Identification of neurodevelopmental organization of the cell populations of juvenile Huntington’s disease using dorso-ventral HD organoids and HD mouse embryos

Reviewer #1 (Public review):

In the manuscript "Identification of neurodevelopmental organization of the cell populations of Juvenile Huntington's disease using dorso-ventral HD organoids and HD mouse embryos," the authors establish a fused dorso-ventral system that mimics cortex-striatum interactions within a single organoid and use this system to investigate neurodevelopmental impairments caused by HD. Specifically, they describe certain phenotypes in 60-day HD organoids and the brains of humanized mouse embryos, utilizing both wet-lab and single-cell sequencing techniques. The authors also develop dorsal/ventral and ventral/dorsal mosaic control/HD organoids, showing a capacity to rescue some HD phenotypes.

The manuscript could be a valuable contribution to the field, however it has relevant drawbacks, the most significant being a lack of clarity regarding the replicates used for each genotype in the sequencing analyses. The lack of information on replicates raises the possibility that only a single replicate was analyzed for each organoid and brain sample. This approach may lead to concerns regarding the reproducibility of the findings, and it may be necessary for the authors to generate additional data to strengthen their conclusions. In addition, the analysis of the HD samples was conducted by pooling distinct cell populations from different brain regions (CTX, HIP, ChP for the dorsal brain, and STR, HYP, TH for the ventral brain). It is unclear why scRNA seq was used on pooled brain regions, which could obscure region-specific insights.

Another issue pertains to their proposed outcome: "Finally, we found that TTR protein, a choroid plexus marker, is elevated in the adult HD mouse serum, indicating that TTR may be a promising marker for detecting HD". This statement appears to lack statistical support, which makes this set of data potentially misleading and inconclusive.

The authors are encouraged to provide evidence of biological replicates, remove outcomes that lack statistical support, and address a series of points as detailed elsewhere.

Reviewer #2 (Public review):

The article titled "Identification of neurodevelopmental organization of the cell populations of juvenile Huntington's disease using dorso-ventral HD organoids and HD mouse embryos" analyses an in vitro human brain organoid model containig dorsal and ventral telencephalum structures derived from human iPSC from Huntington's disease patients or control subjects.

The authors describe differences in the pattern of expression of genes related to proliferation and neuronal maturation, with a slower pattern of differentiation present in HD cells. Moreover, the authors described a higher differentiation capacity of HD cells to generate choroid plexus identity following dorsal telencephalon prime protocol differentiation when compared to control cells. Whereas the claims related to Choroid plexus identity are intriguing, most of the claims made through the manuscript are not sustained by quantitative data or consistent results in the different conditions analysed, or many experiments seem to be missing to reach final conclusions.

In addition, the quality of the organoids used for experiments does not seem to have been assessed or satisfactorily presented in the figures of this paper. Many important details related to the experimental execution are missing in the current version of this manuscript.