Human cell-based models for urea cycle research: From gene regulation and ureagenesis to disease modeling and...

Development of cell-based model systems for studying the urea cycle has been motivated by the urgent need to develop and evaluate treatments for urea cycle disorders, such as gene therapies currently in clinical trials, and to conduct mechanistic studies of the regulation of the rate of ureagenesis and urea cycle gene expression. However, studying the urea cycle in cellular models has historically been challenging. Traditional in vitro models, primarily 2D cell cultures, often fail to fully recapitulate the complex physiological environment and metabolic zonation necessary for accurate urea cycle research. Studies of ureagenesis utilized primary hepatocytes and hepatoma cell lines such as HepG2 and HuH-7. The expression of urea cycle genes in HepG2 and HuH-7 cells is variable, and the currently available cell lines do not express all urea cycle enzymes. However, these cell lines express transcription factors that regulate urea cycle gene expression, making them suitable for investigating urea cycle gene expression and non-coding sequence variants in patients with urea cycle disorders. Primary hepatocytes can survive in culture for only a few days, and expression of urea cycle enzymes in cultured primary hepatocytes ceases even faster. Culturing primary hepatocytes in conditions that recapitulate their interactions with other cells and the extracellular matrix, as well as nutrient and hormone signals, can prolong their survival and ureagenesis. Patient-derived induced pluripotent stem cells (iPSCs) provide a renewable source for generating disease-specific cellular models, enabling the study of genetic mutations and the development of personalized therapeutic approaches. However, in vitro differentiated hepatocytes derived from iPSCs tend to produce and secrete less urea than adult primary hepatocytes due to incomplete maturation. Liver organoids and microfluidic devices represent a significant advancement in modeling the urea cycle, more closely mirroring the intricate cellular architecture, physiological functions, and metabolic pathways of the native liver than conventional methods. These models provide a platform for investigating disease mechanisms, identifying novel biomarkers, and evaluating the efficacy and toxicity of potential therapeutic interventions, including gene therapy and small-molecule drugs. The development and refinement of these sophisticated in vitro systems are crucial for accelerating our understanding of urea cycle disorders and translating basic scientific discoveries into effective clinical treatments.