• 3D cell cultures
• assembloid
• dopaminergic neurons
• forebrain
• human brain development
• modelling human development
• neural stem cells
• organoids
• pluripotent stem cells
• ventral midbrain

The inaccessibility of human brain tissue and the inability of two-dimensional in vitro cultures to recapitulate the complexity of dopaminergic (DA) circuitries have made the study of brain functions and dysfunctions challenging. Despite intensive research efforts in re-cent years, the molecular mechanisms controlling the developmental pro-gram and differentiation of DA neuron subtypes remain largely unknown. Three-dimensional (3D) human brain organoids have rapidly become a widely used system to study brain devel-opment and mature neurons in a dish. Cultured over long periods of time, 3D organoids pro-vide a unique opportunity to model human neural tissue features such as cytoarchitecture and cell-cell interactions reminiscent of human brain complexity.
The aim of the present study was to investigate the formation of authentic and functional DA neurons by differentiating human pluripotent stem cells (hPSCs) into 3D ventral midbrain (VM) patterned organoids. We combined transcriptional and protein analysis and we showed that ty-rosine hydroxylase-positive cells exhibit molecular and electrophysiological properties of ma-ture DA neurons. Comparing 2D versus 3D culture system we were able to show that VM or-ganoids can better recapitulate structural and morphological features of human brain including rosette-like organised structures. However, the use of a conventional 3D methodology resulted in high variability in terms of morphology, size, and cellular composition. We therefore estab-lished a novel technological approach using recombinant silk protein to create a bioengineered scaffold that arranges hPSCs in an organ-like configuration while maintaining their self-organizing property. In order to test silk scaffold properties, we induced hPSCs to differentiate into forebrain organoid following the previously reported protocol (Lancaster et al., 2017 Nat Biotech). In contrast to traditional 3D culture systems, bioengineered silk organoids increase the yield of neuronal differentiation at the expense of mesodermal, endodermal and stem cells populations.