PTCH1-mutant human cerebellar organoids exhibit altered neural development and recapitulate early medulloblastoma tumorigenesis

ABSTRACT Patched 1 (PTCH1) is the primary receptor for the sonic hedgehog (SHH) ligand and negatively regulates SHH signalling, an essential pathway in human embryogenesis. Loss-of-function mutations in PTCH1 are associated with altered neuronal development and the malignant brain tumour medulloblastoma. As a result of differences between murine and human development, molecular and cellular perturbations that arise from human PTCH1 mutations remain poorly understood. Here, we used cerebellar organoids differentiated from human induced pluripotent stem cells combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences of PTCH1 mutations on human cerebellar development. Our findings demonstrate that developmental mechanisms in cerebellar organoids reflect in vivo processes of regionalisation and SHH signalling, and offer new insights into early pathophysiological events of medulloblastoma tumorigenesis without the use of animal models.

sequencing revealed uniform cutting at the intronic target site compared to more heterogeneous cutting at the exonic target site.The genomic DNA region between the target sites is absent in the mutant amplicons (mut).Point mutations at the intronic target site resulting from the Cas9-induced double strand break are detected in heterozygous clones (G>A) (marked in red).

Fig. S1 .Fig. 1 )
Fig. S1.Sanger sequencing of CRISPR target site in the PTCH1 gene (related to Fig. 1) Top: Sequencing of control PTCH1 sequence (reference).Guide RNA (gRNA) sequences (exonic in green, intronic in purple) and respective protospacer adjacent motifs (marked orange in reference sequence) are displayed with the coding strand of the reference sequence on top.Below: Sequencing of mutant clones.The expected cut sites are depicted as vertical dashed lines.Sanger

Fig. S2 .
Fig. S2.PTCH1 mutant clones maintain iPSC morphology and pluripotency marker expression (related to Fig. 1) A) Brightfield images of iPSC clones.Scale bars 1000μm.All clones display normal iPSC morphology with large nucleus-to-cytoplasm ratio and growth in densely packed colonies.B) A total of 50,000 cells were measured by flow cytometry.Density plot shows the distribution of fluorescence intensity along the x-axis.Coloured histograms represent cells stained with antibodies targeting NANOG (left) or TRA-1-60 (right).Grey histograms represent cells stained with isotype control antibodies to assess non-specific binding and autofluorescence.The percentage of cells with fluorescence intensities above the isotype control condition are depicted.

Fig. S3 .
Fig. S3.SNP array to test for chromosomal aberrations in gene edited iPSC clones (related to Fig. 1) For each chromosome, the first dot plot displays the B-allele frequency and indicates whether the SNV is heterozygous (data points fall at around 0.5) or homozygous (data point at around 0.0 of 1.0).The second plot displays the log R value (first dot plot, indicated with R on the left side) is given, representing the probe intensity of individual SNVs.Chromosomal gains are identified by doubling of the log R value while halving the R value indicates loss.The different clones are given in the following order: control line: AH017-3; heterozygous (PTCH1 +/-) clones: A3, C6, B2, B4; homozygous (PTCH1 -/-) clones: H3, B3, A2.

Fig. S4 .
Fig. S4.Sanger sequencing does not reveal off-target CRISPR-Cas activity (related to Fig. 1) Sanger sequencing of the top five off-target sites of each guide RNA.The alignment with each respective gRNA is shown below the wildtype (WT) strand with mismatches shown in red.Sequence traces of both the AH017-3 WT and tested homozygous mutant (H3) were identical.

Fig
Fig. S8.Neuronal marker expression is maintained in Cyclopamine-treated PTCH1-/organoids (related to Fig. 4) Immunofluorescence staining of day 35 organoids with antibodies specific to the pan-neuronal marker NeuN (green).Nuclei are visualised in blue by Hoechst staining.Scale bars 150μm.

Fig. S9 .Fig. 6 )
Fig. S9.PTCH1 heterozygous organoids cluster with human SHH-MB samples (related to Fig. 6) CTRL and PTCH1+/-heterozygous organoids were clustered with human medulloblastoma (MB) RNAsequencing samples using an unsupervised hierarchical clustering method based on the normalized gene expression of 917 subgroup specific MB gene markers.The top subtype-specific markers are indicated on the right of the heatmap.

Table S2 .
Differentially expressed genes in heterozygous clones