Human-mouse Hybrid Brain Add New Progress in Brain Organoid Research

Since the birth of modern science, human research on the brain has never ceased, and it has always been at the forefront of life sciences. However, the scarcity and preciousness of brain samples limit the development of in vitro studies of the brain. The development of brain organoids based on stem cell technology provides new opportunities for brain research.

Neuroscientist Pascal from Stanford University led his team to publish the latest research article on brain organoids in the top international journal Nature online. In this study, the researchers induced human-derived pluripotent stem cells to differentiate into cerebral cortical organoids and then orthotopically transplanted them into the somatosensory cortex of neonatal athymic rats to construct a human-mouse hybrid brain organoid—t-hCO. This organoid can not only grow normally in rats, showing the normal physiological structure of the brain, but also participate in the neural circuits that control behavior in the brain, providing a new strategy for brain neurodegenerative disease research and new drug development.

Human brain development is a complex process in which the mechanisms and processes by which neurons proliferate, differentiate, migrate and connect to form functional neural circuits remain to be studied. In recent years, the development of cerebral organoid culture technology has allowed researchers to study the developmental process and function of specific brain regions. However, existing cerebral organoids do not have neural circuits that generate behavioral outputs, limiting their development in neurological diseases.

In this study, the researchers induced differentiation in vitro to produce intact three-dimensional brain organoids hCO-directed transplantation into the S1 area of the brain (the primary sensory area responsible for processing tactile information called the brain of 3-7 days postnatal athymic rats The somatosensory zone (S1 area), located in the front of the parietal lobe), was used for MRI imaging of the rat brain 2-3 months after transplantation to observe the growth state of t-hCO. The results showed that the volume of t-hCO increased 9-fold within 3 months after transplantation in vivo, and 12 months after transplantation, the transplanted animals had a survival rate of 74%, and there were no obvious motor and memory deficits, glial cells Hyperplasia or abnormal electroencephalogram (EEG).

Subsequently, the co-expression of PPP1R17 (cortical progenitors), NeuN (neurons), SOX9 and GFAP (glial cells) or PDGFRα (oligodendrocyte progenitors) within t-hCO was identified by immunofluorescence and the presence of vascular endothelial cells, indicating the successful induction of differentiation by hCO.

Further, at the eighth month of t-hCOs-induced differentiation, we performed single-cell-level analysis of cell populations within t-hCOs using single-cell nuclear transcriptome sequencing. Single-cell sequencing results identified t-hCOs in cells including deep and superficial glutamatergic neurons, circulating progenitor cells, oligodendrocytes, and astrocytes. In addition, the researchers found certain differences between hCOs cultured in vitro and t-hCOs grown in vivo by transplantation.

The researchers further verified whether the aforementioned differences in single-cell sequencing results were related to the morphological differences between hCOs in vitro and t-hCOs in vivo. It was found that the total dendritic length of t-hCOs increased 6-fold compared to hCO in vitro, and the density of dendritic spines in neurons was also significantly higher than that of hCO neurons. Electrophysiological measurements showed an 8-fold increase in membrane capacitance in t-hCOs, a more hyperpolarized resting membrane potential (~20 mV), and electrical stimulation elicited higher firing rates in t-hCO neurons than in in vitro hCO neurons. These electrophysiological properties are consistent with the aforementioned larger and more complex morphological features of t-hCO. Taken together, t-hCOs neurons exhibited more mature differentiation characteristics compared to hCO neurons in transcriptional, morphological, and functional analyses.

In addition, the researchers used the aforementioned single-cell sequencing data to compare and analyze human fetal and adult cerebral cortex cells and gene expression data during their development. The results indicated that the overall transcriptome maturation status of hCO and t-hCO at 7-8 months of differentiation roughly matched the in vivo developmental time and was equivalent to late fetuses; whereas morphologically, t-hCOs were associated with human L2/3 neural Elements are more similar.

Finally, the researchers verified whether t-hCO can be used for the construction and study of mental disease models. Timothy Syndrome (TS) is an inherited autism-related disorder. The researchers isolated stem cells from three TS patients and three normal patients and induced hCO differentiation. The results after transplantation showed that TS t-hCO had an abnormal dendritic branching pattern compared with the control group, but not in in vitro TS hCO at a similar differentiation stage. Further, the researchers demonstrate that t-hCO can be anatomically integrated into the rat brain, enabling activation by host rat tissues, and further activation by sensory stimuli in the in vivo environment, participating in neural circuit-driven behaviors, To achieve the regulation function of rat activity.

Brain organoid technology is currently a powerful model for studying nervous system development and disease, but the lack of neural circuits limits its development. In this blockbuster study in Nature, the researchers spent seven years developing the t-hCO brain organoid system and demonstrated that it can participate in brain neural circuits in vivo to modulate neuronal activity and behavioral responses in rats.

This study also has the following two shortcomings: First, due to species and brain space-time differences, even early organoid brain implants cannot fully simulate the development of human brain neural circuits. Second, the factors that affect neural circuit connections in transplanted organoids still need to be determined. These are scientific problems that need to be overcome in the future. This study provides ideas for the development of cerebral organoid technology, and also provides a new model for in vitro research, drug development and treatment strategy design for neurological diseases such as Timothy Syndrome (TS).