A recently published paper reports the experiments of scientists at Stanford University in the USA, who inoculated self-organizing brain organoids from human cells into the brains of newborn rats, observing that there was integration of both neuronal tissues — murine and human — that led to changes in the animal’s behavior.

These neural organoids are clumps of human neurons cultured in vitro from adult human cells, reprogrammed to convert them first into induced pluripotent cells (hiPSCs) and then into neurons. According to different studies, they represent a promising in vitro platform with which to model human development and disease (1-5)

However, in the laboratory, they lack the connectivity that exists in vivo, which limits their maturation and makes integration with other brain circuits related to behavior impossible. The newly published paper shows that these human brain organoids transplanted into newborn athymic rats (rats with a limited immune response) develop mature cell types that integrate into the sensory and motivation-related circuits of the rat brain. The main findings are as follows:

  • Magnetic resonance imaging reveals post-transplantation organoid growth across multiple stem cell lines, with progression of corticogenesis and the emergence of activity-dependent transcriptional programs.
  • The transplanted cortical neurons display more complex morphological, synaptic and intrinsic membrane properties than in their “in vitro” state, which, according to the authors, enables the discovery of defects in the neurons derived from individuals with Timothy syndrome.
  • Anatomical and functional analysis shows that sensory responses to thalamocortical and corticocortical stimuli can occur in the transplanted organoids. In addition, they can extend axons throughout the rat brain and their optogenetic activation can induce reward-seeking behavior. Thus, the transplanted human cortical neurons mature and engage the neuronal circuits in which they are inscribed, modifying the behavior of the recipient animal.

The application initially proposed for this technique is to detect phenotypes related to particular neural circuits in patients affected by certain diseases.

Background

The formation of chimeras between species with human pluripotent stem cells (hPSCs) represents a necessary alternative to assess the pluripotentiality of hPSCs in vivo, and could constitute a promising strategy for a variety of regenerative medicine applications, including the generation of human organs and tissues in animals for use in transplantation. So far, studies that have used mouse and pig embryos suggest that hPSCs do not contribute strongly to the formation of chimeras in species that are evolutionarily distant from humans.

Some of these trials have been subjected to ethical scrutiny because of the inability to ensure that the human cells present in the human-animal chimera could not invade other non-target tissues, such as the brain, the consequences of which would be difficult to predict.

Other research published in 2021 by Spanish researcher Carlos Izpisúa’s group studied the chimeric competency (human-animal hybridization) of human extended pluripotent stem cells (hEPSCs) in cynomolgus monkey (Macaca fascicularis) embryos cultured “ex vivo”, in which it was shown that the hEPSCs survived, proliferated and generated several pre- and post-implantation cell lineages within these monkey embryos.

According to the authors, these results may help to better understand early human development and primate evolution, and to develop strategies to improve human chimerism in evolutionarily distant species. In the future, this chimerism would allow humanized organs to be obtained in animals that can be used in transplantation.

Previously, in 2019, a research group used gene editing techniques to perform transgenesis of a gene involved in human brain development in monkeys. They successfully generated 11 transgenic rhesus monkeys (8 first-generation and 3 second-generation) carrying human copies of the MCPH1 gene, which is important for brain development and brain evolution.

Analyses of brain images and tissue sections indicated an altered pattern of neural cell differentiation, resulting in delayed neuronal maturation and neural-fiber myelination of the transgenic monkeys, similar to the known evolutionary change of developmental delay (neoteny) in humans. Further analyses of the brain transcriptome and the tissue section of major developmental stages showed a marked human-like expression delay of neuron differentiation and synaptic signaling genes, providing a molecular explanation for the observed delay in brain development in transgenic monkeys. More importantly, transgenic monkeys exhibited better short-term memory and a shorter reaction time compared to wild-type controls in the delayed-matching-to-sample task. These data were a first attempt to experimentally question the genetic basis of the human brain origin using a transgenic monkey model, and to value the use of non-human primates in understanding unique human traits.

The same year (2019), in an experiment similar to the one now published in Nature, another team led by Vincent Bonin at VIB Neuro-Electronics Research Flanders in Leuven transplanted a soup of human neurons that integrated as single cells, rather than as a group, into the cortex of a newborn mouse.

Bioethical assessment

The experiments for obtaining human-animal chimeras were questioned by the United States National Institutes of Health (NIH) as early as 2015 due to the ethical difficulties that could arise from the introduction of human pluripotent cells into non-human vertebrate animal pre-gastrulation stage embryos. They stated that experiments in this area would not be funded until the Agency considered a possible policy revision.

Alta Charo, a bioethicist based in Washington D.C. and professor emerita at the University of Wisconsin-Madison, said that “[t]he neural combinations touch on what it is that makes us essentially humans – our minds, our memories, our sense of self”. She added that the public finds the idea of a human mind trapped in an animal’s body, or a creature with a semi-human brain, disturbing.

Professor Charo was involved in drawing up a report published in the National Academies of Sciences, Engineering, and Medicine in 2021, according to which, in recent years, researchers have developed new models to better represent and study the human brain. The three models considered in this report, all of which generate and use pluripotent stem cells from healthy individuals and patients, are: human neural organoids, human neural transplants, and human-animal neural chimeras. In this emerging field of human neural organoids, transplants and chimeras, science, ethics and governments must review the status of the research, considering its benefits and risks, examining the associated ethical issues and considering the mechanisms that governments must articulate for this type of research. The third chapter of the report, “Ethical concerns”, states that, for the time being, some of these concerns are alleviated by the fact that human neural organoids are currently very limited in size, complexity and maturity and are likely to remain so. They do not meet any current criteria for developing consciousness. In the future, however, their complexity and that of the circuits they contain will surely increase. It will therefore be essential to revisit these questions as models improve and the understanding of consciousness and awareness changes.

We are thus at a very common crossroads in bioethics: scientific progress — promising in many fields both in research and therapeutics — offers possibilities that are not free from ethical difficulty, i.e., likely to harm rather than cure if measures are not adopted to ensure that they do not exceed their possibilities, leading to unpredictable horizons that may involve setbacks in respect for human dignity.

The current breakthrough, which has overcome many of the difficulties of previous similar experiments, presents a momentous finding as the result: the proliferation of human nerve cells in rat brains contributes to changing their behavior. What degree of modification in animal behavior could be achieved when this human neuronal “colonization” can be intensified in future trials and, even more, in species closer to our own such as primates? We must not dismiss this possibility, which may help to blur certain human-animal boundaries, creating conflicts that are difficult to predict.

The previous experiments, in which 11 rhesus monkeys were genetically modified, pose an even more uncertain scenario. The progressive advance in knowledge of the genes involved in the formation of the human brain and their interaction may lead to the reprogramming of certain animal embryos with the capacity to achieve significant humanization of their brains, as was achieved to a small extent in this experiment. However, it could cause major changes in behavior, with unpredictable outcomes. In addition, these genetic modifications in very early embryos could affect their germ cells and be transmitted to offspring, and could modify the species, although this is not the case with neural transplants.

Prudence and consensual regulation of the limits of these experiments is essential to avoid overstepping, which, beyond the lawful objective of their contribution to research aimed at therapeutics in humans, provide results that involve an attack on the dignity of individuals of our species, as well as unacceptable outcomes in the animal species involved.

Julio Tudela and Lucía Gómez

BIoethics Observatory – Institute of Life Sciences

Catholic University of Valencia

 

References

[1] Kelley, K. W. & Pașca, S. P. Human brain organogenesis: toward a cellular understanding of development and disease. Cell 185, 42–61 (2021).

2 Pasca, A. M. et al. Functional cortical neurons and astrocytes from human pluripotent

stem cells in 3D culture. Nat. Methods 12, 671–678 (2015).

3 Valesco, S. et al. Individual brain organoids reproducibly form cell diversity of the human

cerebral cortex. Nature 570, 523–527 (2019).

4 Qian, X. et al. Brain-region-specific organoids using mini-bioreactors for modeling ZIKV

exposure. Cell 165, 1238–1254 (2016).

5 Yoon, S. J. et al. Reliability of human cortical organoid generation. Nat. Methods 16, 75–78

(2019).

 

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