RSUSSH 2020
IN20-189 Establishment of Inducible SMAD4 Knockout PSC-Derived Cholangiocyte Committed Cells
Presenter: Natthida Kittimawikrom
Chulalongkorn University, Thailand
Abstract
Cholangiocarcinoma (CCA), malignancies of the biliary duct system, is a major public health problem especially in the region of northeastern Thailand. Previous study demonstrated SMAD4 which is a crucial mediator of TGF-β and BMP4 signaling pathway was high mutation frequency in CCA. The mechanism of SMAD4 mutation drive cholangiocarcinogenesis remains unknown. With ethical and reproducible limitation, adult human cholangiocyte still lacks the reliable in vitro model to study the pathogenesis. Pluripotent stem cells (PSCs) have potential for bridge the gap with unlimited cell source. Recently, PSC-derived cholangiocyte differentiation protocols have been reported. The constitutively activation of conventional CRISPR-Cas9 system is not suitable for study the role of SMAD4 by affecting the cell differentiation. To dissecting SMAD4 gene function with precise and less interfere differentiation procedure, an inducible knockout system is indispensable for SMAD4 disruption in this lineage. In this study, we developed an inducible SMAD4 knockout PSC for SMAD4 knockout which is stage-specific inducible gene knockout during cholangiocyte differentiation. The disruption of SMAD4 gene was succeeded in PSC and cholangiocyte lineage-committed cells after doxycycline treatment. T7EI analysis revealed an ∼20% Indel rate in PSC. Indel rate was succeeded in differentiated cell; hepatoblast (10.81%) and cholangiocyte progenitor (6.31%). 3 indel mutation patterns were confirmed by DNA sequencing with frameshift mutation. These results suggest that inducible SMAD4 knockout cassette has potential to generate DSB in several stage of cholangiocyte lineage-committed cells. This study will be applied for developing the model and dissecting SMAD4 gene function in any developmental stage in further study.
Citation format:
Israsena Na-Ayudhaya, N., Ingrungruanglert, P., & Kittimawikrom, N.. (2020). Establishment of Inducible SMAD4 Knockout PSC-Derived Cholangiocyte Committed Cells. Proceeding in RSU International Research Conference, May 1, 2020. Pathum Thani, Thailand.QUESTIONS & ANSWERS
Your work is good idea for establish SMAD4 knockout cell model for further studies on CCA development. I have some questions about SMAD4 knockout cell validation. Why you not validate SMAD4 knockout cells by using Western blotting for check SMAD4 protein expression after induction? In my opinion, the Western blot technique is able to determine SMAD4 protein expression after induction directly. Why you use the T7 endonuclease 1 detection for validate SMAD4 knockout cells? Thank you in advance for your answer.
T7 endonuclease 1 (T7EI) assay is use for preliminary validation at genomic DNA level in this study. As your suggestion, only the SMAD4 knockout result in genomic DNA level are not enough to validate the SMAD4 knockout. Although, the SMAD4 knockout potential of guide RNA in this study was published by previous study in other cell type. In this study, the total cell number was collected after induction and validated by using T7EI. With collecting the total cells, it may not represent the change of protein expression level (not precise) by using the western blotting. So, single clone selection is required for genomic DNA and protein expression validation. However, the protein collection is needed a lot of cell number. We will select single clone, scale-up cell number and use the western blotting assay for SMAD4 KO validation in further study. Thank you for your question and suggestion.
Thank you for your response.