Organ transplantation is the ultimate treatment option for patients suffering from refractory diseases. However, there has been an absolute scarcity of donor organs, and the gap between available donors vs. waitlisted recipients continues to expand (Giwa et al., 2017). In 2017, approximately 114,000 patients in the United States waited for an organ transplant (Sykes and Sachs, 2019). Presently, in the United States, another person is added to an organ transplant list every 10 min, 17 people die each day while waiting for donor organs, and approximately 105,800 patients are waitlisted for an organ transplant according to the health resources and services administration (HRSA). To overcome this significant crisis, researchers are investigating various approaches involving direct xenotransplantation, organoids, decellularization, and recellularization, and more recently, organ bioengineering using blastocyst complementation (BC). Depending on the patient’s medical condition, a refractory disease patient also requires an on-time selective option, such as less invasive cellular therapy options or curative organ transplantation that can function immediately after transplantation. In this regard, whole organ generation via the BC approach holds great promise with a ready resource (livestock) for cellular therapies and as a radical treatment option for most terminal diseases. However, though BC is emerging as a potential organ transplant option, challenges regarding organ size scalability, immune system incompatibilities, long-term maintenance, potential evolutionary distance, or unveiled mechanisms between donor and host cells remain. These challenges can be overcome by a multifaceted approach, especially by filling in the knowledge gaps on the mechanisms of interspecies chimera formation. In this review, we summarize the history of interspecies chimerism in various animal models to find hints for BC application and describe the challenges and prospects of utilizing BC for human organ generation.

In ancient history, humans used the term “chimera” to describe mythical creatures and hybrids. Animals sexually derived from the fusion of gametes from two different organisms, such as mules, are considered “hybrids.” On the other hand, a chimera is defined as an organism in which cells from two or more different organisms have contributed. In nature, hybrids and chimeras are rare due to incompatibilities and developmental divergence during evolution. At the organismal level, evolutionary distance is defined by genome-wide sequence homology between species. Humans, and other species, such as mice, swine, birds, etc., have diverged from one another. A molecular program that causes the interspecific barrier might be independent of genome-wide evolution. Instead, it may depend on the host and donor molecular similarities or distinctions critical for the organogenesis program. For instance, human LIF protein can signal via LIF receptors to help maintain a high degree of stemness in mice and rats, but the converse is ineffective (Dahéron et al., 2004). How evolutionary distance affects the formation of hybrids and chimeras is an open question that may vary from situation to situation.

In 1997, a breakthrough discovery was made: Dolly the sheep. Dolly was the first mammal cloned from an adult cell using somatic cell nuclear transfer (SCNT) (Wilmut et al., 1997). This study proved that a differentiated cell has the ability to produce exact copies of its source animals. SCNT has been successfully applied to clone 24 mammalian species, including mice, cows, pigs, cats, rats, dogs, and monkeys (Rodriguez-Osorio et al., 2012). However, SCNT was not successful in interspecies attempts. The process had been tested through electro-fusion using donor somatic cell nucleus replacement into a host’s enucleated interspecies egg cytoplasm, but failed. The failure appears to be due to reprogramming elimination of the donor mitochondria by the host mitochondria, and inability to activate the donor embryogenesis program after fusion (Chang et al., 2003; Chen et al., 2003; St John and Lovell-Badge, 2007; Jiang et al., 2011). The interspecific SNCT studies imply that there are critical interspecies barriers at the subcellular level of totipotent zygotes, distinct from interspecies chimera that are at an intercellular level (Figure 1A). The study of interspecies SCNT, focusing on intracellular compartments, such as the interspecific reprogramming process, mitochondrial competition between donor and host, and genome-stability post-electrical fusion, may open new avenues to better understand subcellular interspecies barriers in SCNT.

The field of chimera research emerged nearly 50 years ago, with pioneering experiments using a mixture of pluripotent stem cells (PSCs) in an intra- or interspecies manner. Briefly, cells from the Ryukyu mouse (Mus caroli), when transplanted into blastocysts of the house mouse (Mus musculus), showed very high chimerism compared to cells derived from rat blastocysts (Stanny et al., 1980). On the other hand, cells derived from the bank vole (Clethrionomys glareolus), a more evolutionary distant rodent from mice, showed no detectable chimerism even during the late embryonic stages (Mystkowska, 1975). Similar studies were also performed within closely related mammalian species such as sheep and goats. Composite blastocysts from goats and sheep could successfully generate goat-sheep chimeras (Fehilly et al., 1984). Likewise, chimeric calves produced from two closely related cow species—Bos indicus and Bos taurus, were also viable (Williams et al., 1990). These early experiments made a clear distinction between interspecies and intraspecies chimerism. In other words, the evolutionary distance between two species may be inversely correlated with the extent of chimerism (de Los Angeles and Wu, 2022).

This section reviews various interspecies chimera formation rates and chimerism during development. Chimera formation rate refers to the percentage of host animals with donor cells. Assessment of chimera formation requires checking the markers of donor cell survival, proliferation, and differentiation and proof that the donor cells are responsive to the host animal’s developmental program. Chimerism, on the other hand, indicates the % of donor cell contribution to a particular developmental niche or organism. Overall, the efficacy of intraspecies chimerism and chimera formation is far superior to that of interspecies (Table 1). Elucidation of the interspecies barrier incompatibility between host animals and human cells during development is a breakthrough in the field of human organ generation in a host animal.

“Blastocyst complementation (BC)” is a significant concept for organ generation as a regenerative approach. BC utilizes the host developmental program to incorporate donor PSCs. Donor PSCs, when injected into recipient morula or blastocysts lacking critical genes for organogenesis, can replace the defective organ niches and compensate for the functional defects of recipient cells.

Alt and colleagues first illustrated intraspecies BC when they injected wild-type mouse ES cells into RAG2-deficient blastocysts. Mature B and T cells derived from donor PSCs rescued adaptive immune responses in host mice that were niche depleted in terms of lymphocyte diversity (Chen et al., 1993). Approximately two decades later, Nakauchi and colleagues showed the first evidence of interspecies pancreatic organ generation in mice using rat PSCs (Kobayashi et al., 2010). Remarkably, they also showed that the rat pancreatic tissues generated in mice via BC were transplantable into a recipient rat disease model and rescued its phenotype (Yamaguchi et al., 2017).

Recently, this concept has been extended to large-animal interspecies, yet other barriers such as donor cell pluripotency, cell-cell competition, and other potential xenogeneic incompatibility barriers result in low chimeric efficacies (Wu et al., 2017; Garry and Garry, 2019; de Los Angeles and Wu, 2022; Roodgar et al., 2022). Chimerism during and beyond the post-implantation stage of development presents a separate set of challenges (Figure 1). Firstly, the stage-matching of donor cells into a particular tissue niche requires extensive optimization to ensure the proliferation of engrafted cells or tissues (Cohen et al., 2018; Thomas et al., 2021). Secondly, donor cells require well-fitness to survive cell competition (Wu and Barbaric, 2021). Finally, tissues derived from donor cells need to avoid immune rejection within transplant recipients even under treatment with immune-suppressive drugs (Lu et al., 2019). Critical barriers in interspecies chimerism corresponding to its developmental stage have been summarized in Figure 1.

In 1964, Dr. James D. Hardy performed one of the first xenotransplantation surgeries in modern medicine by replacing a 68-year-old patient’s heart with that of a chimpanzee. Although that first patient did not survive beyond 2 h and succumbed to a hyperacute immune response, there has been tremendous progress in the field of xenotransplantation over the last sixty years: earlier this year, the first porcine-to-human heart transplantation was performed in a patient who was not a candidate for conventional allogeneic heart transplantation, and the patient survived for 2 months (Griffith et al., 2022). Much progress may be attributed to a deeper understanding of the immune system and the relative ease of gene editing (Ekser et al., 2017; Niu et al., 2017). Although many large animals have been considered, pigs are more favored for xenotransplantation mainly because of ease of breeding (with large and frequent litters) and physiologic organ and size similarity to humans. The grafts of porcine origin survived for months and, in some cases, even years upon transplantation into non-human primates. However, xeno-organ transplantation to monkeys, such as lungs, islets, heart, and kidneys, requires systemic immune suppressive drugs for the long term, and most organs are ultimately rejected (Sykes and Sachs, 2019). Strikingly, the thymokidney approach can induce tolerance across allogeneic barriers, prolong the life of the xenograft when transplanted across xenogeneic barriers, and may ultimately be used to reduce the intensity of immunosuppression for xenotransplanted organs (Yamada et al., 1999; 2000; Shimizu et al., 2005; Rivard et al., 2018).

If an organ from a wild-type pig is transplanted directly into a human patient or a non-human primate, the organ will be rejected within minutes. This form of rejection, also known as a hyperacute immune reaction, results from interactions between host-preformed natural antibodies (Nabs) with the galactose-α1,3-galactose (Gal) antigens expressed on cells of pig grafts (Platt et al., 1991). Using antibody absorption techniques, acute rejection can be delayed to a few hours, but eventually, the graft is rejected (Kozlowski et al., 1997). To overcome innate responses and non-Gal preformed antibodies in addition to T Cell dependent responses against graft, researchers have continued to modify the pig genome to 1) target non-Gal natural antibodies and 2) correct species incompatibilities between swine and primate coagulation, phagocytosis and complement regulatory proteins. These include coagulation inhibition by overexpression of anti-coagulation factors hCD39 and hCD141, that express on the surface of endothelial cells, as well as the addition of complement regulatory protein human transgenes (Lee et al., 2018; Singh et al., 2019; Kemter et al., 2020). Using serial SCNT, additional mutations have been accommodated in pigs to avoid anti-inflammatory and anti-apoptotic responses (Fischer et al., 2016). For details on various engineering strategies of an ideal humanized pig, please refer to the latest reviews by Qin and colleagues (Lu et al., 2020).

If permissive, the humanized swine model would be an ideal background for generating niche-depleted host models using the BC approach (Figure 2B). Upon complementation by donor human PSCs, the resulting organ would be suitable for humanized organs, the mixture of human cells and humanized host cells, into MHC-matched patients awaiting organ transplantation. Suppose the entire targeted organ niche that involves endoderm, mesoderm, and ectoderm derivatives would be complemented by human iPSC-derived cells. It is an ideal treatment for MHC-compatible patients awaiting organ transplantation, with minimal or no immunosuppressive approaches.

Transplanted organs should theoretically be superior to humanized porcine-derived organs in function and means of evading short- and long-term immune responses since all cells are derived from each individual patient or MHC-matched donor (Figure 2B).

With the introduction of iPSC cell technology, the field of organoid biology expanded to provide simple yet powerful insights underlying human tissue development and, more importantly, disease modeling of pathophysiological conditions (Rossi et al., 2018; Kim et al., 2020). Various organs, such as the liver, lung, kidney, brain, intestine, and even specialized tissues such as the endometrium, have been modeled using human iPSC-derived organoids, histologically similar to human organs (Lancaster et al., 2013; Takasato et al., 2015; Turco et al., 2017; Fujii et al., 2018; Hu et al., 2018; Sachs et al., 2019). With the increasing success of human iPSC-derived organoid grafting studies, exploring organoid engraftment in a non-human host such as a mouse is exciting. Liver and kidney organoids derived from human iPSCs induced vascularization from host mouse tissue and support host tissue function (Takebe et al., 2013; van den Berg et al., 2018). Likewise, human intestinal organoids showed robust viability when implanted into mouse mesenteric tissues (Cortez et al., 2018). In another set of studies, human iPSC and ESC cell-derived brain organoids established subcortical projections and functionally integrated them into the mouse pre-existing neural (Dong et al., 2020). Human neural crest cells differentiate into melanocytes and produce pigmented hair in a c-kit mutant mouse background that lacks melanoblasts (Cohen et al., 2016). Engraftment of human iPSCs-derived neurospheres recover motor function characterized by synapse formation and increased local myelination in a nude mice model of spinal cord injury (Nori et al., 2011). In two models of acute liver failure, human liver organoids derived from hiPSCs rescued hepatic functions and improved survival rates in mice (Nagamoto et al., 2016; Nie et al., 2018). Human ES cell-derived cerebral organoids, when transplanted into injured mouse cerebral cortices, showed high survival efficiency, robust vascularization from host tissues, and recovery of axonal projections along corticospinal tracts (Daviaud et al., 2018; Kitahara T. et al., 2020). Transplantation of human iPSC-derived endothelial-like cells in a mouse model of hindlimb ischemia caused significant improvement in postnatal vascularization (Cho et al., 2007). The engineered human iPSC-derived organoid transplantation into the defective tissue niche, particularly in the immune-privilege niche such as subrenal regions, testes, eyes, liver, and brain, holds great promise for evaluating stemness and mouse disease modeling as a next-generation personalized medicine (Harrison et al., 2014; Paquet et al., 2016; Driehuis and Clevers, 2017; Artegiani et al., 2020). Overall, human iPSC-derived organoids are compelling disease models in vitro and in vivo. However, the main challenges of organoid.

-based approaches are the lack of transplantation methods, scalability to generate whole organs, and reproducibility to generate all cell types to replicate organ function. Unless these issues are resolved, the feasibility of organoids for organ transplantation is low and will depend largely on the stage of the disease; the earlier, the better, but not likely, at the terminal stage.

Tissue engineering is another approach to generating transplantable organs. In such an approach, the goal is to grow an entire or partial organ in vitro. If an engineered organ could be derived from autologous cells of the recipient, the chances of rejection would be close to zero. Creating mechanically, anatomically, physiologically, and biologically compatible organs similar to native organs is ideal. In this regard, decellularization of the extracellular matrix preserves the vascular network and structural framework (Crapo and Wang, 2011). In theory, a decellularized organ scaffold would function as a barcode of cellular repopulation. After an optimal number of cells have populated an organ scaffold, a bioreactor is used to facilitate organ generation. Bioreactors are devices where biological and/or biochemical processes develop under closely monitored and tightly controlled environmental conditions (Martin et al., 2004). Decellularized scaffolds have been made for various organs, including the lung, heart, kidney, liver, intestine, bladder, corneas, limbs, pancreas, and vasculature. (Ott et al., 2008; Hashimoto et al., 2010; Petersen et al., 2010; Yang et al., 2010; Quint et al., 2011; Totonelli et al., 2012; Song et al., 2013a; Gerli et al., 2018; Goh et al., 2019).

For lung tissue engineering, Niklason and colleagues repopulated acellular rat lung scaffolds with neonatal rat lung epithelial cells and microvascular endothelium. They generated viable lung tissue for up to 1 week under in-vitro conditions (Petersen et al., 2010). Ott and colleagues reported that perfusion decellularization of cadaveric lungs yield intact scaffolds seeded with cells to generate bioartificial lung grafts. Upon transplantation of such bioartificial lungs into rats, the grafts maintained respiratory function for up to 7 days (Song et al., 2011). Large animal trials have also been conducted with the hope of pre-clinical applications. Cortiella and colleagues generated porcine lung scaffolds with autologous cells from porcine recipients. Within 2 weeks of transplantation, the lung grafts generated alveolar tissues with supporting vasculature without any sign of immune rejection (Nichols et al., 2018). Ott and colleagues seeded porcine decellularized lung scaffolds with human airway epithelial progenitor cells derived from human donor lungs and banked human umbilical vein endothelial cells. By repopulating porcine extracellular matrix scaffolds with human endothelial cells, they generated pulmonary vasculature with mature endothelial lining supplemented with anti-thrombotic function to enable blood perfusion. They created a functioning gas exchange graft by repopulating the epithelial surface with human epithelial progenitor cells. This graft could withstand physiological blood flow from the recipient’s pulmonary circulation and exchanged gases upon ventilation during the 1-h after transplantation (Zhou et al., 2018).

Initial studies on kidney tissue engineering focused on tissue decellularization methods. Soker and colleagues decellularized porcine kidney scaffolds using a technique that sustained blood pressure up to 2 weeks post-implantation into recipient swine (Orlando et al., 2012). Decellularized monkey kidneys were also functionally effective and biocompatible (Nakayama et al., 2010). Like the lung, kidney tissue engineering also requires the recellularization of decellularized scaffolds, as shown by various studies using swine, monkey, rat, and human organs. Upon transplantation, bioengineered kidney perfused well after orthotopic transplantation into anephric rats and showed urine production (Song et al., 2013b).

Taylor and colleagues reported perfusion decellularization of the whole rat heart, thus setting the stage for other groups to examine this for large animals such as swine (Ott et al., 2008; Wainwright et al., 2010; Weymann et al., 2011; Momtahan 2016). Human-sized decellularized porcine hearts were also developed and showed recellularization of coronary vasculature and myocardium with measurable electrical activity in the decellularized scaffold (Weymann et al., 2014). Shimizu and colleagues reported heterotopic transplantation of a decellularized porcine heart scaffold with mesenchymal stem cells into recipient pigs. The scaffolds could endure surgical procedures and perform short-term coronary artery perfusion by angiography (Kitahara et al., 2016).

Bioengineering techniques of organ generation involving decellularization-recellularization approaches can be challenging in many ways. For instance, existing protocols cannot guarantee the complete removal of residual toxic products within a decellularized tissue. Furthermore, upon transplanting bioengineered tissue into a recipient organism, the degradation rate of decellularized scaffolds should be synchronous with connective tissue remodeling to prevent transplantation failure. Yet, despite its limitations, bioengineering holds excellent promise for organ generation. Further efforts for optimizing recellularization coupled with drug-or genome-editing-mediated approaches for long-term organ maintenance, particularly endothelial components, are essential for organ transplantation into patients suffering from refractory diseases.

Various chimeric, hybrid, and bioengineering studies are being attempted in many initiatives that could be applied to the generation of human organs. Still, most fields are immature, but the successful impact is enormous. Among them, BC is one of the most promising technologies, especially for generating whole organs for transplantation. One of the critical elements for successful entire organ generation using BC is a better understanding of the mechanism of interspecies chimerism between human and host cells during the host developmental program. However, the studies of human-animal chimeras are limited by a lack of resources and ethical concerns. In particular, the contribution of human PSCs to neurogenesis and reproductive tissues within chimeric animals can have profound ethical implications (Bourret et al., 2016; Kwisda et al., 2020). These concerns can be mitigated by engineering cells to be incompetent of ethical-related lineage differentiation.

Another essential step for BC’s success is preparing human iPS cells with organogenic potential. Comparative genome-wide and epigenome-wide analysis of host animal pluripotency with human iPS cells can provide critical guidance. Besides human pluripotency maintenance in human iPSCs, ES cells have also recently been derived from large animals such as pigs, cows, and sheep (Bogliotti et al., 2018; Choi et al., 2020; Vilarino et al., 2020; Kumar et al., 2021). However, except for one study involving pig blastomeres, it has been technically challenging to accurately define naïve state pluripotency and germline competence of large-animal stem cells (Matsunari et al., 2013). To overcome the incompatibilities at the molecular level for efficient interspecies chimerism and subsequent xenotransplantation, humanized large-animal models need to be established, and attempts are already beginning to emerge (Yang et al., 2016; Boettcher et al., 2020). Most recently, Wu and colleagues showed further evidence for the involvement of interspecific cell competition during early embryogenesis. This paper demonstrated the inflammatory-like pathway activation of Myd88-p65-NFkB signaling in donor human iPS cells. The loss of function of Myd88 resulted in overcoming cell competition in early embryogenesis (Zheng et al., 2021). The common feature of outcompeting donor intra- and interspecies PSCs, including ES and iPSC cells that form functional organs in host large animals, is still an open question.

Bioengineered organ transplantation is another option to solve the shortage of donors. Toward human transplantation, the generation of scaffolds based on swine adapted to human organ sizes or human iPSC-derived organoids is promising. However, the fabrication of whole organs that can withstand clinical application has not been realized. Further conceptual and technological breakthroughs are needed beyond the scaffold barcodes of decellularized organs and the self-assembling function of organoids. In this regard, the BC method has perspective advantages over the organoid and decell-recell approaches in terms of scalability, livestock, and a surgical indication of organ transplantation applicable to most end-stage refractory diseases. The advantage of decell-recell-based or organoid systems over the BC approach is the potential for a xeno-free environment and less invasiveness during transplantation. In the future, depending on the recipient’s refractory disease status, the advantages and disadvantages of each technology should complement each other to provide us with multiple beneficial options.