citations

                    Bile acid-FXR signaling facilitates the long-term maintenance of hepatic characteristics in human iPSC-derived organoids
                

Authors:

Taro Shimizu , Masato Miyoshi , Sei Kakinuma , Jun Tsuchiya , Daisuke Yamane , Keiya Watakabe , Tomohiro Mochida , Kento Inada , Kaho Yamada , Kotomi Shinozaki , Ayako Sato , Shun Kaneko , Fukiko Kawai-Kitahata , Miyako Murakawa , Sayuri Nitta , Mina Nakagawa , Mamoru Watanabe , Yasuhiro Asahina , Ryuichi Okamoto

In:

Source: Stem Cell Reports
Publication Date: (2025)
Issue: 44: 5

Research Area:

Gastroenterology

Stem Cells

Regenerative medicine

Toxicology

Drug Discovery

Cells used in publication:

Hepatocyte, human: Species: human; Tissue Origin: liver
Induced Pluripotent Stem Cell (iPS), human: Species: human; Tissue Origin:

Culture Media:

Hepatocyte Culture Medium

Hepatocyte Plating Medium (MP)

Platform:

4D-Nucleofector® X-Unit

Experiment

Establishment of Dox-inducible overexpression iPSCs

We constructed a self-contained, tetracycline-inducible expression vector based on the PiggyBac transposon system. Activation of gene expression in response to Dox can be indirectly monitored by co-incident GFP production. Briefly, we produced a derivative vector expressing human NR0B2 using the Gateway cloning technique. The vector, PB-T-NR0B2-G-ERN, was transfected together with PiggyBac transposase into the P11025 iPSC line using the Lonza P3 Primary Cell 4D-Nucleofector X kit. Transfected cells were selected using media containing G418 for 3 days to generate pooled iPSC cell lines containing genomic transposon integrations. We obtained multiple cell lines, and they were analyzed in this study after the evaluation of NR0B2 expression levels. The resultant cell lines were differentiated into iHOs as described above.

Cryo-preserved hepatocyte culture

Cryo-preserved hepatocytes were purchased from LONZA. Cells were cultured by the sandwich model method according to the manufacturer’s protocols. In this study, we referred to them as cultured primary human hepatocytes (Cultured-PHH). Briefly, 5x10⁵ cells were plated on collagen I-coated 24-well plate and replaced with fresh maintenance medium supplemented with Matrigel after at least 6 h of incubation. Thereafter, maintenance medium was changed daily. Cells from three different lots were cultured and harvested on day 1 (HUM181561A), day2 (HUM221971, HUM182531), day 4 (HUM181561A) and day 7 (HUM182531) for quantitative PCR analysis. Detailed information on each lot is shown in Table S3.

Abstract

Human induced pluripotent stem cells (iPSCs) can be differentiated into hepatocyte-like cells (iPS-Heps); however, maintaining the long-term proliferation and hepatic characteristics of iPS-Heps remains a challenge. In this study, we aimed to develop a human iPSC-derived hepatic organoid (iHO) culture system that effectively retains hepatic characteristics long term. Our original culture strategy, using bile acids and their receptor (farnesoid X receptor [FXR]) agonists, yielded human iHOs capable of long-term culture with a distinctive "grape-like" structure. Comprehensive analysis showed that these iHOs maintained hepatocyte-like phenotypes, even after multiple passages, whose gene expression profiles were consistent with those of fetal hepatocytes. In addition, the overexpression of small heterodimer partner (SHP), a downstream gene of FXR, in iHOs negatively regulated genes related to the intestine and cholangiocytes. Our data demonstrated that bile acid-FXR signaling promotes both the hepatic characteristics and proliferative potential of iHOs, offering promising potential for future applications in regenerative medicine and as a disease model.

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