Single-cell RNA-seq of telencephalic organoids from healthy CTRL and HD (Huntington's disease) hESC (RUES2) grown as mono-culture or co-culture of chimeric organoids

Huntington's disease (HD) causes selective degeneration of striatal and cortical neurons, resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells, grown separately or as mosaics of the two genotypes. We used single-cell transcriptomics to test early HD selective cellular phenotypes by comparing healthy (CTRL) and pathologic (HD) telencephalic organoids at days 45 and 120 of differentiation. To test the influence and the interactions between healthy and HD cells, mosaic organoids composed of CTRL and HD cells juxtaposed within the same organoid were grown and analyzed by scRNAseq at day 120. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids, confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons, while dorsal populations showed milder phenotypes mainly in the maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity, trajectories, synaptic density, and communication pathways upon cell-cell contact while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells, emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment. Overall design: At DIV 45, 6 individual organoids were independently sequenced for scRNAseq (3 per genotype: the CTRL with 20cag and the HD with 56cag). At DIV 120, 4 pools of 10 mosaic organoids each were collected for FACS sorting to separate the GFP and the TOM cell lines. The 4 pools derived from mono-culture organoids where GFP and TOM cells of the same genotype were grown together (20CAG-GFP with 20CAG-TOM and 56CAG-GFP with 56CAG-TOM) and co-culture organoids by co-culturing cells of different genotypes together (20CAG-GFP with 56CAG-TOM and 20CAG-TOM with 56CAG-GFP). After FACS we obtained and sequenced 8 independent samples. We then merged them as 4 biological conditions: “CTRL_mono” (CTRL cells grown alone); “CTRL_co” (CTRL cells that were grown in the presence of HD cells and then isolated); “HD_co” (HD cells that were grown in the presence of CTRL cells and then isolated); and “HD_mono” (HD cells grown alone, as for CTRL_mono).