The last few years have seen a significant increase in the use of technology to manipulate genetic sequences and generate animals as a source of xeno-organs. This has made the generation of genetically bespoke organisms a reality. This paper will analyze the regulatory and practical aspects of such an innovative approach to xenotransplantation on the basis of a hypothetical case study applied to Germany and highlight the gaps in the current regulation. This paper thus provides the basis for legal debate within a specific country. In addition, the identified gaps also pose a barrier toward the harmonization of international regulation. This publication therefore lays the groundwork for guiding the international debate regarding the regulatory framework for solid organ xenotransplantation toward specific issues.

Interspecies blastocyst complementation (IBC) provides a unique platform to study development and holds the potential to overcome worldwide organ shortages. Despite recent successes, brain tissue has not been achieved through IBC. Here, we developed an optimized IBC strategy based on C-CRISPR, which facilitated rapid screening of candidate genes and identified that Hesx1 deficiency supported the generation of rat forebrain tissue in mice via IBC. Xenogeneic rat forebrain tissues in adult mice were structurally and functionally intact. Cross-species comparative analyses revealed that rat forebrain tissues developed at the same pace as the mouse host but maintained rat-like transcriptome profiles. The chimeric rate of rat cells gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late pre-natal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRISPR-based IBC strategy holds great potential to broaden the study and application of interspecies organogenesis.

Orthotopic liver transplantation (OLT) currently serves as the sole definitive treatment for thousands of patients suffering from end-stage liver disease; and the existing supply of donor livers for OLT is drastically outpaced by the increasing demand. To alleviate this significant gap in treatment, several experimental approaches have been devised with the aim of either offering interim support to patients waiting on the transplant list or bioengineering complete livers for OLT by infusing them with fresh hepatic cells. Recently, interspecies blastocyst complementation has emerged as a promising method for generating complete organs in utero over a short timeframe. When coupled with gene editing technology, it has brought about a potentially revolutionary transformation in regenerative medicine. Blastocyst complementation harbors notable potential for generating complete human livers in large animals, which could be used for xenotransplantation in humans, addressing the scarcity of livers for OLT. Nevertheless, substantial experimental and ethical challenges still need to be overcome to produce human livers in larger domestic animals like pigs. This review compiles the current understanding of interspecies blastocyst complementation and outlines future possibilities for liver xenotransplantation in humans.

Millions of people suffer from end-stage refractory diseases. The ideal treatment option for terminally ill patients is organ transplantation. However, donor organs are in absolute shortage, and sadly, most patients die while waiting for a donor organ. To date, no technology has achieved long-term sustainable patient-derived organ generation. In this regard, emerging technologies of chimeric human organ production via blastocyst complementation (BC) holds great promise. To take human organ generation via BC and transplantation to the next step, we reviewed current emerging organ generation technologies and the associated efficiency of chimera formation in human cells from the standpoint of developmental biology.

Generating human organs inside interspecies chimeras might one day produce patient-specific organs for clinical applications, but further advances in identifying human chimera-competent pluripotent stem (PS) cells are needed. Moreover, the potential for human PS cells to contribute to the brains in human-animal chimeras raises ethical questions. The use of non-human primate (NHP) chimera-competent PS cells would allow one to test interspecies organogenesis strategies while also bypassing such ethical concerns. Here, we provide the first evidence for a putative chimera-competent pluripotent state in NHPs. Using histone deacetylase (HDAC) and selective kinase inhibition, we converted the PS cells of an Old World monkey, the African Green monkey (aGM), to an ERK-independent cellular state that can be propagated in culture conditions similar to those that sustain chimera-competency in rodent cells. The obtained stem cell lines indefinitely self-renew in MEK inhibitor-containing culture media lacking serum replacement and FGF. Compared to conventional PS cells, the novel stem cells express elevated levels of KLF4, exhibit more intense nuclear staining for TFE3, and manifest increased mitochondrial membrane depolarization. These data are preliminary but indicate that the key to deriving primate chimera-competent PS cells is to shield cells from the activation of ERK, PKC, and WNT signaling. Because of the similarity of aGMs to humans, the more ethically palatable use of NHP cells, and the more similar gestation length between aGMs and large animals such as sheep, the aGM cell lines described herein will serve as a useful tool for evaluating the efficacy and safety of interspecies organogenesis strategies. Future studies will examine chimera-competency and generalizability to human cells.

The shortage of human organs for transplantation is a devastating medical problem. One way to expand organ supply is to derive functional organs from patient-specific stem cells. Due to their capacity to grow indefinitely in the laboratory and differentiate into any cell type of the human body, patient-specific pluripotent stem (PS) cells harbor the potential to provide an inexhaustible supply of donor cells for transplantation. However, current efforts to generate functional organs from PS cells have so far been unsuccessful. An alternative and promising strategy is to generate human organs inside large animal species through a technique called interspecies blastocyst complementation. In this method, animals comprised of cells from human and animal species are generated by injecting donor human PS cells into animal host embryos. Critical genes for organ development are knocked out by genome editing, allowing donor human PS cells to populate the vacated niche. In principle, this experimental approach will produce a desired organ of human origin inside a host animal. In this mini-review, we focus on recent advances that may bring the promise of blastocyst complementation to clinical practice. While CRISPR/Cas9 has accelerated the creation of transgenic large animals such as pigs and sheep, we propose that further advances in the generation of chimera-competent human PS cells are needed to achieve interspecies blastocyst complementation. It will also be necessary to define the constituents of the species barrier, which inhibits efficient colonization of host animal embryos with human cells. Interspecies blastocyst complementation is a promising approach to help overcome the organ shortage facing the practice of clinical medicine today.

The clinical need for organ replacement therapies has inspired the idea of growing human organs in animal hosts. The injection of human pluripotent stem cells into animal blastocysts provides a possible strategy to accomplish this goal. Subject of Review: A recent study [Wu et al. Cell 2017;168:473-486.e415] tests the feasibility of this approach by creating chimeric embryos between humans and large domestic animals, including pigs and cattle. The study further examines the potential of combining CRISPR-Cas9 gene editing with blastocyst complementation to grow fully foreign organs in chimeric hosts. Second Opinion: Here, we consider what this report and related studies reveal about the likelihood of human-animal chimeras reaching the clinic and translating into therapies. A careful look suggests hope for eventual success in this area but also underscores important challenges that will require dedicated effort to resolve.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.