC organs are only partial chimeras. Although 80% of cells within an IBC pancreas may be derived from the donor species, that still leaves 20% of cells of host species.10,21 These host species cells may present difficulties for transplantation of the whole IBC organ. In particular, the vasculature derives from a distinct germ layer from the pancreatic epithelium, and therefore is host-derived in IBC pancreas. Transplantation of the pancreas into the donor species would necessarily involve this host-derived blood supply. Interactions

between circulating blood cells from the donor species and the endothelial wall of the host species could provoke a “hyperacute” rejection event, such as the rapid clotting of blood that occurs in pig kidneys when transplanted into primates.1,2,23 Pig endothelia provoke this response because they express galactosyl-α1,3,-galactose and other carbohydrate antigens against which humans and other primates have specific, circulating humoral antibodies. Acute immunosuppression might not be sufficient to prevent such a hyperacute rejection event. Thus, there is a certain advantage to purifying islets and transplanting them in the absence of the entire pancreas, in that it avoids direct interaction between the recipient immune system and the interspecies vasculature. Although the graft of islets beneath the kidney capsule is likely to be less functional than an intact pancreas, it may be a less risky approach, and suitable for proof of principle of IBC graft function after transplant.

Potential for IBC vasculature Admittedly, limiting IBC to avascular grafts such as pancreatic islets partially defeats the greatest advantage of doing IBC in the first place, which is to generate a fully functional, intact organ. To generate an IBC organ including a vasculature that is wholly derived from the donor species would therefore be a more ideal therapeutic strategy. To begin to address this possibility, same-species blastocyst complementation experiments have been performed to generate mice with exogenic vasculature.14 In these experiments, a loss-of-function Flk1 mutant was used as the host embryo. Implantation of mouse pluripotent stem cells produced chimeric mice (about 10% of all live births) in which both the vasculature and the hematopoietic lineage appeared to be ~100% donor-­ derived. Other components of the blood vessels, such as the smooth muscle, were a mixture of donor and host cells. In the same study, the authors tested the ability of rat cells to generate IBC endothelium within a mouse. Unfortunately, this resulted in embryonic lethality.14 Thus, it may not be possible to generate a viable IBC chimera with wholly donor vasculature (e.g., a live rat with mouse blood vessels), given the pleiotropic and central role of the vasculature in organizing and nourishing the body. It is notable, however, that IBC vasculature embryos in this experiment did survive until gestational day 9.5. This may be sufficient time for certain embryonic rudiments to form. Thus, one strategy may be to remove embryonic rudiments from IBC embryos at an early time point, while the embryo is still viable, and use these for transplantation. Such a strategy would depend on

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whether the rudiments could mature sufficiently in the context of the new recipient. Alternatively, it is conceivable that there may be subtypes of vascular endothelium that are specific to the pancreas, as has been suggested for other solid organs.24 If so, the pancreas-specific vasculature may have specific genetic vulnerabilities that can be exploited to create a pancreatic vasculature niche. This could be combined in trans with Pdx1 or an equivalent gene to create a donor pancreas with a donor vascular tree within a host species. Such technologies will require substantive advances in our understanding of vascular developmental biology at the organ-specific level and our ability to manipulate those boundaries. Besides the vasculature, there may yet be other types of systemic cells that cannot be made wholly human. For instance, resident blood cells (e.g., lymphocytes or dendritic cells) in a human IBC pancreas would still derive from the nonhuman host. These would need to be depleted from the graft, or else accepted as a contaminant that could introduce safety concerns. Notably, the same genetic mutations that are permissive for the creation of IBC vasculature also generate IBC blood cell populations,14 providing a possible avenue of attack for this lineage.

Autograft tolerance A final, but not insignificant, immune consideration is the allogeneic immunogenicity of the eventual IBC graft. Ideally, an IBC transplant would be an autograft. It is furthermore possible to conceive of an autograft strategy, using induced pluripotent stem (iPS) cells for IBC. iPS cells can be derived from practically any patient’s somatic cells, by reprogramming these cells with genes expressed in embryonic stem (ES) cells and growth ­factors.25,26 An iPS IBC organ would therefore be a true “perfect match” for its recipient. Although in theory we would not expect any adverse reaction to the human cells within an iPS IBC organ, in practice we cannot know for certain whether such cells might provoke some form of immune response. The immune system is highly complex and sensitive, and we do not yet fully understand its intricacies. As the iPS IBC organ has developed outside of the body into which it is being transplanted, there is a chance that its antigen presentation may in some way differ from the recipient. There is some suggestion that such immune responses are a general outcome of iPS cell-derived transplants (e.g., those generated purely in vitro), although most of the data suggests that these are likely to be safe, at least in animal models.27–29 The possibility of autologous rejection is compounded by the consideration that the iPS IBC organ has ­developed

in the presence of external cues from the host species (e.g., the mouse) cells for its entire life. During this time, the organ may have incorporated host antigens, and could present them, even though the organ cells themselves are autologous with the recipient. Such foreign antigen presentation could conceivably provoke an autoimmune response. Thus, even autologous transplantation strategies are not necessarily foolproof in IBC, and need to be tested first in animal models. In this regard, it is encouraging that IBC mouse pancreatic islets were well tolerated in the C57BL/6 inbred background, which was syngeneic with the stem cells used to derive them, and is analogous to an autograft.21 Nevertheless, it is important to demonstrate that the same holds true for other species, which may be more distant evolutionarily than mouse and rat.

Allograft considerations While autograft IBC is attractive as an ideal strategy, in practice it may be very difficult to generate “personalized” IBC grafts tailored for each individual patient. Generating iPS cells can be a challenging and an expensive proposition. Furthermore, every iPS cell line is different with regard to its ability to differentiate, and it is possible that certain ones will work better for IBC than others. Significant quality control effort must accompany the process of generating any individual cell line to ensure that it does not become tumorigenic or otherwise carry mutations that could damage the host.29 Given the low efficiency of IBC to begin with, adding a requirement to optimize the process for each individual cell line may well place the technology out of reach. Even if these considerations can be overcome, if autologously derived, the IBC graft will carry the same mutations that caused disease in the recipient’s pancreas, which may result in the production of an IBC pancreas with disease (or require a costly genetic correction step to avoid this, if the genes involved can be identified). Thus, at least in the short term, it seems unlikely that iPS IBC will be developed at the level of personalized therapy. One possibility is to instead utilize allograft iPS cells, which could be produced from a subset of human leukocyte antigen (HLA)-matched founder lines.30,31 The resultant IBC allografts could be HLA-matched to the patient recipient, to reduce the likelihood of acute or chronic rejection events. Although such an approach might be convenient, and could conceivably improve the supply chain for these organs, it is worth considering whether an IBC allograft strategy could realistically compete with deceased donor pancreas as a source of transplant. After all, such an IBC allograft pancreas would still require HLA matching and immunosuppression, much like a deceased donor pancreas, and would come with all of the added risks associated with IBC, in addition to

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Breeding schemes

its technical difficulty and expense. Pancreatic islets are also immunologically complex, for instance, expressing HLA-G, and the effect this complexity might have on IBC transplantation is not yet clear.32 Thus, allograft IBC may not be a viable approach in today’s market and in lieu of further research. Alternatively, multiple groups are engaged in developing “universal donor” cell lines that are engineered to express a limited set of HLA molecules that enable them to evade the immune system and cannot be readily recognized or rejected by a host.33,34 These could make a useful source of donor pluripotent stem cell IBC. Such organs could have clear superiority over deceased donor organs, in that they would not require HLA matching or immunosuppression. In addition, only one founder pluripotent stem cell line could suffice for thousands of recipients. This would be a true “off the shelf” pancreas for transplant, and a worthy successor to deceased donor pancreases. Attractive as this vision may be, there are also certain risks associated with HLA-engineered cells. For instance, should any cell within the organ become tumorigenic, it is likely that the tumor could evade the immune system of the host, due to its natural invisibility to the immune system. We do not fully understand how the immune system will interact with universal cells over long periods of time, particularly in humans. There is also a substantial risk that the universal cells could become contagious and spread from person to person, like certain cellular cancers found in canines and Tasmanian devils. These risk factors need to be assessed in animal models and mitigated to avoid adverse outcomes of universal cell IBC transplantation, compared to conventional transplant modalities.

Efficiency of IBC The efficiency of IBC is currently very low. In the original report of pancreas IBC, 10 mice with rat pancreas were born, out of a total of 139 implanted embryos.10 In addition, only 2 of the 10 that were born survived to adulthood. While this level of success may be tolerable for certain mouse experiments, where a large litter is born every 3 weeks, it is unlikely to be useful in larger, more clinically relevant species with much longer gestational times. Fundamentally, why the success rate of IBC is so low is not yet understood at the mechanistic level. It is clear that the IBC success rate is substantially lower than same-species blastocyst complementation. Even in the absence of IBC, interspecies chimerism is lower than intraspecies chimerism, and this is true even in rodents, which are relatively tolerant of chimerism between different species.35 This may be attributable to mismatches

between the pluripotent stem cells of the donor species with the host embryo at the stage of the blastocyst, or later stages such as the induction of pancreas. As we have already discussed, the role of cross-species immune rejection is currently poorly understood in blastocyst complementation, and may also contribute to the generally low success rate of IBC. Attempts have been made to produce better matching between host embryo and donor IBC cells, in the hopes of increasing the efficiency of IBC. One issue has been that pluripotent stem cells from different species have strikingly different properties. Mouse ES cells, for instance, tend to stabilize in vitro in a “naïve” pluripotent state, which is highly compact and do not depend on fibroblast growth factor signaling. In contrast, human ES cells grow in a “primed” state that is closer to the epiblast stage of the embryo, depend on fibroblast growth factor, and forms disc-like colonies in culture. As might be predicted, these two types of cells do not mix particularly well in the setting of a blastocyst.16–20 Recent work has attempted to identify culture conditions for hPSC (including both embryonic and iPS cells) that enhance chimerism during blastocyst complementation in rodents, possibly by mimicking the naïve state.18,36–38 Although most of this work has been performed on mice, finding appropriate match is particularly important in non-rodent species. In one recent study, the potential for chimerism between human stem cells and two large domestic species, cattle and pigs, was explored.13 These studies suggested that the pig could in some cases exhibit a very low degree of chimerism from human cells, although this likely represented less than one-tenth of 1% of all cells in the embryo. Such experiments have only been performed in the absence of IBC. A critical experiment, which has not yet been performed, is to evaluate human chimerism in these species in the context of IBC. Whether IBC could be successfully performed using human cells in a pig embryo is not yet clear, from a technical standpoint.

Breeding schemes The genetics of producing blastocyst complementation offspring are rather complicated. Due to the nature of the complementation-associated mutations, which prevent the formation of essential organs, the host animals can rarely be bred as homozygotes. The simplest approach is to breed heterozygotes (e.g., Pdx1+/−), but due to the Mendelian rules only ~25% of all resultant embryos are homozygous nulls and appropriate hosts for IBC. To identify appropriate host embryos, one strategy is to screen for homozygous null embryos using b ­ lastocyst

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polymerase chain reaction (single cell biopsy of the blastocyst, followed by PCR (polymerase chain reaction) of the mutant locus), which could then be selected for IBC. Although this approach can reduce the number of embryos needed for implantation, it would need to be timed carefully to coordinate it with transferring of the embryos into the pseudopregnant mother. It should also be noted that heterozygous breeders may also manifest disease phenotypes, due to haploinsufficiency. For instance, heterozygous Pdx1 rats exhibit a diabetic phenotype.21 The impact of such illnesses on breeding efficiencies must therefore also be taken into consideration when heterozygotes are utilized. An alternative breeding scheme to increase efficiency of IBC incorporates blastocyst complementation within the host species to generate suitable parents. For instance, to generate rat pancreas in mouse, the male parent was a chimeric Pdx1−/− mouse that had received complementing wild-type cells as a blastocyst. This mouse could be mated with multiple Pdx1+/− female mice to obtain litters, each of which was 50% Pdx1−/−. If a female chimera could be obtained, the ratio could be increased to 100%. This illustrates how creative application of blastocyst complementation can be used to circumvent some of the limitations associated with the technique. In a similar vein, tetraploid complementation can be used as an alternative to conventional Mendelian breeding schemes to generate a host soma of the desired genotype. This begins by intentionally fusing together the cells of a two-stage wild-type blastocyst into a single cell, using electrical current. Without complementation, the resultant tetraploid cell will proceed through the early stages of embryonic development, and will be capable of generating extra-embryonic tissues of the embryo, but will not be able to generate soma in the longer term. To perform tetraploid complementation, the tetraploid blastocyst or morula can be injected with ES cells of the desired genotype. The resulting soma will derive entirely from the injected ES cells. To generate chimeras (e.g., for IBC), ES cells carrying the host knockout mutation can be injected together with the complementing cells.39 In an IBC setting, the resultant embryo would be guaranteed to be a knockout, circumventing the inefficiency issue that plagues Mendelian breeding schemes. Although this is an interesting approach, there are certain limitations. Tetraploid complementation is a specialized technique that may not be readily available to many laboratories. It is typically performed only in mice, and may not work in other species with more clinical relevance for human IBC. Blastocyst complementation via tetraploid complementation has only been demonstrated within the mouse species, using mouse donor and mouse host cells in a tetraploid embryo—IBC has not yet been demonstrated.39

Another possible methodology that could potentially be used to increase the efficiency of IBC breeding is somatic cell nuclear transfer (SCNT), in which a somatic nucleus is implanted into an enucleated egg to produce a zygote. SCNT, which is also known as reproductive cloning, offers complete control over the donor genome, and is used relatively frequently in pigs to generate mutants.40,41 It is compatible with blastocyst complementation techniques, as a means of generating a knockout host, as was demonstrated for same-species blastocyst complementation of pancreas in pigs.15 The drawback of this approach, however, is that SCNT is itself less efficient than straightforward breeding schemes, and comes with a substantial risk of embryo loss or disease. It is not difficult to imagine, however, that the process of using SCNT to generate IBC hosts might be optimized in a choice species, by selecting the most compatible cell lines for this process as nuclear donors, which would result in a streamlined approach for the large-scale “farming” of organs.

Gene editing with IBC A relatively new approach to circumvent classical breeding schemes is to utilize genome editing in host embryos to target a gene or genes of interest. The CRISPR (clustered regularly interspaced short palindromic repeats) gene editing system has been used to accomplish this in the context of IBC in the mouse.13 The CRISPR system uses a nuclease (Cas9) that can be directed to specific loci in the genome by a short RNA sequence, called a guide RNA.42,43 Mismatch repair of the resultant double-stranded break frequently introduces short insertions or deletions (indels) at the targeted locus. This is a powerful methodology for editing the genome, and particularly for disrupting genes. To test the potential of CRISPR for IBC, a guide RNA targeting a gene required for organogenesis of the pancreas, heart, or eye was injected into mouse zygote-stage embryos, together with the Cas9 enzyme.13 This produced knockout host blastocysts, which were then injected with rat iPS cells that had been transiently exposed to a fluorescent label, into elicit IBC. The resultant chimeras contained high contribution of rat cells in the organ of interest, with substantially lower contribution elsewhere in the body. Although the CRISPR approach has substantial advantages in terms of its ease of use, there are some concerns regarding its reproducibility and efficiency.44 In particular, the likelihood of mosaicism in such embryos is a concern that needs to be more fully addressed. Because the mutation is introduced at the zygote stage, there is a substantial risk that knockout will occur only in a proportion of the embryo’s cells. This could lead to a false positive rate

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Ethical concerns

of apparent chimerism, which would be enhanced by the presence of host species cells. Indeed, transplantability experiments designed to show clinical relevance have eschewed using the first generation of gene edited rat hosts, in favor of using “F1” second-generation animals with confirmed germline mutations.21

Suitability of large animal hosts To date, IBC has only been demonstrated in rodent species. For the approach to work for humans, however, it will likely to be necessary to successfully execute IBC with human donor cells in a large animal species. This is for two reasons: first, hPSC have consistently been unsuccessful in engrafting in rodent embryos; and second, a rodent host is unlikely to be of sufficient size to produce functional organ for a human being. To date, however, no one has been successful in using human cells for IBC in any animal species. Practically speaking, it is important to find a species in which human IBC might actually work, that is, the embryos should be sufficiently compatible to successfully produce a human IBC organ in the host. Nonhuman primates would seem to be a logical choice from an evolutionary point of view, but the ethical and financial challenges of performing experiments in such species, outside of their natural habitat, makes them less than suitable for these types of applications. Thus, a domesticated species typically farmed and consumed by our society would be a far better choice, if IBC could be made to work. From a xenogenicity standpoint, the same considerations that make a host more suitable for xenograft also apply for IBC. Pigs, for instance, have certain advantages for xenotransplantation, including their wide availability, domesticated nature, and organ structure similar to humans.1,2 However, pigs also have specific drawbacks including endogenous viruses that might spread to humans,40,45 and antigens that provoke a hyperacute rejection response in primates.1,2 To some degree these concerns can be ameliorated using genetic strains designed to improve cross-species xenotolerance or safety.40,45–47 It might be possible to perform IBC in such strains, as well, to reduce the risk of graft rejection or recipient infection.

Ethical concerns There are significant ethical concerns surrounding the creation of animals containing human cells. Although IBC enriches for chimerism in a particular organ, a low degree of chimerism may also arise in non-IBC organs.13 IBC donor hPSC can potentially differentiate into any of

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the body’s somatic cell types. In particular, there is a concern that human brain or germ cells would be present in an IBC chimera that was implanted with hPSC.44,48 The presence of human brain cells being generated inside animals would raise questions about the consciousness of the organism and whether such a chimera would have human rights. The brain is a particularly sensitive organ for pancreas IBC, because Pdx1 is expressed not only in the pancreas but also in certain types of cells in the brain.49 Thus there is a risk that generating Pdx1 chimeras would create a niche in the brain, as well as in the pancreas. In addition to the brain, there is also concern over the potential of human cells to enter the germline of another species. This could occur, for instance, if a mutation were present that enabled the generation of human eggs or sperm within an animal. This could create a situation where human reproduction becomes uncoupled from speciation, and perhaps even lead to hybrids between humans and other species. Human germ cells, even very rare ones, would potentially enable chimeras to mate with one another and conceive wholly human embryos inside their reproductive organs, which would raise contentious questions regarding the rights of those embryos. Due to concerns such as these, it is currently illegal to create human-animal chimeras in Japan. The Japanese group that invented IBC was therefore compelled to transfer its studies to the United States to perform experiments in pigs with human cells. Even in the United States, the National Institutes of Health has instituted a funding moratorium on these types of experiments, due to the ethical uncertainties surrounding this research strategy. Thus, while not illegal in the United States, financial support for IBC studies is limited to private funds.44,48 One proactive approach to reduce the ethical risk is to utilize genetic tools to direct differentiation of the donor cells into the lineage of choice, rather than off-target lineages. This has been tested in mouse same-species blastocyst complementation, using donor stem cells that were genetically engineered to express Mixl1, a transcription factor that biases early embryonic cells toward an endodermal fate. When Mixl1 was forcibly expressed during the first several days of embryonic development, either using a doxycycline controllable promoter or expression from the OCT4 promoter, the resultant embryos showed reduced contribution of the donor stem cells to non-endodermal germ layer derivatives, such as the fur coat.39 However, the extent to which this skewed the differentiation away from off-target lineages was not carefully quantified, and it is possible that some cells escaped selection. Other strategies may also be possible. For instance, rather than implant hPSC, it might be possible to instead implant a more differentiated stem cell specific to

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32.  Interspecies blastocyst complementation

the organ of interest (for instance, pancreatic progenitor cells). These would be restricted to a specific cell fate and would unlikely contribute to other organs such as brain or gonads. It is also worth noting that the levels of chimerism between humans and domestic large animals in the absence of IBC is extremely low to date, and therefore may ultimately be negligible at the functional level.13 When embryos from different species have been combined into one in the absence of IBC, chimerism has only been successfully achieved between certain closely related species such as mouse and rat, or goat and sheep.10,50,51 Although there are examples of hPSC contributing to rodent embryo formation, chimerism levels are very low and do not produce viable animals.16–20 As a result, in IBC, donor cells contribute mainly to niche cell types but not in a significant way to the rest of the embryo. The formation of gallbladder in chimeric animals also suggests a strong need for a survival-promoting niche to produce mature structures. Mice naturally possess gallbladders while rats do not. Although rat blastocysts can tolerate a significant degree of chimerism from mouse cells (>20% of cells in certain lineages), gall bladders were never observed in such interspecies chimeras.10 Thus, an empty organ niche is necessary, but may not be sufficient, to drive IBC in the absence of a biological need for the organ in question. There is also a strong ethical argument to be made in favor of the potential benefits of IBC for patients in need of transplantation, many of whom may never receive an allograft transplant due to the restrictions of current waiting lists and the global organ shortage. If IBC can be made fully immunocompatible, without the need for chronic immunosuppression, for instance via an autograft strategy as described above, this would also represent a major advantage over existing organ replacement therapies, which are associated with strong side effects of the immunosuppressive medications. Thus, the ethical concerns surrounding IBC may be surmountable, either from a technical or a philosophical perspective, and development of IBC in a safe and responsible manner through preclinical research should continue. Nevertheless, as this work proceeds toward more clinically relevant experiments with human cells, it is important that it do so carefully, to avoid outcomes that traverse ethical lines. Such experiments could be widely perceived as “going too far,” and could usher in a backlash against scientific research.

Outlook Demonstration that IBC pancreas can be generated and used for islet transplantation in the mouse is an important advance and proof of principle experiment.

Such experiments have not yet been performed to generate an IBC human pancreas. At this time, there remain more challenges in this field than solutions. To achieve the grand vision of IBC in a therapeutic context, critical issues, both technical and ethical, need to be addressed in preclinical models. Creative solutions using gene editing and other disruptive technologies, combined with trial-and-error approaches, are likely to be required to advance IBC into a viable option for the clinic. As this work develops, presumably there will come a point at which the benefits of having a fully formed, functional, and mostly human IBC pancreas available for transplant will outweigh the risks of not receiving such a transplant, at least for certain patients. At that point, IBC pancreas will be ready for clinical trials. Such trials must be performed very slowly and carefully, initially with islets and subsequently with whole pancreases, with abundant monitoring of the patients for both acute and chronic complications, including rejection and infection. As with allograft transplantation of solid organs, this is likely to be a long process, which will require patience and persistence to achieve successfully. The reward may be a far more functional system for pancreas transplant, with substantial increases in availability, safety, and efficacy, than is currently possible.

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