Abstract Details

Title Editing of IDH1 R132H Mutation in Human Induced Pluripotent Stem Cells to Investigate Tumor Genesis in Glioma

Topic Neuro-oncology

Presentation(s) P1 - Poster Session 1 (12:00 PM-1:00 PM)

Poster/Presentation
Number 13-008

Objective Low grade gliomas and secondary glioblastomas are characterised by a hot-spot mutation in the Isocitrate dehydrogenase 1 (IDH1). The mutation causes a new catalytic function of the enzyme resulting in the production of 2-Hydroxyglutarat (2-HG), which is known as an oncometabolite. To this day, the pathomechanism by which IDH1 mutation promotes tumorigenesis is not completely understood. Moreover, reliable cell models reflecting the patient's situation are not available. Thus, we aim at creating human induced pluripotent stem cells (hiPSC) carrying the IDH1 R132H mutation. We will utilize this cell model to investigate how the mutation influences stem cell properties and cell differentiation into cerebral organoids.

Background NA

Design/Methods For designing the cell model, we applied the novel CRISPR/Cas9 based genome editing tool Base Editor 3 and 4. To determine editing efficiency, cells were investigated using the T7 Endonuclease 1 assay and Next Generation Sequencing. Single cell clones were picked to analyse allele status using allele-specific PCR with subsequent Sanger Sequencing. Differentiation into cerebral organoids is based on a protocol from Lancaster et al..

Results

Next generation sequencing analysis revealed a base transition in about 1 % of the reads. We confirmed expression of mutated IDH1 R132H by Western Blot. Measurement of the tricarboxylic acid cycle metabolites using liquid chromatography tandem mass spectrometry (LC-MS/MS) showed a forty times increased concentration of 2-HG in IDH-mutated compared to the wildtype iPSCs. To investigate effects of the IDH1 R132H on cell differentiation, we generated cerebral organoids from our iPSC-models. The IDH1 R132H mutation did not inhibit the cell differentiation in maturation of cerebral organoids. On the transcriptional level, pathways for ECM-receptor interaction were upregulated and pathways regarding the spliceosome were downregulated.

Conclusions We successfully established a heterozygous IDH1 R132H mutated hiPSC cell line, which represents a promising cell model for future studies of early glioma genesis.

Authors/Disclosures
PRESENTER	No disclosure on file
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