RESUMEN
Recent amendments to regulatory frameworks have placed a greater emphasis on the utilization of in vitro testing platforms for preclinical drug evaluations and toxicity assessments. This requires advanced tissue models capable of accurately replicating liver functions for drug efficacy and toxicity predictions. Liver organoids, derived from human cell sources, offer promise as a reliable platform for drug evaluation. However, there is a lack of standardized quality evaluation methods, which hinders their regulatory acceptance. This paper proposes comprehensive quality standards tailored for liver organoids, addressing cell source validation, organoid generation, and functional assessment. These guidelines aim to enhance reproducibility and accuracy in toxicity testing, thereby accelerating the adoption of organoids as a reliable alternative or complementary tool to animal testing in drug development. The quality standards include criteria for size, cellular composition, gene expression, and functional assays, thus ensuring a robust hepatotoxicity testing platform.
RESUMEN
A human cell-based liver model capable of long-term expansion and mature hepatic function is a fundamental requirement for pre-clinical drug development. We previously established self-renewing and functionally mature human pluripotent stem cell-derived liver organoids as an alternate to primary human hepatocytes. In this study, we tested long-term prolonged culture of organoids to increase their maturity. Organoid growing at the edge of Matrigel started to deteriorate two weeks after culturing, and the expression levels of the functional mature hepatocyte marker ALB were decreased at four weeks of culture. Replating the organoids weekly at a 1:2 ratio in fresh Matrigel, resulted in healthier morphology with a thicker layer compared to organoids maintained on the same Matrigel and significantly increased ALB expression until three weeks, although, it decreased sharply at four weeks. The levels of the fetal hepatocyte marker AFP were considerably increased in long-term cultures of organoids. Therefore, we performed serial passaging of organoids, whereby they were mechanically split weekly at a 1:3∼1:5 ratio in fresh Matrigel. The organoids expanded so far over passage 55, or 1 year, without growth retardation and maintained a normal karyotype after long-term cryopreservation. Differentiation potentials were maintained or increased after long-term passaging, while AFP expression considerably decreased after passaging. Therefore, these data demonstrate that organoids can be exponentially expanded by serial passaging, while maintaining long-term functional maturation potential. Thus, hepatic organoids can be a practical and renewable cell source for human cell-based and personalized 3D liver models.
RESUMEN
Emerging evidence has emphasized the importance of cancer therapies targeting an abnormal metabolic state of tumor-initiating cells (TICs) in which they retain stem cell-like phenotypes and nicotinamide adenine dinucleotide (NAD⁺) metabolism. However, the functional role of NAD⁺ metabolism in regulating the characteristics of TICs is not known. In this study, we provide evidence that the mitochondrial NAD⁺ levels affect the characteristics of glioma-driven SSEA1⁺ TICs, including clonogenic growth potential. An increase in the mitochondrial NAD⁺ levels by the overexpression of the mitochondrial enzyme nicotinamide nucleotide transhydrogenase (NNT) significantly suppressed the sphere-forming ability and induced differentiation of TICs, suggesting a loss of the characteristics of TICs. In addition, increased SIRT3 activity and reduced lactate production, which are mainly observed in healthy and young cells, appeared following NNT-overexpressed TICs. Moreover, in vivo tumorigenic potential was substantially abolished by NNT overexpression. Conversely, the short interfering RNA-mediated knockdown of NNT facilitated the maintenance of TIC characteristics, as evidenced by the increased numbers of large tumor spheres and in vivo tumorigenic potential. Our results demonstrated that targeting the maintenance of healthy mitochondria with increased mitochondrial NAD⁺ levels and SIRT3 activity could be a promising strategy for abolishing the development of TICs as a new therapeutic approach to treating aging-associated tumors.