Dysregulated development of cerebellar granule neurons (CGNs) underlies many cerebellar diseases. However, human models that recapitulate progenitor states of the CGN lineage are still lacking. In this study, Peter Dirks and colleagues establish a new CGN model generated from human hindbrain neuroepithelial stem (hbNES) cells. Fist, the authors differentiate hbNES cells to CGNs in vitro using soluble growth factors. Then, the authors use bulk RNA-sequencing and single cell Multiome sequencing to show that the differentiating hbNES cells transit through key human-specific progenitor states, resembling cerebellar neurogenesis in vivo. The authors observe that at day 7, the hbNES cells transit through a rhombic lip (RL) progenitor state that includes human-specific sub-ventricular cell identities. This RL state is followed by the granule neuron progenitor state at day 14 and mature CGNs are observed by 8 weeks. Using electrophysiology, the authors demonstrate that the neurons at this time point are functional. Finally, making use of their in vitro CGN system, the authors investigate the role of sonic hedgehog (SHH) during granule neuron differentiation and find that SHH may be involved in lineage determination and CGN progenitor proliferation. Overall, this study presents a new model for studying human cerebellar development and disease.
