Directed differentiation of human hindbrain neuroepithelial stem cells recapitulates cerebellar granule neurogenesis

Abstract

Cerebellar granule neurons (CGNs) are the most abundant neurons in the human brain and modulate cerebellar output to the motor cortex. Dysregulation of CGN development underlies movement disorders and medulloblastomas. It is suspected that these disorders arise in progenitor states of the CGN lineage, for which human models are lacking. Here, we have differentiated human hindbrain neuroepithelial stem (hbNES) cells to CGNs in vitro using soluble growth factors, recapitulating key progenitor states in the lineage. We show that hbNES cells are not lineage committed and retain rhombomere 1 (r1) regional identity. Upon differentiation, hbNES cells first transit through a rhombic lip (RL) progenitor state at day 7, demonstrating human specific sub-ventricular cell identities. This RL state is followed by an ATOH1+ CGN progenitor state at day 14. By the end of a 56-day differentiation procedure, we obtain mature neurons expressing CGN markers GABAAa6 and vGLUT2. These neurons generate spontaneous and evoked action potentials. A small fraction of endpoint neurons were unipolar brush cells (UBC). We noted maintenance of a RL population throughout differentiation, as is consistent with human development. We show that sonic hedgehog (SHH) promotes {gamma}-aminobutyric acid (GABA)-ergic lineage specification and is a positive regulator of CGN progenitor proliferation. Interestingly, we observed that functional neuronal maturation is impaired by either elevated or absent SHH signaling. Impaired maturation under high SHH levels represents the potential of our system to model cerebellar tumorigenesis. Further, our data suggest a potential pro-differentiation role of SHH within a certain concentration range. Our work is, to our knowledge, the first detailed temporal characterization of the complete human CGN lineage in vitro. Our system recapitulates developmentally relevant progenitor states and is a new tool to model this specific cerebellar lineage, and how it may be disrupted to cause human disease.