Generation of Functional Endodermal Hepatic Organoids

Soheil Akbari; Nevin Ersoy; Alper Bagriyanik; Nur Arslan; Tamer Tevfik Önder; Esra Erdal

doi:10.3791/65027

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Method Article

Generation of Functional Endodermal Hepatic Organoids

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DOI:

10.3791/65027

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May 2nd, 2025

Soheil Akbari^*¹ , Nevin Ersoy^*¹^,²^,³ , Alper Bagriyanik¹^,²^,⁴ , Nur Arslan¹^,⁵ , Tamer Tevfik Önder⁶ , Esra Erdal¹^,⁷

¹Izmir Biomedicine and Genome Center, ²Department of Histology and Embryology, Health Science Institute, Dokuz Eylul University, ³Department of Histology and Embryology, Faculty of Medicine, Izmir Tinaztepe University, ⁴Department of Histology and Embryology, Faculty of Medicine, Dokuz Eylul University, ⁵Department of Pediatric Gastroenterology and Metabolism, Faculty of Medicine, Dokuz Eylul University, ⁶Department of Medical Biology, Faculty of Medicine, Koc University, ⁷Department of Medical Biology, Faculty of Medicine, Dokuz Eylul University

^* These authors contributed equally

Summary

This protocol describes the generation of fast and reproducible endodermal hepatic organoids (eHEPOs). With this protocol, eHEPOs can be produced within 2 weeks and expand long-term (more than 1 year) without losing their differentiation and functionality.

Abstract

Organoid technology has allowed us to generate a variety of human organ-like mini structures, such as for the liver, brain, and intestine, in vitro. The remarkable advances in organoid models have recently opened a new experimental era for various applications in disease modeling, developmental biology, and drug discovery. Adult stem cells or induced pluripotent stem cell (iPSC)-derived liver organoids govern the generation of hepatocytes to use for diverse applications. Here, we present a robust and reproducible protocol for generating hepatic organoids from pluripotent stem cells. This protocol is applicable to healthy and patient-derived cells. To achieve 3D endoderm-derived hepatic organoids (eHEPOs), iPSCs were directly first differentiated into endodermal cells, and then FACS-enriched EpCAM-positive (EpCAM+) cells were used to establish hepatic organoids using the expansion medium. We provide a fast and efficient method to generate hepatic organoids within 2 weeks. The generated organoids mimic the essential properties and functions of hepatocytes, such as albumin secretion, glycogen storage, and cytochrome P450 enzyme activity. Besides the liver-specific gene expression similarities, eHEPOs comprise polarized epithelial cells with bile canaliculi in between. In addition, eHEPOs can be expanded and serial passages long term (1 year) without losing their capacity to differentiate into mature hepatocytes. Thus, eHEPOs provide an alternative source to produce functional hepatocytes.

Introduction

Organoids are miniaturized organ-like structures grown in 3-dimensional culture conditions that mimic the organ microenvironment and include the intrinsic factors necessary for self-organization and self-renewal in organ development itself. Organoids can be derived either from pluripotent stem cells (PSCs) or adult tissue-derived cells (stem or progenitor cells)¹. Although their accurate organ-like organization and functional similarity to the specific organ make them valuable tools for disease modeling, they still need further improvements in terms of standardization in culture. In particular, several protocols have been published for the gene....