The resultant cells were combined to generate all iPSC-LBs, whose hepatic functions were equivalent to those of LBs generated using the conventional media both in vitro and in vivo. We examined the generation of hepatic, endothelial, and mesenchymal lineages from iPSCs using the CD-AOF medium. In this study, we optimized the protocol for generating iPSC-LBs and developed a CD-AOF medium for clinical use. 375 2014) during therapeutic applications. While these approaches will facilitate the large-scale production of iPSC-LBs for treating liver failure, which requires 1 × 10 8–1 × 10 10 hepatocyte-equivalents, the use of serum and animal-derived products must be reduced or completely eliminated during the production of all iPSC-LBs to meet the Standard for Biological Ingredients (The Japanese Ministry of Health, Labour and Welfare Notification No. In addition to hepatic lineages, endothelial cells (ECs) and stage-matched mesenchymal cells (MCs), such as septum transversum mesenchymal cells (STM), have been derived from clinically applicable Ff-iPSCs. Furthermore, we have recently reported the mass production of iPSC-LBs derived entirely from human Ff-iPSCs, which we have termed all iPSC-LBs 13. Single-cell RNA sequencing has revealed that the iPSC-LBs exhibit a clear resemblance to fetal liver tissues compared to the 2D-differentiated hepatocytes 12. We have recently succeeded in generating multicellular liver organoids from human iPSCs which we have defined as iPSC-liver buds (LBs) these LBs exhibit therapeutic potential in mouse disease models 11. Undifferentiated iPSC culture conditions were also improved with the development of feeder-free (Ff) culture conditions and chemically defined, animal origin-free (CD-AOF) iPSC medium for use in clinical applications 6, 7, 8, 9, 10. Retrovirus and lentivirus used to transduce iPSCs into host cells in initial experiments have been replaced by methods that do not require integration into host chromosomes, thereby avoiding insertion mutations and potentially malignant transformations 4, 5. Important improvements have been achieved in recent years to ensure the safety and quality of iPSC culture systems for clinical application. Human induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine 1, 2, and in the last ten years iPSC-derived cells have been successfully transplanted into patients 3. The protocol developed in this study will facilitate the clinical applicability of all iPSC-LBs in the treatment of liver diseases. Taken together, these results demonstrate the successful development of a CD-AOF medium suitable for all iPSC-LBs. Furthermore, we found that this CD-AOF medium could be used in several cell culture settings. The hepatic functions of all iPSC-LBs generated using the CD-AOF medium were equivalent to those of all iPSC-LBs generated using the conventional medium both in vitro and in vivo. We next generated all iPSC-LBs by incubating individual cell types in ultra-low attachment micro-dimple plates. We first developed a CD-AOF medium for hepatocytes, ECs, and stage-matched MCs, i.e., septum transversum mesenchyme (STM), in 2D cultures. Therefore, we developed a CD-AOF medium to generate all iPSC-LBs. However, in previous studies we used media originating from animals for differentiation except for the maintenance of undifferentiated iPSCs. We previously generated liver organoids from iPSCs, namely iPSC-liver buds (iPSC-LBs), by mimicking the organogenic interactions among hepatocytes, endothelial cells (ECs), and mesenchymal cells (MCs) and recently reported the mass production of iPSC-LBs derived entirely from iPSCs (all iPSC-LBs), which should facilitate their large-scale production for the treatment of liver failure. CD-AOF media and supplements ensure the quality and reproducibility of culture systems by lowering lot-to-lot variations and the risk of contamination with viruses or toxins. Advances in organoid technology have broadened the number of target diseases and conditions in which human induced pluripotent stem cell (iPSC)-based regenerative medicine can be applied however, mass production of organoids and the development of chemically defined, animal origin-free (CD-AOF) media and supplements are unresolved issues that hamper the clinical applicability of these approaches.
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