Construction of human 3D striato nigral circuitoids to recapitulate medium spiny neuronal projection defects in Huntington disease

Striato nigral circuit is composed of medium spiny neuronal projections that were mainly sent from striatum to midbrain substantial nigra (SN), which is essential for regulating motor behaviors. Dysfunction of striato-nigral circuitry may cause a series of motor disabilities which are associated with neurodegenerative disorders, such as Huntington disease (HD). Although the etiology of HD is known as abnormally expanded CAG repeats of the huntingtin gene (HTT), treatment of HD remains tremendous challenges. One possible reason is lack of effective HD model which resembles striato nigral circuitry deficits for the pharmacology studies. Here we first differentiate striatum-like organoids from human pluripotent stem cells, containing functional medium spiny neurons (MSN). We then generated 3D striato nigral circuitoids by assembling striatum-like organoids with midbrain substantial nigra like organoids. With AVV hSYN GFP viral tracing, the extensive MSN projections from striatum to SN are established, which form synaptic connection with dopaminergic neurons and showed the electronic field potentials by labeling striatum like organoids with optogenetic virus. Furthermore, this striato nigral circuitoids exhibited improved calcium activity than individual striatal organoids. Importantly, we further demonstrated the reciprocal projection defects of the HD iPSC derived circuitoids, which could be reversed with the treatment of brain derived nerve factors. Altogether, the striato nigral circuitoids could be used for identifying MSN projection defects, which would be applied as a potential drug test platform for HD. For the scRNA-seq, striatal organoids and striato-nigral circuitoids were dissociated by 1 ml of trypLE (Life Technology Inc) for 30 min in the condition of 37°C and 5% CO2 into single cell when differentiated in vitro for 80 days. The cell debris and other aggregates were removed by additional low-speed centrifugation. Droplet-based single-cell RNA sequencing was performed using the 10X Genomics Chromium Single Cell platform, version 3. Approximately 5,000 cells were captured for each sample onto a Chromium Single Cell 3’ Chip (10X Genomics, PN-120236), and the released RNA in each cell was barcoded through reverse transcription in individual gel beads in emulsions (GEMs).