Tissue-Specific Microparticles Improve Organoid Microenvironment for Efficient Maturation of Pluripotent Stem-Cell-Derived Hepatocytes.

Ensieh Zahmatkesh,Zahra Heydari,Roberto Gramignoli,Marc Ruoß,Zahra Farzaneh,Mohammad Hossein Ghanian,Ibrahim Zarkesh,Mustapha Najimi,Amnah Othman,Saeed Yakhkeshi,Hossein Baharvand,Andreas Nüssler,Majid Halvaei,Massoud Vosough,Farideh Moeinvaziri,Abbas Piryaei

doi:10.3390/cells10061274

Abstract

Liver organoids (LOs) are receiving considerable attention for their potential use in drug screening, disease modeling, and transplantable constructs. Hepatocytes, as the key component of LOs, are isolated from the liver or differentiated from pluripotent stem cells (PSCs). PSC-derived hepatocytes are preferable because of their availability and scalability. However, efficient maturation of the PSC-derived hepatocytes towards functional units in LOs remains a challenging subject. The incorporation of cell-sized microparticles (MPs) derived from liver extracellular matrix (ECM), could provide an enriched tissue-specific microenvironment for further maturation of hepatocytes inside the LOs. In the present study, the MPs were fabricated by chemical cross-linking of a water-in-oil dispersion of digested decellularized sheep liver. These MPs were mixed with human PSC-derived hepatic endoderm, human umbilical vein endothelial cells, and mesenchymal stromal cells to produce homogenous bioengineered LOs (BLOs). This approach led to the improvement of hepatocyte-like cells in terms of gene expression and function, CYP activities, albumin secretion, and metabolism of xenobiotics. The intraperitoneal transplantation of BLOs in an acute liver injury mouse model led to an enhancement in survival rate. Furthermore, efficient hepatic maturation was demonstrated after ex ovo transplantation. In conclusion, the incorporation of cell-sized tissue-specific MPs in BLOs improved the maturation of human PSC-derived hepatocyte-like cells compared to LOs. This approach provides a versatile strategy to produce functional organoids from different tissues and offers a novel tool for biomedical applications.

Highlights

End-stage liver diseases account for almost two million deaths per year worldwide [1].drug-induced liver injury, as a public health concern, remains a potential health challenge [2]
To determine the gene expression profile in Liver organoids (LOs) and bioengineered LOs (BLOs) derived from hESC and hiPSC cultures after 12 days, we evaluated the mRNA expression of the following hepaticspecific genes: α-fetoprotein (AFP), albumin (ALB), tyrosine aminotransferase (TAT), tryptophan 2,3-dioxygenase (TDO), multidrug resistance-associated protein 2 (MRP2), multidrug resistance (MDR), asialoglycoprotein receptor1 (ASGPR1), carbamoyl phosphate synthase 1 (CPS1), glucose-6-phosphatase (G6PC) and phase I enzymes, cytochrome p450 subunit 1A2 (CYP1A2), subunit 3A4 (CYP3A4), subunit 3A7 (CYP3A7), subunit 2C9 (CYP2C9), and subunit 2B6 (CYP2B6)
The results indicated that there were more ALB+, cytochrome p450 subunit 3A4 (CYP3A4)+, CYP1A2+, ZO-1+, and E-cadherin+ cells in the BLOs group compared to the LOs group

Summary

Introduction

End-stage liver diseases account for almost two million deaths per year worldwide [1]. Drug-induced liver injury, as a public health concern, remains a potential health challenge [2]. Animal models are used for drug screening, discovery, and toxicity testing, and they offer numerous advantages; they are often limited in relevance, time-consuming, and expensive and raise concerns [4,5]. In vitro culture of tissue-specific cells has attracted considerable attention as a promising approach to develop biomimetic systems for the prediction of potential hepatoxicity of drugs [6]. Traditional two-dimensional (2D) monolayer cells, cultured on flat and rigid substrates, are usually used for cell-based assays in drug development. Since almost all cells in the in vivo environment are surrounded by other stromal cells and ECM in a 3D condition,

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Conclusion