Figures and data
Scheme of the culture system generation of fused dorso-ventral HD and control forebrain organoids.
A) The neurospheres were formed from iPSC control and HD juvenile lines and were primed either by SHH inhibition by smoothened inhibitor cyclopamine A for obtaining dorsal brain patterning and by chemical inhibition of WNT by IWP2 and activation of SHH by agonist SAG for obtaining ventral brain cell lineages. On DIV 16, the dorsal and ventral primed spheres were fused and cultivated to model the early pseudo tissue mimicking the cerebral cortex (CTX) assembled with the striatum (STR). To model early brain development, we established day 60 as the maturing endpoint for our control and HD organoids. The mosaic organoids from dorsal and ventral priming of HD and control lines were generated to produce a compensatory model to assess the contribution of dorsal and ventral parts in the development of HD phenotypes in fused organoids. B) A representative slice of HD fused brain organoid stained for TBR1 protein, a marker of the dorsal forebrain, and the scheme of the coronal embryonic brain section showing the dorsal-ventral gradients and their respective small molecules used.
Macroscopic organization of 60-day-old HD fused organoids shows their less regular structure and increased sizes.
A) Macroscopic 2D images of the fused dorsal/ventral control, HD organoids, and mosaic HD/control organoids from the iPSC lines used to generate the organoids; B) Area measurement graphs demonstrated data collected from 2D images of the organoids. The sizes of dorsal/ventral fused HD vs. control organoids were significantly greater and is also depicted as C) aggregated data panel where all control organoid sizes were plotted against HD organoid sizes (Levene’s test p > 0.05: variance examination: equal; ANOVA p.val = ∼ 0.0086; posthoc: T-test, *p < 0.05, **p < 0.01***p < 0.001). The sizes of the mosaic organoids vs control and HD organoids did not reach statistical significance; however, on average, 21D/71V was close in size to control organoids, and the opposite configuration of mosaic 71D/21V was close in size to HD organoids. The area [mm2] of each organoid was measured in ImageJ software.
Prominent occurrence of neuronal rosettes containing TBR1 and PAX6 positive neural stem cell populations in 60-day-old fused dorso-ventral HD organoids
The internal structure of the organoids and characteristic PAX6-positive (early progenitors, NSC) and TBR-positive (early-born neurons) dorsal populations in organoids were detected by immunostaining protocol. The panels of representative micrographs are designated by HD (71D/71V, 77D/77V), control (17D/17V, 21D/21V), and mosaic organoid (21D/71V, 71D/21V) types. The panels contain a co-staining experiment with PAX6 (green) and TBR1 (red) and a merged image of both stainings for each organoid. The micrographs revealed the occurrence of a large number of PAX6-positive neural rosettes lying close to each other and the occurrence of the separate layers of TBR1 expression in HD fused organoids (71D/71V & 77D/77V) while in control fused organoids (17D/17V & 21D/21V) only several scattered rosettes and lower expression of PAX6 and TBR1 is present, indicating less neural stem cells therefore less proliferative profile. In turn, HD organoids are directed towards the proliferative phase characteristic for early neural development with a vast number of progenitors in rosettes. Importantly, in the mosaic organoids, there is a regression in the HD phenotype represented by reduced neural rosette occurrence.
Evaluation of the mRNA expression levels of neurodevelopmental marker genes of selected types of neuronal and non-neuronal cell populations in 60-day-old juvenile HD fused brain organoids.
The panels are designated with names of cell types and show graphs of mRNA levels of marker genes in 60-day-old fused dorso-ventral HD and control organoids originating from several iPSC lines. The graphs of marker genes are grouped by Inhibitory neurons, Choroid plexus and tanycytes, Astrocytes and oligodendrocytes, Nitrergic neurons, Progenitors and Excitatory neurons. The expression level of an analyzed gene in each type of fused organoid was compared to 21Q control organoids as basal expression level. The mRNA expression was normalized using ACTβ as a reference gene. (Levene’s test p > 0.05: was used for variance examination.
Identification of deregulated expression of genes in pooled cell transcriptomes of juvenile HD, control and mosaic juvenile HD/control organoids, and HD embryonic brains.
The cell transcriptomes from scRNAseq experiments were first pooled by genotypes indicated on volcano plots, namely by types of organoids and ventral or dorsal parts of HD embryonic brains, (YAC128/null and Hu128/21) or control brains (Bac21/null) for screening of DEGs in these datasets. A) The analysis of DEGs for organoids has shown the most striking upregulation of the TTR expression in HD organoids, which is closely related to the occurrence of the ChP population. This deregulation was not significant in both mosaic organoids. B) The analysis of mouse embryonic brains has shown upregulation of the TTR in both models (YAC128/null and Hu128/21). The analysis is extended by GSEA [Gene Set Enrichment Analysis], available as supplementary figures 2 and 3.
Indication of the main differences in all cell populations for HD (71Q dorsal/71Q ventral; 77Q dorsal/77Q ventral), control (17Q dorsal/17Q ventral; 21Q dorsal/21Q ventral), and mosaic (21Q dorsal/71Q ventral; 71Q dorsal/21Q ventral) organoids.
A) The UMAP presentation of the composition Choroid Plexus (ChP) population in HD, control, and mosaic organoids. There are noticed occurrence of ChP populations the most in HD (71Q dorsal/71Q ventral; 77Q dorsal/77Q ventral) and mosaic (71Q dorsal/21Q ventral) organoids. B) Percentage representation of cells belonging to ChP populations compering to the rest populations in HD, control, and mosaic organoids. There are also noticed the occurrence of ChP populations the most in HD (71Q dorsal/71Q ventral; 77Q dorsal/77Q ventral) and mosaic (71Q dorsal/21Q ventral) organoids and less percent in control (21Q dorsal/21Q ventral) and complete lack of occurrence in control (17Q dorsal/17Q ventral) organoids.
Indication of the main differences in all cell populations for full HD (YAC128/null – dorsal & ventral brain), semi HD (Hu128/21 dorsal & ventral brain) and control (BAC21/null - dorsal & ventral brain) embryo brains.
A) The UMAP presentation of the composition of the Choroid Plexus (ChP) population in HD and control embryo brains. There are noticed occurrences of ChP populations just in HD cell populations (YAC128/null – dorsal, Hu128/21 - dorsal) with dorsal lineages. B) Percentage representation of cells belonging to ChP populations compared to the rest populations in full HD, semi HD, and control embryo ventral and dorsal parts of brains. There are also approved occurrences of ChP populations only in HD (YAC128/null – dorsal, Hu128/21 - dorsal) and a complete lack of occurrence in control dorsal and ventral brain parts (BAC21/null - dorsal & ventral brain) and also in ventral parts of HD brains (YAC128/null – ventral, Hu128/21 - ventral).
In single-cell RNA sequencing (scRNAseq) data, we observed the co-expression of genes related to the mitotic spindle, cilia, and genes that regulate differentiation in selected cell populations across various datasets:
A) In the dataset for fused brain organoids, including healthy (17D/17V, 21D/21V), mosaic (17D/17V, 21D/21V), and disease (17D/17V, 21D/21V) conditions, we identified the co-expression of these genes, B) In the dataset for the dorsal part of mouse embryonic brains, encompassing healthy (BAC21/null) and disease (Hu128/21 & YAC128/null) conditions, these genes were found to be co-expressed, C) Similarly, in the dataset for the ventral part of mouse embryonic brains, including healthy (BAC21/null) and disease (Hu128/21 & YAC128/null) conditions, the co-expression of these genes was observed. These genes exhibited mutual expression across various cell populations in both normal and Huntington’s disease (HD) affected brains. They are involved in essential processes such as differentiation, migration, and maturation, primarily through their influence on the mitotic spindle and cilia.
Molecular validation of scRNA-seq results of TTR upregulation and choroid plexus impairment results using immunofluorescence and Western blotting.
(A) Immunofluorescence staining for TTR in 60-day-old fused dorso-ventral 21Q/71Q mosaic organoids and 21Q dorso-ventral control organoids. IF results show an abundance of TTR in 71Q dorsal/ 21Q ventral mosaic organoids compared to the other organoids, which corresponds to the scRNA-seq results. (B) Western blot analyses of blood serum from adult 6-8-months mice show non-significant but yet an elevation level of TTR monomer and tetramer in YAC128Q/null mice compared to BAC21Q/null mice. The level of TTR monomer protein (16 kDa) was normalized to the level of the reference protein LAMIN B1. The graph was generated in GraphPad Prism. The statistical test used was the t-test for independent samples (n=3). (B) The level of TTR tetramer protein (55 kDa) was normalized to the total amount of protein in lanes stained with Ponceau S solution. The graph was generated in GraphPad. The statistical test used was the t-test for independent samples (n=5).
Sequences of primers used for qRT-PCR analyses.
