Project: 1a

Title: Comparative analyses of DNA damage response pathways in hepatocytes and neurons differentiated from healthy and NBS-patient derived iPSCs

PI: Prof. Dr. James Adjaye

Institute: Institute for Stem Cell Research and Regenerative Medicine, Medical Faculty


Abstract

Background: Nijmegen Breakage Syndrome (NBS) is a rare autosomal recessive genetic disorder, first described 1981 in Nijmegen, the Netherlands. NBS is caused by mutations in the gene coding for NIBRIN (NBN) which is involved in DNA damage repair. The MRE11-RAD50-NBN (MRN) complex binds directly to DNA double-strand breaks (DSBs).

Characteristics of NBS include genomic instability, premature aging, microcephaly, immune deficiency, extreme radiosensitivity, higher cancer susceptibility and insulin resistance. A common denominator of these conditions could be chronic oxidative stress caused by endogenous ROS overproduction and impairment of mitochondrial homeostasis. Preliminary experiments have demonstrated that patient derived induced pluripotent stem cells (iPSCs) are suitable for modelling the disease and studying DNA damage during 2D and 3D in vitro differentiation processes.

Aim: Every cell type reacts differently to genotoxic noxes. Therefore, we aim to comparatively analyse DNA damage response pathways in hepatocytes and neurons differentiated from healthy and NBS-patient derived iPSCs after subjecting them to genotoxic stress.

Experimental procedure / working programme: We will analyse the influence of ionizing radiation, Benzo(a)pyrendiolepoxid (BaP), and Aflatoxin B1 (AB1) on the iPSCs during and after differentiation. The efficiency of DNA repair and the associated stress response will be compared between stem-, progenitor-, and mature cells derived from NBS patients and healthy donors. We will analyse how genotoxic stress influences the process of neurogenesis and hepatogenesis as well as the phase I and II metabolism of hepatocytes. Transcriptome analyses will indicate cell type specific changes in signalling pathways and Gene Ontologies. Key factors of DNA damage repair, apoptosis, and proliferation will be analyses on RNA and protein level, as well as with functional assays. In addition, we will focus on neuronal migration, mitochondrial damage and hepatocyte function (CYP450 activity). The project will be accomplished in close cooperation with other members of the GRK 2578.

Graphical abstract

iPSCs derived from NBS patients and healthy donors will be differentiated into neuro- and hepatocyte like cells. After submission to genotoxic agents, DNA damage repair (A), mitochondrial dysfunction (B), neuronal migration (C), and the detoxification capacity of hepatocyte like cells (D) will be analysed.

Requirements

The ideal candidate should have a strong background in toxicology, biochemistry, molecular biology and/or stem cell biology. High motivation and commitment to the project is expected. Ideally, the candidate should have experience in human iPS cell culture and 3D cellular differentiation models.

Application

Interested candidates are welcome to apply for this project. Please note to fill in the project number 1a in the application form.

Recommended literature for further information

  1. Mlody B et al. Nijmegen breakage syndrome fibroblasts and iPSCs: cellular models for uncovering disease-associated signalling pathways and establishing a screening platform for antioxidants. Sci Rep. 7(1):7516, doi: 10.1038/s41598-017-07905-2 (2017).
  2. Matz P et al. Footprint-free human fetal foreskin derived iPSCs: A tool for modelling hepatogenesis associated gene regulatory networks. Sci Rep. 7(1):6294, doi: 10.1038/s41598-017-06546-9 (2017).
  3. Lorenz C, et al. Human iPSC-derived neural progenitors are an effective drug discovery model for neurological mtDNA disorders. Cell Stem Cell 20(5):659-674.e9, doi: 10.1016/j.stem.2016.12.013 (2017).
  4. Halevy T, et al. Chromosomal instability and molecular defects in induced pluripotent stem cells from Nijmegen breakage syndrome patients. Cell Rep. 16(9):2499-511, doi: 10.1016/j.celrep.2016.07.071 (2016).
  5. Mlody B and Adjaye J Generation of iPSC lines from a Nijmegen Breakage Syndrome patient. Stem Cell Res.15(3):629-32, doi: 10.1016/j.scr.2015.10.013 (2015).


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