Why it is important to study human–monkey embryonic chimeras in a dish

Abstract

The study of human–animal chimeras is fraught with technical and ethical challenges. In this Comment, we discuss the importance and future of human–monkey chimera research within the context of current scientific and regulatory obstacles.

In recent years, the development of methods for generating new types of human stem cells, culturing embryos in vitro, genetically altering embryos, and producing stem-cell-derived embryo models have substantially advanced the field of developmental biology. One such important methodology has been the creation of embryonic chimeras — organisms whose cells come from two or more different zygotes or species. This advance has received international attention and laid the foundation for understanding human development and evolution, creating humanized disease models, and generating transplantable human organs in animals.

The utility and potential of interspecific chimeras for generating stem-cell-derived organs from one species within another species was first revealed by combining donor stem cells with normal or organogenesis-disabled host embryos, and with closely related or disparate species^1–4. For example, these model studies demonstrated the feasibility of growing a rat pancreas within the related rodent species of mouse and vice versa^2,3. Researchers now aim to similarly grow human tissues or organs in animals by injecting human pluripotent stem (PS) cells into genetically engineered animal embryos in which the ability to produce a particular organ has been disabled by genetic manipulation. Although some progress with generating human tissue inside pigs was recently achieved, the levels of chimerism were still too low to support the generation of human organs inside livestock species, highlighting the need for additional studies^5,6. If achieved, the generation of human tissues and organs by forming interspecies chimeras could generate scalable sources of organs for clinical transplantation, provide a transformative basic scientific tool for studying comparative evolutionary biology, human physiology, human development, and disease modeling, and serve as a vehicle for the development of pharmacological and cell therapies. Notably, if the PS cells can be derived from a patient’s own cells, the resulting patient-specific organ may survive for the long term in the patient without the complications of prolonged post-transplant immunosuppression².

The successful generation of the first in vitro human–monkey embryonic chimeras was recently reported¹. Although the in vivo generation of gestated human–monkey chimeras or the production of human organs inside monkeys is ethically problematic, we believe that the generation of early-stage human–monkey embryonic chimeras in a dish can be justified and could provide detailed basic developmental insights into the full differentiation potential of human PS cells. Such a disruptive technology could also ultimately enable new basic scientific applications. For example, in vitro human–monkey chimera systems could facilitate powerful research tools for the study of the comparative and evolutionary developmental biology of human and non-human primates (NHPs)⁷, or for the study of human developmental oncogenesis⁸.

Here, we elaborate the salient scientific, ethical, and policy challenges for studying chimeras generated by combining human PS cells with preimplantation-stage monkey embryos and suggest a path forward using in vitro chimera developmental systems.

Experimental challenges

The generation of a complete, transplantable human organ in an animal currently remains ambitious and unrealized. Although several groups have attempted to generate human PS-cell-derived interspecies chimeras using mouse, pig and rabbit blastocysts, only small numbers of human cells have been observed in host embryos^4–6,9,10.

We propose that several technical caveats will need to be overcome before efficient human–animal chimerism is achieved¹¹. These technical obstacles, at a minimum, include: (1) lack of developmental stage synchrony between human PS cells and host animal embryos; (2) lack of full developmental competence of injected human PS cells; (3) kinetic differences in the developmental progression between human and host species cells; and (4) interspecific differences in cell–cell interactions.

To address some of these challenges and enhance the chimera-forming potential of human PS cells, scientists have attempted to adjust the developmental stage of human PS cells to a ‘naive’ preimplantation epiblast-like stage that better matches that of the host embryo^9,11. Alternatively, approaches that activate pro-survival genes to help human cells thrive in a xenogeneic milieu have been described, but such an approach was still insufficient to achieve efficient human–animal chimerism¹⁰.

It is worth noting that some human naive PS cells possess high rates of genomic instability, loss of imprints, and low rates of competitive cell fitness that might collectively compromise their full developmental potential within interspecific chimeric hosts^10–15. Moreover, currently available human naive PS cells lack the robust proliferation characteristic of rodent naive PS cells^10,11. Thus, it remains unclear whether such human stem cells (which are adapted to propagate in current serum-free or serum-replacement-containing culture media) are competent to re-enter a normal developmental progression following transfer into a xenogeneic preimplantation embryo.

More generally, the factors that prevent the formation of high-degree interspecific chimeras may also relate to the vast evolutionary distance between humans and mice, pigs, and rabbits, the lineages of which diverged from that of humans more than 90–95 million years ago (http://timetree.org/). The use of less evolutionarily distant host embryos (for example, derived from NHPs), may inform us about interspecific cell–cell crosstalk that is required at the embryonic stage and may lower the threshold for the formation of chimeras with human PS cells. For example, the use of NHP hosts may reduce species–specific differences in the primary amino acid sequences of growth factor ligands, receptors, cell adhesion molecules and other regulators that facilitate cell integration and cell differentiation. The unique ability to assess the developmental potential of human PS cells in human–monkey embryonic chimeras would allow investigators to test experimental conditions aimed at overcoming each of the variables listed above. Such research will require the use of either natural embryos or embryo models derived from old world (macaque) and new world (marmoset) monkeys. These species are already frequently used as experimental models for assisted reproductive technologies and vaccine research. The potential insights gained from such experiments could be profoundly useful for guiding efforts to generate human organs inside more evolutionarily distant host species such as pigs; if successful, such efforts would revolutionize the fields of comparative developmental biology and regenerative medicine.

A recent study reporting the generation of human–monkey embryonic chimeras suggested the feasibility and utility of studying chimerism between human cells and more closely related species. In the in vitro study by Tan et al., monkey embryo culture was adapted for analyzing the earliest stages of formation of human–monkey embryonic chimeras^16,17 (Fig. 1a). In brief, the authors used a distinct human PS cell type known as human extended pluripotent stem (EPS) cells, which showed a higher level of putative chimerism than conventional human PS cells in mouse embryos. Human EPS cells labeled with a fluorescent protein were injected into early-stage monkey pre-implantation embryos, and the resulting chimeric embryos were cultured to early post-implantation developmental stages. The developmental fates of injected human extended PS cells were traced in peri- and post-implantation embryos. Human and monkey cell lineage markers in chimeric embryos were assessed by immunofluorescence staining and single-cell RNA sequencing. The authors reported reproducible human cell chimerism in both embryonic and extraembryonic lineages, and also identified signaling events underlying interspecific crosstalk that may influence the developmental trajectories of human cells in monkey embryos.

Fig. 1 |. Scheme of representative proposed in vitro experiments.

a, Fluorescent-protein-labeled human pluripotent stem cells would be introduced into monkey pre-implantation embryos (early blastocysts) by microinjection. Injected embryos would be cultured to the late blastocyst stage for analysis. Chimeric embryos would be cultured in vitro up to the gastrulation stages, similarly to Tan et al.¹ In such an embryo culture system, the zona pellucida is removed and the resulting chimeric blastocysts attach to the culture dish for development to the gastrulation stages. The fates of human Ps cells (PsCs) can be traced at peri- and post-implantation stages. To determine the developmental trajectory of human–monkey chimeric embryos, single-cell RNA sequencing analysis is conducted to capture the transcriptome of human and monkey cells at the different embryonic stages. b, Putative chimeric stem cell embryo models generated using human and monkey Ps cells. ICM, inner cell mass; TE, trophoectoderm; PgCs, primordial germ cells. Figure by C.P.-A.

Although important, this and other in vitro studies had several limitations. One issue related to the technical limitations posed by currently available in vitro embryo culture methods. For example, culture of natural monkey embryos beyond 20 days results in poor long-term in vitro cell survival^16,17; this caveat is likely to limit the ability to assess the developmental potential of human–monkey embryonic chimeras. Another group similarly reported failure to efficiently generate human–monkey chimeras in vitro owing to inherent proliferative and differentiation roadblocks of injected human PS cells, although they did not use EPS cells¹⁰. Nonetheless, these data collectively suggest that a more detailed analysis of the cell cycle status of injected human PS cells and their progeny is warranted, as in vitro cultured embryos may depart from their normal in vivo developmental capacities.

Another challenge pertains to the lineage identities of differentiating human PSCs within in vitro human–monkey embryonic chimeras. To evaluate this variable, Tan et al. conducted a cross-species comparison with NHP gastrulating embryos to infer whether the human cells differentiated into putative gastrulating-like cellular states. However, little is known about the normal expression profiles of pre- and post-gastrulation developing human embryonic cells. Indeed, a recent study reported the first single-cell gene expression data from a single human gastrulating embryo¹⁸; such studies of normal post-implantation human embryonic development will aid molecular benchmarking of in vitro systems. However, such analyses are rare owing to ethical constraints that limit the availability and use of human embryonic samples.

Tan et al. did not observe an increase in apoptosis or programmed cell death in the chimeric embryos, and noted differentiation to putative human gastrulation-like states. However, the decreasing proportion of human chimeric cells during in vitro developmental progression, and their differentiation delay, suggest that human-specific growth factors may be required within the monkey embryonic environment for efficient chimerism. Overcoming this challenge may require humanized stage-specific growth factor editing in monkey embryos. To this end, investigations should focus on the species specificity of ligand–receptor interactions, cell–cell communication in directed differentiation, developmental stage, responsiveness and synchronization, cell adhesion signaling, cell cycle, gestational length, and the biology of implantation. Regulators of efficient integration of human PS cells in NHP embryos may further serve as promising candidates for promoting increased levels of chimerism in non-human embryos. For example, it may be possible to humanize key factors in host animal species to confer improved compatibility with human cells.

We also propose that experimental refinements using alternative types of earlier-stage human PS cell¹¹, as well as further elucidation of the cell-dependent stem cell milieu interactions, will facilitate more efficient interspecies chimerism irrespective of host species. A deeper understanding of the roadblocks to human PS cell differentiation could also provide strategies to enhance the ability of human donor cells to integrate with other animal host embryos. In sum, future in vitro studies of human–monkey chimeras may yield a clearer understanding of cell competition and cooperation not only in the normal developing human embryo, but also in embryos of our distant evolutionary relations.

Some may argue that in vitro chimera systems involving established NHP PS cell lines and stem-cell-derived embryo models can provide a sufficient research method that obviates the need to use natural embryos (Fig. 1b). High-throughput approaches with natural embryos are generally not feasible, owing to their relative unavailability. In addition, the scalability of in vitro embryo models could help to provide insights into the global transcriptional, epigenetic, translational, and metabolic processes that govern embryonic development, and could improve our understanding of the cross-communication between human and monkey embryonic cells. Despite these advantages, in vitro embryo models are not equivalent to natural embryos, as their potential to give rise to a fully developed organism remains unclear. Even the capacity of pre-implantation stage embryo models to undergo early gastrulation has not yet been compellingly demonstrated^19–21. Observations with these embryo models may not readily extrapolate to interspecies chimera formation in vivo. Thus, given the uncertainty surrounding the developmental fidelity of in vitro embryo models at this time, we believe that the study of human–monkey chimeric embryos using natural embryos will continue to be of considerable value in the foreseeable future.

A summary of the methods by which different human–monkey embryonic chimeras might be produced, along with their advantages and disadvantages, is provided in Table 1.

Table 1 |.

Different types of in vitro human–monkey embryonic chimera and their advantages and disadvantages

	In vitro chimeras
	Natural embryos	Stem cell models
Method	Introduce human PS cells into natural monkey embryos obtained from monkeys.	Introduce human PS cells into monkey PS-cell-derived embryo models.
Advantages	Close fidelity to in vivo system, including epigenetic features that are often off-center in human PS cells and the known developmental potential of the NHP component of the chimera.	Availability, scaleability (the ability to conduct experiments with a large number of embryo models), ease of introducing multiple gene edits and ability to test human–ape interspecies chimerism.
Disadvantages	Difficulty of obtaining large numbers, difficulty of multiplexed gene editing and inability to test human–ape interspecies chimerism.	Lack of clarity of to what extent stem cell models recapitulate natural embryos and unknown developmental potential of the NHP component of the chimera.
Ethical concerns	Permissible within ISS CR guidelines, where allowed by local regulations with appropriate oversight and public engagement. Additional animal care and use concerns associated with acquisition of NHP embryos. Provenance of human PS cells should include specific consent for the creation and early embryonic in vitro study of human–monkey chimera embryos.	Not specifically addressed within ISS CR guidelines, may be permissible where allowed by local regulations with appropriate oversight and public engagement. Provenance of human pluripotent stem cells should include specific consent for the creation and early embryonic in vitro study of human–monkey chimera embryos.

Different types of human–monkey embryonic chimera might be generated by introducing human Ps cells into natural monkey embryos or by introducing human Ps cells into monkey Ps-cell-derived embryo models.

Ethical and regulatory considerations

We believe that the potential benefits of human–monkey embryonic chimera research are clear, as are the substantial ethical challenges. The ethics of generating human–animal chimeras has been explored^22–24; however, the specific ethics of human–monkey chimeras have been less discussed²⁵. Some of the issues raised, such as those related to appropriate donor consent for the use of human stem cells in such controversial experiments, can be managed through appropriate oversight using either existing or specialized review and approval processes. Other more difficult challenges are mired in unknowns and moral disagreements; for example, how to understand and avoid unacceptable levels of chimeric animal suffering, and how to think about the potentially shifting moral status of the chimera²⁶.

The former may be the more tractable. We may be able to gain sufficient understanding about the well-being and care needs of new chimeras to justify their humane use in research with appropriate oversight by animal care and use committees. For the latter, there is less room for optimism. A primary constraint is the lack of consensus around a philosophical account of moral status. Even if a widely acceptable account were to emerge, we are still left with the substantial empirical challenges of reliably evaluating the sorts of traits, such as sentience and cognition, that are likely to be relevant. Identifying and evaluating these traits would be a daunting challenge with animals we know well, and would be much harder still in new chimeric species. These and other ethical challenges associated with the gestation and live birth of human–monkey chimeras are formidable, and it is unclear whether they can ever adequately be addressed.

The pace of recent scientific advances in this field stands in contrast to its limited governmental funding and the uncertainty around international and state regulatory issues. Whereas some countries have enacted relatively permissive regulations, human–animal chimera research has been banned and criminalized elsewhere. For example, Japan revised regulations in 2019 to allow the review and approval of human–animal chimera research, potentially including transfer into an animal uterus for full-term gestation²⁷. In the United Kingdom, most human–animal chimera research can proceed with existing oversight. Research that involves adding human genes or cells to NHPs requires specialized review and approval, and, for now, research involving the formation of chimeric embryos through the mixing of NHP embryos and human stem cells is limited to 14 days of development or primitive streak formation^28,29.

Although the United States does not limit a broad range of human–animal chimera research at a federal level, the National Institutes of Health has implemented and upheld a moratorium on the funding of human–animal embryonic chimera research since 2015. Notably, in May 2021, federal legislation that would ban chimera research narrowly failed to pass in the US Senate, thereby highlighting the relevance and contentiousness of this discussion³⁰. The US states of Louisiana and Arizona have enacted state legislation prohibiting human–animal chimera research, with criminal penalties that include fines and even imprisonment with hard labor^31,32. Many other US states have not enacted any laws specifically addressing chimera research.

Such uncertainty caused by policy variations is problematic but not unique to human–animal chimera research^33,34, leading scientists to navigate a complex and sometimes punitive policy landscape. Scientific bodies have undertaken interdisciplinary efforts to reduce the uncertainty and develop global ethical guidance for best practices, and these can serve as a basis for formulating national guidelines^35–37. Important common elements include considering research in three categories: permissible with existing oversight; permissible with specialized review and approval; and not permissible at this time. They also include calls for a cautious, incremental approach and for public engagement efforts.

For example, the 2021 guidelines issued by a committee convened by the US National Academies of Sciences, Engineering, and Medicine state that while the generation of chimeras in which human PS cells are introduced into animal blastocysts and maintained solely in vitro can proceed under existing oversight mechanisms, scientists should not pursue research in which human PS cells are introduced into nonhuman primate blastocysts followed by transfer into a uterus. It remains unclear, however, whether these guidelines pertain only to neural chimeras³⁵. National efforts to adopt clear research policies and reduce this uncertainty should therefore be encouraged.

ISSCR guidelines

The International Society for Stem Cell Research (ISSCR) has recently updated its guidance to include human–monkey chimera research^36,37: in vitro embryonic chimera research should be reported to the relevant bodies to determine whether planned studies require specialized review and approval. While this applies to in vitro human–monkey embryonic chimeras produced using natural monkey embryos, the guidelines do not explicitly comment on the generation of in vitro human–NHP chimeras using NHP stem cell embryo models. Future revisions of the ISSCR guidelines should aim to address this gap.

Contemporary international societies, including the ISSCR, advise that chimeric embryos should be studied for the minimal amount of time necessary to answer important and well-defined research questions^36,37. Moreover, the ISSCR encourages the deployment of strategies to constrain human cell contributions to target lineages and organs and to prevent unwanted systemic contributions. For example, one strategy may involve the use of stem cells in which key neural and germ cell transcription factors have been ablated by genome editing, to prevent substantial cellular contributions to the brain and gonads.

Notably, upon specialized review and approval, the ISSCR condones the gestation of human–NHP chimeras following transfer into a uterus, but explicitly forbids the use of great apes or lesser apes (chimpanzees, gorillas, orangutans, bonobos, gibbons, and siamangs) as NHP host species. The use of apes is severely limited or banned for invasive research in many jurisdictions owing to ethical considerations. Additionally, even for in vitro embryonic chimera experiments that do not involve intrauterine transfer, it remains technically challenging and ethically problematic to obtain natural pre-implantation embryos from apes^36,37.

As a result, for studies involving natural NHP embryos, we propose that guidelines should be clarified to avoid ambiguity in interpretation. At this time, we support the generation and study of early-stage in vitro embryonic chimeras between humans and NHPs that are more distantly related to humans than apes using natural embryos and stem cell models. While the use of natural ape host embryos remains forbidden, the ISSCR guidelines do not comment on in vitro human–ape chimeric embryo models achieved by using ape stem cell-derived embryo models, a potential platform for studying human–ape interspecies chimerism in a dish (Table 1). This is a topic that should be addressed in future guidance discussions.

Outlook

The research proposed in this Comment includes necessary constraints to avoid the substantial ethical challenges to human–monkey chimera research. By limiting research to the in vitro study of early embryonic development, research on human–monkey chimeric embryos can be ethically justified in a similar way to in vitro research on early human embryos. For example, justification for this research relies on conditions that preclude sufficient neural development to potentially establish any capacity for individuality or suffering. At present, there is active debate over revisiting and extending prior commitments to a 14-day rule as the appropriate limit for this sort of research, as reflected in the revised guidelines for stem cell research from ISSCR. The specific limits for this research proposed in this paper should mirror the outcomes of this debate.

Despite defining constraints to avoid the most important ethical challenges, this research remains controversial, and this makes certain obligations held by researchers more pressing. Scientists must clearly and transparently engage with diverse communities about their research proposals before launching into studies. Deliberative engagement can facilitate better understanding of the research: knowns, unknowns, risks, benefits, methods, aims, and limits. At the same time, engagement can help scientists to better identify and understand the most pressing concerns held by members of different communities. Navigating ethically controversial terrain will remain difficult, but engagement can provide an essential forum for members of different communities, including the scientists themselves, to deliberate what ought to be done and to establish better mutual understandings of intentions, values, and concerns.

A cautious, constrained, incremental approach with ongoing engagement and appropriate oversight is crucial for justifying in vitro research on the early embryonic development of human–monkey chimeras that can answer important scientific questions. The findings from this research can provide evidence to better inform future debates if further, more ethically challenging, study is ever warranted.

In summary, although the production of human–monkey embryonic chimeras is ethically controversial, we believe that the potential benefits of such experiments performed in vitro, as outlined herein, can justify their execution. However, this research must proceed in a cautious, step-wise manner, with local regulatory compliance and community engagement, and subject to appropriate ethical oversight.

We continue to welcome a consultative approach toward guidance for our global community of collaborative researchers that will enable the performance of this beneficial research at the same time as ensuring adherence to international ethical standards. Thus, ongoing efforts in this field should focus on not only technological advances, but also on harmonizing guidance and developing interim practices for horizon scanning; with the ultimate goal of identifying and addressing newly developing ethical challenges and initiating public engagement efforts. Furthermore, guidance can be strengthened through inclusion of additional stakeholders such as research funders, scientific journal editors, and other key entities who together can encourage adherence where research is allowed. In light of the promise of in vitro human–monkey chimera research, we anticipate that there will be further robust discussions of this pivotal area of biomedical research.