Unbiased Profiling of Isogenic Huntington Disease hPSC-Derived CNS and Peripheral Cells Reveals Strong Cell-Type Specificity of CAG Length Effects

Ooi, Jolene; Langley, Sarah R.; Xu, Xiaohong; Utami, Kagistia Hana; Sim, Bernice; Huang, Yihui; Harmston, Nathan; Tay, Yi Lin; Ziaei, Amin; Zeng, Ruizhu; Low, Donovan; Aminkeng, Folefac; Sobota, Radoslaw M.; Ginhoux, Florent; Petretto, Enrico; Pouladi, Mahmoud A.

doi:10.1016/j.celrep.2019.02.008

Cited by 68 publications

(107 citation statements)

References 63 publications

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“…Similarly, accumulation of single and double-strand DNA breaks in response to mutant HTT expression has been shown to cause increased ATM activation [56]. Consistent with these observations, we observe CAG repeat lengthdependent increases in reactive oxygen species as well as activation of ATM in response to DNA damage in neural progenitors derived from the IsoHD hESC panel [31].…”

Section: Discussionsupporting

confidence: 84%

“…Human ESC and iPSC lines. The IsoHD female hESC lines and HD hiPSC lines (18Q, 71Q, and 109Q) were previously reported [31,36,61]. The lines were grown and expanded in feeder-free conditions using mTeSR1 medium (STEMCELL Technologies), at 37°C with 5% CO2.…”

Section: Methodsmentioning

confidence: 99%

“…To understand the effect of HTT CAG repeat length on early neurodevelopment and organization, we generated cerebral organoids from a panel of isogenic HD (IsoHD) hESC lines harboring HTT CAG lengths of 30, 45, 65 and 81 repeats, alleles that are associated with control, adult-, adolescent-, and juvenile-onset HD, respectively [1,2]. For each line, we chose 2-3 independent hESC clones with confirmed pluripotency, normal karyotype, and stable maintenance of the HTT CAG repeat tracts [31]. The hESC lines were induced to form cerebral organoids using an established protocol [30] whereby suspended hESCs are reaggregated to form embryoid bodies (EB) that grow to form neuroectoderm and contain neuroepithelium that expands over time ( Figure 1A).…”

Section: Expanded Htt Cag Repeats Lead To a Reduced Number Of Large Nmentioning

confidence: 99%

“…Human IsoHD lines 30Q, 45Q, 65Q, and 81Q were generated in-house and were fully characterized [31]. Cell lines were maintained with mTeSR medium.…”

Section: Maintenance Of Isohd Hesc Linesmentioning

confidence: 99%

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Expanded huntingtin CAG repeats disrupt the balance between neural progenitor expansion and differentiation in human cerebral organoids

Zhang

Ooi

Utami

et al. 2019

Preprint

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Huntington disease (HD) manifests in both adult and juvenile forms. Mutant HTT gene carriers are thought to undergo normal brain development followed by a degenerative phase, resulting in progressive clinical manifestations. However, recent studies in children and prodromal individuals at risk for HD have raised the possibility of abnormal neurodevelopment.Although key findings in rodent models support this notion, direct evidence in the context of human physiology remains lacking. Using a panel of isogenic HD human embryonic pluripotent stem cells and cerebral organoids, we investigated the impact of mutant HTT on early neurodevelopment. We find that ventricular zone-like neuroepithelial progenitor layer expansion is blunted in an HTT CAG repeat length-dependent manner due to premature neurogenesis in HD cerebral organoids, driven by cell intrinsic processes. Transcriptional profiling and imaging analysis revealed impaired cell cycle regulatory processes, increased G1 length, and increased asymmetric division of apical progenitors, collectively contributing to premature neuronal differentiation. We demonstrate increased activity of the ATM-p53 pathway, an up-stream regulator of cell cycle processes, and show that treatment with ATM antagonists partially rescues the blunted neuroepithelial progenitor expansion in HD organoids. Our findings suggest that CAG repeat length regulates the balance between neural progenitor expansion and differentiation during early neurodevelopment. Our results 1/26further support the view that HD, at least in its early-onset forms, may not be a purely neurodegenerative disorder, and that abnormal neurodevelopment may be a component of HD pathophysiology.

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Section: Discussionsupporting

confidence: 84%

Section: Methodsmentioning

confidence: 99%

Section: Expanded Htt Cag Repeats Lead To a Reduced Number Of Large Nmentioning

confidence: 99%

“…Human IsoHD lines 30Q, 45Q, 65Q, and 81Q were generated in-house and were fully characterized [31]. Cell lines were maintained with mTeSR medium.…”

Section: Maintenance Of Isohd Hesc Linesmentioning

confidence: 99%

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Expanded huntingtin CAG repeats disrupt the balance between neural progenitor expansion and differentiation in human cerebral organoids

Zhang

Ooi

Utami

et al. 2019

Preprint

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“…Gene expression of prioritized genes was assessed using RNA-seq data from isogenic allelic hPSC lines for four different HTT CAG lengths (CAG lengths: 30, 45, 65, 81; n =4 per length) that had been differentiated into five cell line types: pluripotent stem cells, hepatocytes, neural progenitor cells, post-mitotic neurons and skeletal muscle cells. Further details are described in Ooi et al 59 The expression for each gene (transcripts per kilobase million) and cell line combination were assessed as a continuous trait with linear regression for different CAG lengths.…”

Section: Methodsmentioning

confidence: 99%

Gene expression profiles complement the analysis of genomic modifiers of the clinical onset of Huntington disease

Wright

Caron

et al. 2019

Preprint

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Huntington disease (HD) is a neurodegenerative disorder that is caused by a CAG repeat expansion in the HTT gene. In an attempt to identify genomic modifiers that contribute towards the age of onset of HD, we performed a transcriptome wide association study assessing heritable differences in genetically determined expression in diverse tissues, employing genome wide data from over 4,000 patients. This identified genes that showed evidence for colocalization and replication, with downstream functional validation being performed in isogenic HD stem cells and patient brains. Enrichment analyses detected associations with various biologically-relevant gene sets and striatal coexpression modules that are mediated by CAG length. Further, cortical coexpression modules that are relevant for HD onset were also associated with cognitive decline and HD-related traits in a longitudinal cohort. In summary, the combination of population-scale gene expression information with HD patient genomic data identified novel modifier genes for the disorder.

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Introducing an Expanded Trinucleotide Repeat Tract into the Human Genome for Huntington's Disease Modeling In Vitro

et al. 2020

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In neurodegeneration studies, researchers are faced with problems such as limited material availability and late disease manifestation. Cell models provide the opportunity to investigate molecular mechanisms of pathogenesis. Moreover, genome editing technologies enable generation of isogenic cell models of hereditary diseases. Our protocol outlines an approach for introducing an expanded CAG repeat tract into the first exon of the HTT gene, the Huntington's disease causing mutation. The protocol allows modeling the disease at various severity levels by introducing different numbers of CAG repeats. Furthermore, the protocol can be applicable for modeling other diseases caused by trinucleotide repeat expansion. It is important to note there are many difficulties with cloning repeated sequences and amplification of GC‐rich regions. Here, we also propose troubleshooting options, which overcome these problems. The protocol is based on CRISPR/Cas9‐mediated homologous recombination with a uniquely designed donor plasmid harboring an expanded CAG tract flanked with long homology arms. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Design and assembling donor and CRISPR/Cas9‐expressing plasmidsBasic Protocol 2: Transfection of cells with plasmids and sorting GFP‐positive cellsBasic Protocol 3: PCR screening single‐cell clones and validation of the mutant HTT expression

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Unbiased Profiling of Isogenic Huntington Disease hPSC-Derived CNS and Peripheral Cells Reveals Strong Cell-Type Specificity of CAG Length Effects

Cited by 68 publications

References 63 publications

Expanded huntingtin CAG repeats disrupt the balance between neural progenitor expansion and differentiation in human cerebral organoids

Expanded huntingtin CAG repeats disrupt the balance between neural progenitor expansion and differentiation in human cerebral organoids

Gene expression profiles complement the analysis of genomic modifiers of the clinical onset of Huntington disease

Introducing an Expanded Trinucleotide Repeat Tract into the Human Genome for Huntington's Disease Modeling In Vitro

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