Human brain organoids thrive in living rats’ brains
Chapter 7 of Principles of Neurobiology, titled ‘Constructing the Nervous System’, explores a fascinating topic in neurobiology – nervous system development and wiring. In Box 7-1, we learn that human brain organoid and assembloid technologies have been increasingly utilized to model early brain development and disease since their emergence in the early 2010s. While these technologies hold great promise, a major obstacle is that organoids lack circuit connectivity such as sensory inputs and behavioral outputs seen in the real animal. This precludes neurons in the organoid from experience-dependent maturation and thus limits their applications to study the advanced stage of brain development.
Figure 1. (A) Generation of human cortical organoids (hCOs) from human induced pluripotent stem cells (hiPS) and transplantation of hCOs into the primary somatosensory cortex (S1) of newborn rats. (B) A transplanted hCO (t-hCO) in the rat cortex is outlined. (C) t-hCO neurons derived from patients with Timothy syndrome show dendritic morphology defects. (D) t-hCO neurons show increased activity following whisker stimulation. (E) Activated t-hCO neurons elicit licking after training. Adapted from Revah et al., 2022.
To overcome this issue, Revah et al. took a novel approach – transplanting human cortical organoids (hCOs) into newborn rats (Figure 1A). Note that newborn rats were used because, at early postnatal stage, their neural circuits are not fully formed yet. This approach should maximize the integration of the transplanted hCOs (t-hCOs) into the rat brain circuitry. Using this in vivo model, the authors asked four major questions: (1) Do t-hCOs develop mature neurons? (2) Do t-hCO neurons derived from patients show disease phenotypes? (3) Do t-hCO neurons receive sensory inputs? (4) Does activation of t-hCO neurons evoke behavioral outputs?
Over the next few months after transplantation, the authors observed that the t-hCOs covered a large area of the rat’s hemisphere (Figure 1B). This substantial growth was likely facilitated by vascularization, which is absent in dish-cultured hCOs [unless fused with endothelial cells to form vascularized hCOs (Cakir et al., 2019)]. Through transcriptional, morphological and electrophysiological analyses, the authors found that t-hCO neurons were more mature than their in vitro counterparts. Notably, these mature neurons, when derived from patients with Timothy syndrome (a multisystem disorder caused by a mutation in the gene encoding calcium channel CaV1.2), displayed dendritic morphology defects that were not seen in the less mature hCO neurons (Figure 1C).
Next, the authors recorded and stimulated the activity of t-hCO neurons, using the calcium indicator GCaMPs and the light-gated cation channel channelrhodopsin-2 (ChR2), respectively, and assessed if the t-hCOs interacted with the rat circuits anatomically and functionally. Using a whisker stimulation assay (in which you move rat’s whiskers to elicit a sensory stimulus), the authors found that GCaMPs-expressing t-hCO neurons displayed increased activity in response to whisker deflection (Figure 1D). In a behavioral assay, rats transplanted with ChR2-expressing hCOs were trained to receive water as a reward only if the rats licked during optogenetic activation with blue light but not with red light. After training, the rats appeared to associate blue light stimulation with delivery of the reward as they showed increased licking whenever blue light was on (Figure 1E). These findings, along with others, indicate that the human neurons connected with rat neurons to receive sensory inputs and drive behavioral outputs.
Overall, Revah et al.’s study demonstrates that t-hCOs can develop mature neurons that are well integrated into the rat brain circuitry, offering a powerful platform for investigating advanced aspects of human brain biology. However, t-hCOs still fail to recapitulate the human cortex completely apropos cell type diversity and cytoarchitecture. Also, human neurons develop at a slower pace than rodent neurons, due at least in part to slower mitochondrial development in human neurons (Iwata et al., 2023). Whether these species-specific features influence connectivity is not known. Future t-hCO research combining incorporation of missing cell types (to improve diversity) with enhancement of mitochondrial metabolism (to accelerate maturation) should further unlock the potential of organoid and assembloid technologies.
References
Revah, O. et al. (2022) Maturation and circuit integration of transplanted human cortical organoids. Nature. https://doi.org/10.1038/s41586-022-05277-w
Cakir, B. et al. (2019) Engineering of human brain organoids with a functional vascular-like system. Nature Methods. https://doi.org/10.1038/s41592-019-0586-5
Iwata, R. et al. (2023) Mitochondria metabolism sets the species-specific tempo of neuronal development. Science. https://doi.org/10.1126/science.abn4705