Recent Advances in Interspecies Chimeras and Organogenesis

Abstract

Interspecies chimeras have served as a crucial tool for understanding the mechanisms of tissue regeneration and repair, offering promising potential to address the global organ shortage crisis. Through a technique known as interspecies blastocyst complementation, researchers can cultivate tissues and organs of one species within the body of another species. This approach involves injecting donor pluripotent stem cells (PSCs) into a host blastocyst that lacks critical developmental genes, allowing the donor cells to compensate for the missing organs or tissues in the host and thereby produce organs derived from the donor species. This review consolidates key findings from studies published in the past two years, highlighting advancements in techniques that enable the development of functional organs across species, as well as the remaining challenges.

Introduction

The global shortage of transplantable organs remains a critical challenge in modern medicine. Every year, thousands of patients die while waiting for organ transplants due to the scarcity of suitable donors (https://www.organdonor.gov/learn/organdonation-statistics). In response to this crisis, researchers have turned to interspecies chimaeras as a promising solution for generating human-compatible organs in large animals. Interspecies blastocyst complementation, which involves the introduction of PSCs from one species into the genetically modified blastocyst of another species, enables the growth of functional organs that could potentially be used in human transplantation.

Recent advancements in identifying genes suitable for interspecies blastocyst complementation and overcoming interspecies chimerism barriers have led to an increasing number of reports on successful intra- and inter-species organogenesis. While significant barriers remain, innovative approaches in synthetic biology and genetic engineering are progressively overcoming these obstacles. At the same time, the ethical and regulatory challenges surrounding interspecies chimeras must not be overlooked. The potential for human cells to contribute to the brain or germline in animal hosts raises complex moral questions that need to be addressed before these technologies can be applied clinically. This review aims to provide a comprehensive overview of the latest developments in interspecies chimerism, focusing on organ generation and tissue repair, while discussing the technical, ethical, and regulatory challenges that accompany these advancements.

Lung

Yuri, Murase, and Isotani [1] explored the generation of mouse and rat lung tissue in Fgfr2b-deficient mice using the tetraploid-based organ complementation (TOC) method. Their approach involved first injecting Fgfr2b-knockout (KO) mouse embryonic stem cells (ESCs) into wild-type (WT) tetraploid mouse embryos at the E2.5 stage, followed by the introduction of either mouse or rat WT ESCs at the E3.5 stage, with subsequent embryo transfer. Their findings revealed that both mouse and rat WT ESCs successfully contributed to lung epithelial cell formation in the Fgfr2b-KO mouse model via TOC. However, while mouse ESC-derived lungs were functional after birth, lungs derived from rat ESCs were not. Interestingly, the study revealed that rat ESC-derived lung epithelium maintained its intrinsic developmental timing despite developing within the Fgfr2b-KO mouse environment—an observation that contrasts with findings in brain tissues, where interspecies blastocyst complementation led to developmental synchronization [2,3].

Wen et al. [4] investigated the feasibility of generating fully functional mouse lungs in a rat model by employing CRISPR-Cas9 to disrupt the Nkx2–1 gene, which is critical for lung and thyroid development, in rat embryos. By complementing Nkx2–1⁻/⁻ rat blastocysts with mouse ESCs, they successfully restored pulmonary and thyroid structures in mouse-rat chimeras. Remarkably, at embryonic day 20.5 (E20.5), mouse ESCs contributed to 98.5% of all respiratory cell lineages, including epithelial, endothelial, stromal, and hematopoietic cells, within the chimeric rat-mouse lungs. The engineered lung tissues exhibited proper lineage differentiation and functional gene expression profiles, closely mirroring normal mouse lung development. However, despite the presence of peripheral lung and thyroid structures in the Nkx2–1⁻/⁻ mouse-rat chimeras, the study identified defects in tracheoesophageal separation and mispatterning of the proximal trachea—phenotypes consistent with Nkx2–1⁻/⁻ mouse mutants [5]. An intriguing difference between Nkx2–1⁻/⁻ mouse-rat chimeras and Nkx2–1⁻/⁻ mouse-mouse chimeras was the rarity of endogenous thoracic cyst formation in rat hosts, whereas such cysts were commonly observed in mouse embryos[3]. Another notable observation was that Nkx2–1⁻/⁻ mouse-rat chimeras at E20.5 had smaller body and lung sizes compared to wild-type rat embryos. This contrasts with findings from interspecies blastocyst complementation for generation of several other tissues, such as pancreas and forebrain, where the body and organ sizes of chimeric animals closely resembled those of the host species[3,6–8].

Miura et al. [9] introduced an innovative strategy for generating fully functional lungs alongside the thymus using Foxa2 promoter-driven conditional blastocyst complementation in Fgfr2-deficient mice. Through lineage tracing, they demonstrated that the gastrulating Foxa2 lineage contributes to both lung mesenchyme and epithelium formation. By selectively targeting Fgfr2 within the Foxa2 lineage, they successfully ablated both lung and thymus developmental niches, creating a permissive environment for donor cell complementation. To rescue this phenotype, WT donor mouse PSCs were injected into the Fgfr2-deficient blastocysts, effectively repopulating the lung epithelium, mesenchyme, and thymic epithelium. Notably, donor-derived cells replaced nearly the entire lung epithelial compartment and a significant portion of the mesenchymal niche, leading to the formation of a fully structured and functional lung. Importantly, these lungs exhibited normal respiratory function in adulthood, and the regenerated thymus was operational. While effective for the same species, it remains unclear whether donor rat PSCs can achieve similar levels of complementation. Addressing this question will be crucial for extending the applicability of interspecies lung generation and advancing regenerative medicine.

Wang et al. [10] mainly used blastocyst complementation as a tool to test how regulatory enhancers control lung blood vessel development, rather than focusing on generating lungs across species. In short, they used CRISPR to delete conserved FOXF1 enhancers in mouse ESCs and then tracked these modified cells through blastocyst complementation. This allowed them to connect the loss of these enhancers to features of Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins phenotypes.

Heart and Vascular System

Coppiello et al. [11] conducted a study demonstrating the feasibility of simultaneous heart and vascular system complementation in rodents using both intraspecies and interspecies blastocyst complementation. The authors employed selective cell ablation in Nkx2.5-Cre; Tie2-Cre; R26-DTA mouse embryos, making them incapable of forming a functional heart or vascular system. Through intraspecies blastocyst complementation with mouse donor PSCs, they successfully rescued both cardiac and vascular development, generating fully complemented hearts composed of donor-derived cardiomyocytes and endothelial cells. The resulting chimeras were viable, reached adulthood, and exhibited normal cardiac function without histopathological abnormalities. For interspecies applications, the authors introduced rat PSCs into mouse embryos, successfully generating structurally complete and functional hearts at the embryonic stage. However, these rat-mouse chimeras encountered challenges in vascular integration, leading to developmental arrest at E10.5.

Founta et al. [12] generated allogeneic hearts in chimeric mouse embryos using induced blastocyst complementation, a technique designed to overcome the limitations of traditional blastocyst complementation for heart development. Their method selectively ablates “host” cells within the developing cardiac niche by leveraging a diphtheria toxin expression system controlled by the AR1 cardiac enhancer of Nkx2.5. This targeted ablation creates an emptied cardiac developmental niche, allowing exclusive repopulation by “donor” cells. Through this strategy, the authors successfully generated chimeric embryos in which the entire heart was composed solely of donor-derived cells. Notably, a similar Cre-mediated conditional cell ablation and blastocyst complementation approach has previously been employed to investigate the embryonic heart’s response to fractional cell loss [13]. While this technique has proven effective in mouse models, its applicability to xenogeneic heart generation remains uncertain.

Yuri et al. [14] used a Mesp1/2-deficient mouse model to explore the possibility of generating functional hearts through interspecies blastocyst complementation. They found that injecting rat PSCs into Mesp1/2-deficient mouse blastocysts led to heart formation, with rat cells mostly making up the developing heart. However, the rat hearts generated were not functional after E12.5, chimeras showed heart defects and died around day 14.5. This highlights a major barrier in growing hearts across species. Compared to earlier approaches using the Nkx2.5-Cre/R26-DTA system, where interspecies chimeras survived only until E10.5, the Mesp1/2-DKO model supported chimeras up to E12.5. This extended survival may reflect the broader contribution of rat cells in the Mesp1/2-DKO model, where they populated both cardiac tissue and the cardiac vasculature, unlike in the Nkx2.5-Cre/R26-DTA model, where rat cells complemented only cardiac tissue without vascular integration. A notable limitation of Yuri et al.’s study is the incomplete understanding of the molecular and cellular mechanisms underlying the observed rat heart abnormalities within the mouse environment. Future studies, especially using single-cell RNA sequencing, could help uncover the key cellular interactions and molecular signals behind these interspecies barriers—knowledge that could be crucial for improving organ generation strategies in more complex models.

Brain Tissue

Huang et al. [3] successfully generated functional rat forebrain tissue within mice using interspecies blastocyst complementation, enhanced by C-CRISPR—a gene-editing technology that enables rapid knockout screening. Through this approach, they identified Hesx1 as a critical gene whose knockout in mouse embryos creates a permissive niche, allowing rat ESCs to develop into forebrain structures. The rat-derived forebrains in the chimeric mice exhibited proper structural and functional integration, maintaining electrophysiological properties and rat-specific gene expression profiles. Interestingly, comparative analyses revealed that the brain size and developmental pace of rat-mouse chimeras closely aligned with those of their mouse hosts, suggesting the influence of non-cell-autonomous regulatory mechanisms in interspecies brain development. However, a notable limitation emerged during mid-to-late prenatal development. Unlike in mouse controls, the contribution of rat cells in the developing Hesx1-deficient mouse forebrain, initially high (90–100% between E9.5 and E12.5), gradually declined to approximately 60% by E15.5 before stabilizing until birth. This progressive decrease indicates the presence of xenogeneic barriers that impede sustained rat cell integration during later developmental stages. This study underscores the potential of interspecies blastocyst complementation as a powerful tool for investigating brain development in an evolutionary context. At the same time, it highlights the need to address species-specific developmental barriers that arise during mid-to-late gestation—an essential step for advancing xenogeneic brain generation and its applications in neuroscience and regenerative medicine.

Throesch et al. [2] investigated the formation and functional viability of neural circuits in interspecies brain chimeras by integrating rat neurons into mouse brains through conditional blastocyst complementation. The study aimed to determine whether neurons from two species that diverged approximately 10–20 million years ago could successfully develop, synchronize, and function within a heterologous brain environment. By injecting rat PSCs into mouse blastocysts, the authors generated chimeric mice with rat-derived neurons contributing to various brain regions, including sensory circuits involved in olfaction. Using whole-brain imaging, genetic silencing, and electrophysiological recordings, they demonstrated that rat neurons were capable of forming synapses with mouse neurons and functionally integrating into neural networks. Remarkably, these rat neurons restored olfactory-driven food-seeking behaviors in anosmic (odor-impaired) mice, providing compelling evidence of cross-species neuronal functionality. These findings underscore the potential of interspecies blastocyst complementation as a tool for studying neural plasticity and evolutionary brain development. However, achieving precise control over donor cell contributions remains a critical challenge for future applications in neuroscience and regenerative medicine.

Other tissues

The generation and transplantation of whole salivary glands hold promise as potential therapies for salivary gland dysfunction. However, identifying the precise cellular lineage required to engineer fully functional salivary glands has remained a challenge. To address this, Tanaka et al. [15] identified the Foxa2 lineage as a key determinant in salivary gland development. Through conditional knockout (cKO) of the Fgfr2 gene within this lineage, they demonstrated that Fgfr2 deletion resulted in complete salivary gland agenesis. To restore salivary gland formation, they introduced donor mouse PSCs into these Fgfr2 cKO mouse blastocysts, generating chimeric mice with fully developed salivary glands. These donor PSC-derived glands exhibited normal size, morphology, and secretion capabilities, closely resembling those of healthy control mice. Importantly, the chimeric mice survived to adulthood with fully functional salivary glands, highlighting the feasibility of blastocyst complementation for regenerating complex exocrine organs.

Parathyroid gland (PTG) transplantation represents a promising therapeutic approach for patients with permanent hypoparathyroidism, a condition requiring lifelong calcium and hormone replacement therapy. However, current efforts to generate PTG cells from PSCs in vitro have failed to produce cells capable of responding physiologically to extracellular calcium, a critical function for maintaining calcium homeostasis. To overcome this limitation, Kano et al. [16] employed single-step blastocyst complementation to generate fully functional PTGs from mouse ESCs. They genetically engineered mouse embryos to lack endogenous PTGs by knocking out the Gcm2 gene using CRISPR-Cas9. Since Gcm2 is essential for early PTG development, its deletion created a developmental niche that allowed donor mouse ESCs to exclusively populate and form the PTGs. The resulting ESC-derived PTGs were able to regulate calcium homeostasis through parathyroid hormone (PTH) secretion, effectively preventing neonatal mortality in Gcm2-deficient hosts and ameliorating hypocalcemia in surgically induced hypoparathyroidism models. Extending their approach to interspecies PTG generation, the researchers attempted to generate mouse ESC-derived PTGs in Gcm2-knockout rats. While initial success was achieved, the survival rate of rat chimeras was low due to developmental complications, highlighting challenges in interspecies PTG complementation.

Wang et al. [17] tackled the critical issue of kidney donor shortages by demonstrating the feasibility of generating a humanized mesonephros in pigs through induced pluripotent stem cells (iPSCs) and a gene-edited nephric-deficient niche. Their approach optimized interspecies chimerism by enhancing human iPSC viability and integration using a specialized PSC culture medium (4CL) and overexpression of pro-survival genes MYCN and BCL2. Using blastocyst complementation in SIX1/SALL1-null pig embryos, the researchers successfully generated mesonephric structures containing human cells, reaching E28. This achievement provides compelling evidence that human cells can organize and sustain nephric tissue development in a xenogeneic environment, representing a significant advancement in the blastocyst complementation field. Despite these promising results, several challenges remain. It is still unclear whether the human iPSCs enriched in the E28 pig mesonephros can persist through later developmental stages and contribute to the fully functional metanephros. Secondly, this study improved human chimerism by genetically modifying donor human iPSCs. However, this strategy is impractical for clinical applications. A more viable approach would be enhancing the survival and integration of unmodified human iPSCs by optimizing the host embryo—a concept that remains in its early stages of exploration[18,19]. Additionally, given that a chimeric kidney would not be composed entirely of human cells, the risk of immune rejection remains a major concern for transplantation into human patients. Addressing these challenges will be critical for translating interspecies organ generation into viable clinical applications.

Hepatocyte transplantation (HT) is a promising therapy for liver failure, but its clinical application is severely limited by the global shortage of liver donors. To address this challenge, Huang et al. [20] explored the feasibility of using interspecies chimera-derived hepatocytes as an alternative source for liver disease treatment. By injecting mouse ESCs into rat blastocysts, they generated chimeric livers, from which functional hepatocytes were isolated. These mouse ESC-derived hepatocytes exhibited essential hepatic functions—including lipid accumulation, glycogen storage, and urea synthesis—closely resembling those of endogenous hepatocytes. To evaluate their therapeutic potential, they transplanted the chimera-derived hepatocytes into immunodeficient mice with chronic liver fibrosis induced by carbon tetrachloride (CCl₄). The treatment significantly reduced fibrosis markers and liver damage indicators, bringing them to levels comparable to healthy controls. These findings suggest that interspecies chimera-derived hepatocytes could serve as a viable and scalable alternative for HT, offering a potential solution to the hepatocyte shortage in liver disease treatment.

Overcoming Early Developmental Barriers to Interspecies Chimerism

One of the biggest challenges in creating functional interspecies chimeras is overcoming cell-cell adhesion barriers, which prevent donor cells from efficiently integrating into the host embryo. Ballard et al. [21] investigated this issue by studying adhesion incompatibility between human and animal PSCs. To address this, they developed a synthetic biology strategy using membrane-tethered nanobodies, small and highly stable single-domain antibodies with strong antigen-binding properties, to enhance interspecies cell adhesion. In this approach, PSCs from one species were engineered to express a membrane-bound nanobody, while PSCs from the other species expressed the matching antigen, creating strong, specific binding between them. The authors designed mouse and human PSCs to express either GFP or an anti-GFP nanobody tethered to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. This system enabled nanobodies to quickly bind GFP and present it on the cell surface, promoting efficient adhesion. Both in vitro and in vivo experiments confirmed that this approach enhanced adhesion between human and mouse PSCs, leading to improved human cell integration in early-stage mouse embryos. However, while the nanobody-based adhesion system was effective in single cells and small aggregates, its efficiency declined in larger cell aggregates, limiting its ability to achieve robust interspecies chimerism. To address this, further optimizations—such as testing nanobodies with different antigen affinities and exploring alternative membrane anchoring strategies—could improve interspecies cell adhesion and enhance chimera formation.

Shetty et al. [22] propose an in-silico approach to improve interspecies chimerism by matching embryonic development stages across humans, marmosets, mice, and pigs. Since developmental mismatches hinder viable chimera formation between distant species, the authors used single-cell RNA sequencing (scRNA-seq) and unsupervised clustering to identify optimal transcriptomic similarities across species. Their analysis revealed that human blastocysts (E6/E7), marmoset late inner cell mass (ICM), mouse gastrulating embryos (E6–E6.75), and pig late blastocysts were the best stage-matched for effective chimerism. Further validation using Spearman’s correlation and pathway analysis identified shared pathways critical for cell adhesion and metabolism, suggesting their role in supporting organ development. These findings provide a framework for improving interspecies chimerism in both in vivo and in vitro studies, guiding donor-host embryo matching for organogenesis. Future in vivo validation will be needed to confirm these predictions.

Nagaya et al. [23] investigated the potential of insulin-like growth factor 1 receptor (IGF1R)-knockout pigs to improve interspecies chimerism. Building on previous research in mice, which showed that disabling Igf1r in host embryos provides donor cells with a competitive growth advantage from mid-gestation onward [24], the study aimed to enhance donor cell integration across multiple tissues and organs. By generating IGF1R-KO pigs, the researchers successfully recreated the growth-deficient phenotype observed in mice. These pigs are expected to serve as improved hosts for interspecies organ generation through blastocyst complementation, potentially advancing the development of human-compatible organs for transplantation.

Future perspectives

The field of interspecies chimerism and blastocyst complementation have made significant progress in recent years, offering promising solutions to the global shortage of transplantable organs and advancing tissue regeneration technologies. Breakthroughs in generating lung, heart, neural, and kidney tissues through blastocyst complementation, along with strategies to overcome xenogeneic barriers, have fueled growing interest and optimism in this research. Despite these advancements, major challenges remain. Functional integration of donor cells from distantly related species is still a hurdle, and ethical concerns persist, particularly regarding the extent of human cell contribution in animals. Unforeseen developmental consequences, especially in the brain and germline, raise critical questions that must be carefully addressed.

Moreover, species-specific differences in developmental timing can affect the success of interspecies organogenesis. For example, rat ESC-derived lung epithelium keeps their original (rat) developmental pace within Fgfr2b-deficient mouse hosts, whereas rat forebrain cells synchronize with the mouse embryonic timeline but their contribution to the mouse embryo declines during mid-to-late gestation [2,3]. It is possible that rat cells able to adjust to mouse’s developmental pace can survive and integrate, while those that cannot are gradually lost. Such timing mismatches may lead to incomplete tissue development or give mouse cells a competitive advantage over donor cells. In silico transcriptomic comparisons have shown that careful matching of donor and host developmental stages is important—for instance, human blastocysts (E6–E7) align most closely with mouse gastrulating embryos (E6–E6.75) to support better chimerism [22]. Moving forward, it will be important to apply precise staging and, when possible, adjust the host’s developmental niche to better synchronize growth between species.

Findings from rodent models—where both intra- and inter-species blastocyst complementation between mice and rats have highlighted the importance of host developmental niche editing and precise stage-matching—have laid the foundation for advancing human-animal chimerism [1,3,11,14]. Studies using human–mouse chimeras have further identified key molecular barriers, e.g. cell competition [18,25] and mismatched cell adhesion [21], to xenogeneic integration. These insights are directly relevant to human–pig chimera models, and strategies developed in rodent and human–mouse systems—such as transcriptomic stage alignment [22], synthetic cell-cell adhesion [21], and selective growth advantages via host IGF1R deletion [23]— now serve as a blueprint for refining both donor PSC engineering and host embryo design in pigs. Moving forward, integrating these approaches will be essential for improving human cell contribution in porcine embryos and progressing toward clinically relevant human–pig chimeras.

Figure 1:

Key Challenges and Solutions in Generating Xenogeneic Organs via Blastocyst Complementation. (A). Schematic workflow of blastocyst complementation. Embryos with organ developmental agenesis are generated by disrupting critical genes required for specific organ development. These genetically modified blastocysts are transferred into surrogate mothers to produce embryos lacking the target organ. Following injection of xenogeneic induced pluripotent stem cells (iPSCs) or Embryonic Stem Cells (ESCs) into these organ-deficient blastocysts, the iPSCs may engraft into the vacant developmental niche, enabling the generation of functional organs from one species within the body of another. (B). Critical barriers to interspecies chimerism and potential strategies to overcome these challenges. Image created with biorender.com

Table 1.

Recent advances in interspecies chimeras and organogenesis.

Organ System	Host (Gene Defect)	Donor Cells	Complementation Method	Developmental Stage	Outcome	Reference
Lung	Mouse (Fgfr2b−/−)	Mouse ESC	Tetraploid-based organ complementation (TOC)	Postnatal	Fully functional lungs	Yuri et al. [1]
Lung	Mouse (Fgfr2b−/−)	Rat ESC	TOC	Postnatal	Maintains rat-intrinsic timing; non-functional after birth	Yuri et al. [1]
Lung	Rat (Nkx2-1−/−)	Mouse ESC	Blastocyst complementation	E20.5	98.5% respiratory lineage restoration; tracheal mispatterning and smaller size	Wen et al. [2]
Lung	Mouse (Foxa2-lineage Fgfr2−/−)	Mouse PSC	Conditional (Foxa2-Cre) complementation	Adult	Fully structured, functional lung and thymus	Miura et al. [3]
Heart & Vasculature	Mouse (Nkx2.5-Cre; Tie2-Cre; R26-DTA)	Mouse PSC	Intraspecies blastocyst complementation	Adult	Viable, normal cardiac function and vasculature	Coppiello et al. [4]
Heart & Vasculature	Mouse (Nkx2.5-Cre; Tie2-Cre; R26-DTA)	Rat PSC	Interspecies blastocyst complementation	E10.5	Complete heart structure; vascular integration fails, arrest at E10.5	Coppiello et al. [4]
Heart & Vasculature	Mouse	Mouse PSC	Induced (Nkx2.5-AR1-DTA) blastocyst complementation	Embryonic	Entire heart composed of donor cells	Founta et al. [5]
Heart & Vasculature	Mouse (Mesp1−/−,Mesp2−/−)	Mouse or Rat PSC	Blastocyst complementation	E12.5–E14.5	Mouse ESCs rescue the lethal phenotype of cardiac deficiency in Mesp1/2-DKO mice. Rat cells populate heart; defects and lethality by E14.5	Yuri et al. [6]
Brain	Mouse (Hesx1−/−)	Rat ESC	CRISPR enhanced blastocyst complementation	Adult	Functional rat forebrain; contribution declines mid-to-late gestation	Huang et al. [7]
Brain	Mouse	Rat PSC	Conditional blastocyst complementation (OMP-Cre)	Adult	Rat neurons form synapses and restore olfactory behavior	Throesch et al. [8]
Salivary Gland	Mouse (Foxa2-lineage Fgfr2−/−)	Mouse PSC	Conditional (Foxa2-Cre) complementation	Adult	Fully developed, functional salivary glands	Tanaka et al. [9]
Parathyroid	Mouse (Gcm2−/−)	Mouse ESC	Blastocyst complementation	Neonatal	Functional PTG; prevents neonatal hypocalce mia	Kano et al. [10]
Parathyroid	Rat (Gcm2−/−)	Mouse ESC	Blastocyst complementation	Embryonic	Low survival of chimeras due to developmental complications	Kano et al. [10]
Kidney	Pig (SIX1/SALL1−/−)	Human iPSC	Blastocyst complementation	E28	Humanized mesonephros formed; persistence and full maturation unknown	Wang et al. [11]
Hepatocyte	Rat	Mouse ESC	Blastocyst complementation	Adult (isolated cells)	Functional hepatocytes that rescue liver fibrosis in mouse models	Huang et al. [12]