Int J Stem Cells.  2021 Feb;14(1):119-126. 10.15283/ijsc20152.

Production of Functional Hepatobiliary Organoids from Human Pluripotent Stem Cells

Affiliations
  • 1Department of Central Laboratory, Shenzhen Hospital, Beijing University of Chinese Medicine, Shenzhen, China
  • 2Department of Hepatology, Shenzhen Hospital, Beijing University of Chinese Medicine, Shenzhen, China

Abstract

The research on human hepatobiliary development and disorders has been constrained by minimal access to human fetal tissue, and low accuracy of animal models. To overcome this problem, we have established a system for the differentiation of human pluripotent stem cells (hPSCs) into functional hepatobiliary organoids (HBOs). We have previously reported that our 45-d approach closely mimics key stages of hepatobiliary development, starting with the differentiation of hiPSC into endoderm and a small part of mesoderm, and subsequently into hepatoblast-like cells, followed by the parallel generation of hepatocyte-like cells and cholangiocyte-like cells, formation of immature HBO expressing early hepatic and biliary markers, and mature HBO displaying hepatobiliary functionality. In this study, we present an updated version of our previous protocol, which only needs 35 days to achieve maturation in vitro. Furthermore, a hepatobiliary culture medium is developed to functionally maintain the HBOs for more than 1.5 months. The capacity of this approach for producing large amounts of functional HBOs and enabling long-term culture in vitro holds promise for applications on developmental research, disease modeling, as well as screening of therapeutic agents.

Keyword

Hepatobiliary; Organoids; Differentiation; Pluripotent stem cells

Figure

  • Fig. 1 Molecular phenotype and differentiation efficiency at key stages of HBO differentiation. (A∼F) At the end of each key stage, characteristic markers and differentiation efficiency were examined by immunofluorescence and flow cytometry, respectively. Scale bars, 100 μm.

  • Fig. 2 Differentiation of hPSC into hepatobiliary organoids (HBOs). (A) Schematic presentation of the protocol for the generation of hPSC-derived HBOs. (B) Bright-field images of cells at key stages of HBO differentiation. The procedure steps and day numbers corresponding to each image are noted for reference. Scale bars, 150 μm. EM, expansion medium; EMM, endoderm and mesoderm medium; HBCM, hepato-bi-liary culture medium; HBDM, hepato-biliary differentiation medium; HBMM, hepato-biliary maturation medium; PM, progenitor medium.

  • Fig. 3 Functional properties of hPSC-derived HBOs. (A) Production of albumin and urea in day-35 HBOs. Cryopreserved adult hepatocytes (CAHs) were used as positive controls; hPSCs were used as negative control. *p<0.05; **p<0.01; n=3. (B) CYP3A4 induction in day-35 and day-80 HBOs. The HBOs were treated with 25 μM rifampin (RIF), followed by assessment of CYP3A4 activity using bioluminescent substrates. CAH were used as positive control. *p<0.05; **p<0.01; n=3. (C) The biliary structures in HBOs demonstrating characteristic ALP staining. Scale bars, 100 μm. (D) GGT activity in day-35 and day-85 HBOs. Human intrahepatic biliary epithelial cells (IBEC) was used as positive control; substrate only was used as negative control. *p<0.01; n=3.


Reference

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