Generation of human induced pluripotent stem cell-derived liver buds with chemically defined and animal origin-free media

Keisuke Sekine , Shimpei Ogawa, Syusaku Tsuzuki, Tatsuya Kobayashi , Kazuki Ikeda, Noriko Nakanishi , Kenta Takeuchi , Eriko Kanai , Yugo Otake , Satoshi Okamoto , Tsuyoshi Kobayashi , Takanori Takebe , Hideki Taniguchi 
Source: Scientific Reports
Publication Date: ()
Issue: 10: 17937
Research Area:
Stem Cells
Drug Discovery
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Culture Media:

Differentiation of human iPSC-DE, iPSC-HE, iPSC-IHs, and iPSC-MHs using the conventional method

HBM (Lonza Bioscience) supplemented with the Single Quotes™ kit without EGF (HCM without EGF), 5% fetal bovine serum (FBS), dexamethasone, and OSM was used to derive MHs.

Generation of iPSC-LBs

The LBs were cultured with 50% HCM without EGF and 50% KBM-VEC1 basal medium (Kojin Bio), for the conventional medium 50% HCM without EGF supplemented with 5% FBS and 50% EGM was used.


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. 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. 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. However, in previous studies we used media originating from animals for differentiation except for the maintenance of undifferentiated iPSCs. Therefore, we developed a CD-AOF medium to generate all iPSC-LBs. We first developed a CD-AOF medium for hepatocytes, ECs, and stage-matched MCs, i.e., septum transversum mesenchyme (STM), in 2D cultures. We next generated all iPSC-LBs by incubating individual cell types in ultra-low attachment micro-dimple plates. 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. Furthermore, we found that this CD-AOF medium could be used in several cell culture settings. Taken together, these results demonstrate the successful development of a CD-AOF medium suitable for all iPSC-LBs. The protocol developed in this study will facilitate the clinical applicability of all iPSC-LBs in the treatment of liver diseases.