Activating Hepatic Progenitor Cells

Abstract

Background and aims – Studies in HepaRG cells have shown that DYRK1A inhibition causes a shift in cell cycle distribution, both by siRNA mediated gene silencing and chemical inhibition. This effect has not yet been described before in liver cells and warrants follow up study in primary liver cells. The aim of this part of the project was to validate the results found in HepaRG cells in a primary liver stem cell culture. Materials and methods – Mouse Organoid Derived Progenitor Cells (ODPCs) are used as an in vitro model for HPCs. Dyrk1A was knocked down by siRNA mediated gene silencing and chemically inhibited with harmine. Knockdown was verified on mRNA level with qPCR and on protein level with Western blot. Proliferation was measured by assessing EdU incorporation and pH3 phosphorylation. Cell cycle distribution was measured using flow cytometry and subsequently analysed using FlowJo software. HepaRGs were taken up in this experiment as a positive control, as it has already been shown that a phenotype can be established in this cell type. Results – In ODPCs knockdown and chemical inhibition of Dyrk1A did not affect proliferation, nor was there an effect on cell cycle distribution. Knockdown was confirmed on mRNA and protein level on 48 hours and on mRNA level on 72 hours. Knockdown of DYRK1A in HepaRGs by siRNA mediated gene silencing resulted in a shift in the cell cycle distribution. Chemical inhibition by harmine did not have an effect on the cell cycle. Conclusion – The effect on cell cycle distribution of siRNA mediated gene silencing of DYRK1A as found in the high throughput screen and part A of this project was confirmed by flow cytometry and FlowJo analysis. This effect is not found in chemically inhibited wells in HepaRGs. In ODPCs there is no effect of Dyrk1A knockdown on the cell cycle, nor is there an effect of chemical inhibition. The fact that the phenotype found in HepaRGs cannot be validated in a primary HPC culture needs to be further investigated. Neither cell types showed an effect of chemical inhibition on the cell cycle distribution.