Generation and Differentiation of Human Olfactory Epithelial Organoids from Adult Stem Cells

The human olfactory epithelium possesses remarkable neurogenic capacity, sustained by a resident population of basal stem cells; however, establishing a dynamic in vitro model that faithfully recapitulates this regenerative process has been proven challenging. Here, we present an established protocol for generating olfactory organoids from progenitor cells isolated from adult human superior turbinate tissue. These organoids maintain stemness, as evidenced by SOX2 expression, and display a spatially organized differentiation pattern, characterized by uniform expression of the immature neuronal marker GAP43 and peripheral localization of the mature olfactory sensory neuron marker OMP. Subsequent targeted differentiation with the Notch inhibitor LY411575, the Wnt activator CHIR-99021, and retinoic acid generated marker-positive olfactory sensory neuron-like cells that co-expressed OMP and the neuronal marker Tuj1 (class III β-tubulin). This human-derived model circumvents the limitations of murine systems and provides a robust in vitro platform for studying olfactory neurogenesis, COVID-19-associated anosmia, and other olfactory-related neurodegenerative disorders.