Maturation and circuit integration of transplanted human cortical organoids

Sometimes, scientists make ancient myths come alive. That is the case for the generation of chimeric organisms. During the evolution of human species, a common myth in many cultures was the existence of mixed creatures, monsters made of several species bound together. The chimera was one of them, described by Homer in the Iliad as a tremendous fire-breathing creature that looked like a lion, with a goat’s head growing out its back, and a snake as a tail. Through history, the term chimera has come to be associated with any creature where parts were taken from various animals. Science was not a stranger to this, and chimeric experiments started: human cells were transplanted into different animal species [1,2]. This type of experiment was also used to study one of the most fascinating and complex of our organs, the brain.

The complexity of our brain is the basis of our uniqueness. To study such an elusive part of the human body, scientists needed to open and deconstruct it, but that is quite difficult because of ethical reasons. For that reason, in vitro human neuronal models have been used instead. By differentiating human pluripotent stem cells, we can generate several types of neurons, giving the possibility to closely study that fascinating system [3,4].

Moreover, a revolutionary technology, which first appeared in 2013, allowed the generation of human brain tissue in vitro: the self-assembling capability of pluripotent stem cells (stimulated by specific cocktails of signalling molecules) was used to generate complex 3D neural tissues [5]. These so-called brain organoids emulate the neurodevelopmental process in generating complex structures similar to the ones found in the brain, such as rudimental cortical layers or specific brain regions. Brain organoids are useful tools that have quickly entered the scientist’s toolkit for a great variety of applications: to study complex interactions between different brain cell types such as neurons, astrocytes, and oligodendrocytes; to understand similarities and differences between primate brain development; to develop highly efficient therapeutic screening with a reduced use of animal models; and to unravel molecular mechanisms of cerebral disorders [6]. These are just few of the research applications where brain organoids have given a significant contribution. The understanding and development of this complex and useful 3D model is further rapidly evolving in order to tackle some of its limitations. One of the major aspects to improve is its size and complexity because even though brain organoids can be a model of the early stages of neurodevelopment, the size of the brain organoids is limited to 4 mm in diameter. The size limit is due to the lack of nutrient diffusion that leads to a necrotic core within the organoids, in the region where most of the neural progenitors are located, causing a subsequent growth arrest. In addition, the neural population in brain organoids lack a proper maturation likely due to the absence of stimuli from an external in vivo-like environment. To overcome those problems, Revah et al. described a novel method to implant brain organoids into developing rat brains, thus generating chimeric organisms [7].

Previous studies have reported the successful transplant of human neurons into the developing mouse cortex, showing intrinsic features of human neuronal cells regulating their maturation, and the successful integration of human cortical neurons into functional neural circuits of the mouse brain [8,9]. Brain organoids have also been successfully transplanted, partially overcoming the lack of nutrient diffusion thanks to the vascularization of the brain organoid tissue [10]. To improve the maturation of human neurons into the brain organoids, Revah and his colleagues (Revah et al., Nature, 2022) described a novel approach: the integration of transplanted human cortical organoids into the somatosensory cortex of newborn rats lacking a functional immune system.

Human cortical organoids were transplanted during a specific developmental window of the rat brain (3-to-7-day-old rat pups) which allowed the proper maturation and integration of human neurons into the developing rat brain circuitries. The efficiency of successful integration they observed is around 80%, and the survival rate one year after transplant is almost 75%. Integration of cortical organoids was also successful in terms of vascularization and infiltration of microglia from the rat brain, as well as the increased growth of transplanted cortical organoids compared to age-matched non-transplanted ones. By performing meticulous morphological, functional, and transcriptional analysis of the transplanted human cortical organoids, Revah et al. reported an increased maturation of human neurons inside the rat brain compared to neurons in age-matched in vitro differentiated cortical organoids. Moreover, human neurons derived from transplanted cortical organoids were more similar to postnatal human neurons than neurons derived from their in vitro counterparts.

This enhanced neural differentiation was used to model Timothy Syndrome, a multisystemic syndrome characterised by severe neurodevelopmental defects caused by a mutation in the gene encoding the calcium-ion channel protein CaV1.2. Human cortical organoids derived from patients with Timothy Syndrome were transplanted into rat brains for a study and the authors observed a peculiar phenotype of patient neurons compared to controls: an altered dendritic spines morphology and electrical activity present only in patient neurons of transplanted cortical organoids but not in in vitro cortical organoids. Their study further showed that neurons of transplanted cortical organoids in the rat brain formed functional synapses with rat neurons, and that they can be integrated into the host neural circuits and communicate with rat neurons. Furthermore, they used optogenetics to demonstrate that neurons from transplanted cortical organoids could influence the host behaviour. Optogenetics uses a specific blue light that drives the activation of engineered light-sensitive proteins called channelrhodopsins, and thus the activation of neuronal cells. By using this approach, they trained rats transplanted with light-activated cortical organoids to lick a spout and get water as a reward. They observed that when human neurons were specifically activated by using blue light in trained animals, rats were licking the spout to get the reward, while using different coloured lights in transplanted rats or blue light in non-transplanted rats, there was no stimulation to trigger the licking response to get a reward. This observation demonstrates how human neurons were integrated into rat brain and could influence the rat´s reward learning behaviour.

Nevertheless, an experiment where a cerebral organoid is transplanted and matures together with the rat brain raises some ethical questions and concerns. Ethics discussion on brain organoids transplantation is part of a broader discussion about chimeric organisms and about the transplantation of human neural cells into non-human animals [11]. Different from past works, currently these cerebral organoids are transplanted as a single “organized” structure inside the rat brain, possibly leading to a more organized and more significant alteration of the rat brain function than a non-organized transplant of single human neuronal cells. On the other side, the limited spatial localization of the cerebral organoids could influence only a specific local neural circuit and thus a local function of the host brain. This means that the implantation of cerebral organoids inside the developing rat brain would unlikely lead to alteration of more broad and complex brain functions like self-awareness and consciousness, excluding the possibility that these chimeric organisms could became more “human” - a human-like organism with more advanced cognitive capacities and complex emotions as these characteristics are not specific of humans since they are shared with non-human animals or primate and artificial intelligence [12]. The debate is still ongoing and experimental behavioural and molecular data should be carefully interpreted and monitored for preserving the welfare of the animals, avoiding unnecessary suffering and pain.

Taken together, Revah et al. showed that transplanting cerebral organoids inside a developing rat brain could be a good idea for studying more advanced and mature features of human neurons, opening the possibility to study behaviour of human neurons in a more physiological environment, and modelling complex aspects of neurological diseases; all with the aim that one day this new model could be used to find and test new therapies for human neurological diseases.

 

References

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