A Human Induced Pluripotent Stem Cell-Derived Isogenic Model of Huntington's Disease Based on Neuronal Cells Has Several Relevant Phenotypic Abnormalities.

Tuyana Malankhanova,Elena Grigor’Eva,Anastasia Malakhova,Elena Kiseleva,Suren Zakian,Julia Minina,Sergey Medvedev,Ksenia Morozova,Lyubov Suldina

doi:10.3390/jpm10040215

Tuyana Malankhanova, Elena Grigor’Eva + Show 7 more

Open Access

https://doi.org/10.3390/jpm10040215

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Abstract

Huntington’s disease (HD) is a severe neurodegenerative disorder caused by a CAG triplet expansion in the first exon of the HTT gene. Here we report the introduction of an HD mutation into the genome of healthy human embryonic fibroblasts through CRISPR/Cas9-mediated homologous recombination. We verified the specificity of the created HTT-editing system and confirmed the absence of undesirable genomic modifications at off-target sites. We showed that both mutant and control isogenic induced pluripotent stem cells (iPSCs) derived by reprogramming of the fibroblast clones can be differentiated into striatal medium spiny neurons. We next demonstrated phenotypic abnormalities in the mutant iPSC-derived neural cells, including impaired neural rosette formation and increased sensitivity to growth factor withdrawal. Moreover, using electron microscopic analysis, we detected a series of ultrastructural defects in the mutant neurons, which did not contain huntingtin aggregates, suggesting that these defects appear early in HD development. Thus, our study describes creation of a new isogenic iPSC-based cell system that models HD and recapitulates HD-specific disturbances in the mutant cells, including some ultrastructural features implemented for the first time.

Highlights

Huntington’s disease (HD) is an actively investigated neurodegenerative disorder caused by an expansion of CAG triplets (>36) in the first exon of the huntingtin fragment (HTT) gene encoding the huntingtin protein
To introduce an expanded CAG tract into HTT, we used a donor plasmid for homologous recombination at the HTT locus and a plasmid expressing elements of the CRISPR/Cas9 system, which were previously constructed in the Laboratory of Developmental Epigenetics (Institute of Cytology and Genetics, SB RAS) [9]
We generated a novel isogenic HD model based on an induced pluripotent stem cells (iPSCs) line carrying 69/22 CAG repeats in the first exon of the HTT gene with a control isogenic iPSC line without this mutation

Summary

Introduction

Huntington’s disease (HD) is an actively investigated neurodegenerative disorder caused by an expansion of CAG triplets (>36) in the first exon of the HTT gene encoding the huntingtin protein. A solution to this problem is the creation of isogenic cell lines [3]. The latter have an identical genetic background and differ from each other only by the disease-causing mutation. Genome-editing tools such as the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system can be used to create isogenic cell lines. An isogenic pair of cell lines can be obtained in two ways: the first method is to correct the mutation in patient-specific cells, and the second one is to introduce the mutation into “healthy” cells.

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