• 3D neural in vitro models
• cortical organoids
• spinal organoids
• corticospinal assembloids

The central nervous system (CNS) development is a complex and highly regulated process that begins early in the embryo and continues through postnatal stages. The process initiates with the formation of the neural plate, which subsequently folds, leading to the formation of the neural tube. Guided by gradients of morphogens along its rostro-caudal and dorso-ventral axes, the neural tube gives rise to brain vesicles rostrally and the spinal cord caudally. Within the telencephalon, the neural stem/progenitor cells undergo differentiation, resulting in the generation of neurons which then migrate toward the pial surface, orchestrating the formation of cortical layers through an "inside-out" mechanism. An important cell progenitor population is the outer radial glial cells, whose proliferative activity allows the increase in size and complexity of the human cortex. Simultaneously, in the spinal cord, progenitor cells differentiate based on the dorso-ventral gradient of various morphogens, giving rise to distinct domains of interneurons and motor neurons. During development, axons primarily from the primary motor and somatosensory cortex, elongate toward the spinal cord forming the cortico-spinal tract, a structure essential for the control of voluntary movements.
In vitro models of human brain development have significantly improved with the advent of hiPSC-derived organoids. With these 3D systems, it is possible to recapitulate the cytoarchitectonic and functional characteristics of specific areas of the CNS. Recently, the implementation of 3D culture systems has led to the generation of assembloids obtained by fusing CNS organoids with different identities. Together, these approaches represent a promising tool for study CNS developmental mechanisms and diseases.
This thesis aims to improve the current protocols for the generation of cortical and spinal organoids with the final goal of creating a corticospinal assembloids that could model the cortico-spinal tract in vitro. We achieved an enhancement in organoid viability by culturing them under dynamic conditions utilizing a peristaltic pump. Additionally, we improved the cellular complexity of cortical organoids by enriching for outer radial glia cells. Regarding spinal organoids, we successfully implemented a protocol that resulted in a high percentage of cervical motor neurons within 28 days. Finally, we established an in vitro model of the cortico-spinal tract by assembling cortical and spinal organoids.
Together, the combination of region-specific organoids and cortico-spinal assembloids holds great promise for advancing our understanding of traumatic and genetic insults of the CNS.