Development of in-vitro hepatic organoid model

The biggest obstacle to modeling and the development of treatment strategies for liver-related diseases from cancer to metabolic diseases is that hepatocytes can not replicate efficiently in the cell culture. In recent years, knowledge learned from developmental biology research has been the basis of in-vitro differentiation protocols to produce liver cells from pluripotent cells such as embryonic stem cell (ESC) or induced pluripotent stem cells (iPSC). However, the current problem is that two dimension (2D) hepatocytes are not functionally equivalent to primary hepatocytes. At this point, three-dimensional (3D) cell culture models have become more important because environmental factors such as cell-cell and/or cell-matrix interaction are similar to tissue. In this thesis, healthy human iPSCs obtained by episomal reprogramming and hepatocytes were generated by using 2D and 3D organoid culture systems. The cells were characterized in vitro and in vivo conditions at each step. To acheive 3D hepatic organoids, iPSCs were first differentiated into endoderme and FACS purified EpCAM+ cells of endoderm step were used to establish organoid by using an expansion medium containing various growth factors used in the Huch's protocol. Finally, the organoids were differentiated toward mature hepatocytes under appropriate conditions. Organoids from citrullinemia-derived iPSCs exhibited phenotypes such as increased ammonia and citrulline accumulation, similar to the disease pathology observed in patients. These phenotypes could be rescued by ectopic expression of the wild-type ASS1 gene in patient derived organoids. Thus, in this study, we report for the the first time the generation of liver organoids from patient-specific human iPSCs for citrullinemia disease. These personalized iPSC derived-organoid cultures have the potential to be utilized in disease modeling, drug screening, personalized and regenerative medicine. Key Words: Organoid, İPSC, Citrullinemia