In this research, the authors from the United Kingdom used a droplet-based printing technique with human induced pluripotent stem cells to fabricate tissues containing simplified cerebral cortical columns. The main focus was on implanting tissues made with a special 3D printing technique, and only one human induced pluripotent stem cell line was tested. Human induced pluripotent stem cells have the potential to generate the cell types that make up all human tissues. The results show that a simplified two-layer cerebral cortical tissue can be produced using a droplet-based 3D-printing. Implantation of these constructs into ex vivo mouse brain explants demonstrated the structural integration of the implants and their correlated Ca2+ oscillations.
Brain injuries, such as traumatic brain injury, stroke, or surgical resection for cancer and epilepsy can cause significant damage to the cerebral cortex, resulting in severe disabilities. The authors, therefore, focused on the generation of neural tissues for potential applications involving implantation. The cerebral cortex consists of layer-specific neurons organized into vertical columns, but, current tissue engineering techniques are incapable to produce such structures.
The original illustration from the article of Jin, Y., et al. Nat Commun 14, 5986 (2023).
About the study
The cerebral cortex typically consists of six layer-specific neurons. The essential first step towards fabrication of the two-layer cortical tissue was the generation of layer-specific cerebral cortical progenitor cells from human induced pluripotent stem cells. Scientists first differentiated human induced pluripotent stem cells into two subtypes of neural progenitors, upper- and deep-layer neural progenitors.
In order to produce two-layer cortical tissues, the upper- and deep-layer neural progenitors, instead of their mature descendants, were harvested for printing. Progenitors were used instead of mature neurons because they were less sensitive to the dissociation procedure from 2D cultures than mature neurons and were compact for printing. The cerebral cortical tissues with a two-layer organization were formed using 3D droplet printing technique, which enables the production of structurally defined and scaffold-free soft tissues composed of cells and extracellular matrix.
The printed progenitor cells underwent maturation, including terminal differentiation, neuronal process outgrowth and migration. Deep-layer cortical neurons, which are the early product of cortical neurogenesis, were first fabricated from deep-layer neural progenitors. This deep-layer cortical tissue was sectioned and immunostained to reveal its tissue structure and the cellular composition, which were subsequently visualized with neural markers. Later, radial glia cells produced neurons that migrated radially into the cortical plate, and passed through the deep-layer neurons to become upper-layer neurons. In order to further confirm the identities of the deep-layer neurons and upper-layer neurons cells, researchers conducted gene expression analysis using a real-time quantitative polymerase chain reaction.
The printed cortical tissues remained in the desired two-layer architecture after two-weeks of culture. A magnified view showed that the upper-layer neurons were projected toward the deep layer. Neuron migration between the layers was also observed. Neuronal process outgrowth and migration are two important developmental phenomena in cortical neurogenesis.
The printed tissues were then implanted into lesions in ex vivo mouse brain explants to assess their capacity for tissue repair. Over a week, the cellular morphology, structural integration and calcium ion activity were monitored. Implantation of printed cortical tissues into ex vivo mouse brain explants resulted in substantial structural implant-host integration across the tissue boundaries. Neuronal process outgrowth and migration of neurons from the implant towards the host were confirmed by confocal fluorescence imaging, indicating that the printed tissues had integrated into the brain explant. A high magnified view showed that individual neurons migrated across the implant-host boundary. Live/dead staining showed that cells of the brain explants were 86 % viable five days after implantation.
To assess the activity of the implanted cortical tissue, researchers performed Ca2+imaging with Fluo-4, a fluorescent calcium indicator. Network analyses demonstrated the existence of multiple neuronal communities with correlated firing patterns, and a group of regions of interest with correlated Ca2+traces between the implant and the host.
In conclusion, this study shows that a simplified two-layer cerebral cortical tissue can be produced using a droplet-based 3D-printing. During in vitro culture after printing, the identity, and structure of the deep and upper layers were maintained. Neuronal process outgrowth, migration of neurons and maturation were observed during this period. Implantation of printed cortical tissues into mouse ex vivo brain explants demonstrated the formation of structural connections and correlated Ca2+ oscillations between the implant and the host.
This technique represents a significant advance in tissue engineering, particularly for future individualized implantation treatments that use 3D tissues made from a patient’s own induced pluripotent stem cells. The authors suggested that employing droplet-printing technology, an implant can be designed to mimic the dimensions, orientation, cellular composition and structure of the lost tissue.
This article was published in Nature Communications.
Jin, Y., et al. Integration of 3D-printed cerebral cortical tissue into an ex vivo lesioned brain slice. Nat Commun 14, 5986 (2023). https://www.nature.com/articles/s41467-023-41356-w