RESTORING CELLULAR HETEROGENEITY FOLLOWING TRAUMATIC BRAIN INJURY VIA TRANSPLANTATION OF HUMAN PLURIPOTENT STEM-CELL DERIVED CORTICAL NEURONS

Following traumatic injuries, the adult brain fails to regenerate neural circuits, resulting in permanent functional deficits. This work aims to explore the fundamental question of whether neuronal development and bona fide synaptic integration can succeed in the damaged neocortex. This is done by cell transplantation, introducing cells dissociated from dorsalized human brain organoids, into the mouse somatosensory cortex following a traumatic brain injury (TBI). We aim to investigate projection neuron subtypes present in the graft and whether a heterogeneous population self-organizes in cortical layers. This is achieved by utilizing immunohistochemical staining for transcription factors specific to discrete populations of cortical projection neurons and cortical layers, complimented by single nuclei RNA sequencing (snRNA-seq) of grafted cells. Our results show that, while cortical layering is not reconstructed during early stages, the graft reintroduces cortical projection neuron heterogeneity. At two-month post-transplantation, the majority of the neuronal population expresses a transcriptional signature of immature neurons, indicating that the maturation of the graft follows the tempo of human neuron development. While there are cell populations expressing layer specific transcription factors already at early developmental timepoints, there are also cells co-expressing transcription factors such as ctip2 and satb2. Later analysis will reveal the mature and truly discrete molecular identities. Despite their immaturity, we observe long-range axonal projections and formation of axonal boutons, indicating the ability to grow axons through the mature brain. This project will enhance our understanding of the potential of stem cell transplantation to regenerate complex cortical circuits in the adult mammalian brain.