Article
The generation of transformed human induced pluripotent stem cells – towards the generation of synthetic in vitro models for molecular subtypes of brain tumours
Herstellung von transformierten humanen induzierten pluripotenten Stammzellen zur Erzeugung von synthethischen in vitro Modellen für molekulare Subtypen von Hirntumoren
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Authors
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Constanze Uhlmann
- University Hospital Düsseldorf, Department of Neurosurgery, Düsseldorf, Deutschland
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Dilaware Khan
- University Hospital Düsseldorf, Department of Neurosurgery, Düsseldorf, Deutschland
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Ann-Christin Nickel
- University Hospital Düsseldorf, Department of Neurosurgery, Düsseldorf, Deutschland
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Julia Tigges
- IUF – Leibniz Research Institute for Environmental Medicine, Düsseldorf, Deutschland
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Sajjad Muhammad
- University Hospital Düsseldorf, Department of Neurosurgery, Düsseldorf, Deutschland
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Hans-Jakob Steiger
- University Hospital Düsseldorf, Department of Neurosurgery, Düsseldorf, Deutschland
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Andrea Rossi
- IUF – Leibniz Research Institute for Environmental Medicine, Düsseldorf, Deutschland
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Daniel Hänggi
- University Hospital Düsseldorf, Department of Neurosurgery, Düsseldorf, Deutschland
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Ellen Fritsche
- IUF – Leibniz Research Institute for Environmental Medicine, Düsseldorf, Deutschland
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Ulf Dietrich Kahlert
- University Hospital Düsseldorf, Department of Neurosurgery, Düsseldorf, Deutschland
| Published: | June 4, 2021 |
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Outline
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Objective: Glioblastoma (GBM) and medulloblastoma (MB) are the most common malignant brain cancer in adults and children, respectively. Patient-derived brain cancer models have been used over the last years for oncological research but show limitations in genetic stability. Further, some subgroups cannot be cultured under laboratory conditions. We recreated GBM and MB subgroups with human induced pluripotent stem cells (hiPSC) through genetic manipulation. Generated models were used for OMICs analysis and a drug screen to identify new treatment approaches.
Methods: Lentiviral transduction was used to generate the subgroup specific GBM and MB models. Known genes to be involved in the malignant transformation were used for model generation: TP53, EGFRvIII, c-Myc and Gli1. Cell lines were antibiotic selected and protein over-expression was confirmed by western blot. Stem cell marker expression of hiPSC models was verified with FACS and immunocytochemistry (ICC) staining. hiPSC models were utilized for a semi-automated drug screen with 167 FDA approved compounds. Candidates were chosen to validate their treatment potency in a smaller set-up. RNA and DNA of all lines was collected for transcriptome and methylome analysis, respectively.
Results: The development of GBM and MB models was achieved for the classical GBM (EGFRvIII), SHH MB (Gli1) and Group 3 MB (c-Myc) subgroup. Overexpression of the marker genes was confirmed in comparison to the wild type and empty vector control. Stem cell expression of Nanog, Sox2 and Oct3/4 could be verified. All markers showed an expression of over 70%. ICC staining with Tra-1-60 and Oct3/4 was positive for all generated models. The drug screen identified compounds effective on the generated models and one compound that is only effective in MB models. Methylome analysis showed a demethylation for all genes expect for EGFR.
Conclusion: We could successfully generate hiPSC models with gene activation similar to GBM and MB subgroups. These models can be used for cancer research. Neural differentiation of hiPSC allows the usage of the cells for further screenings. GBM subgroup specific neurospheres are currently under investigation for the implementation into embryonic chicken brain to verify the tumour forming potential.
