Three-dimensional hydrogel culture conditions promote the differentiation of human induced pluripotent stem cells into hepatocytes

doi:10.1016/j.jcyt.2017.08.008

Cytotherapy

Volume 20, Issue 1, January 2018, Pages 95-107

https://doi.org/10.1016/j.jcyt.2017.08.008 Get rights and content

Highlights

•
Cell behavior was analyzed after single-cell embedding into three-dimensional (3D) hydrogel during differentiation.
•
Hepatocyte-like cells were generated from human induced pluripotent stem cells in completely synthetic hydrogel.
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The 3D hydrogel culture condition enhances the functions of differentiated hepatocytes.
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A conformational 3D protocol for hiPSCs differentiation into hepatocytes is proposed.

Abstract

Background aims

Human induced pluripotent stem cells (hiPSCs) are becoming increasingly popular in research endeavors due to their potential for clinical application; however, such application is challenging due to limitations such as inferior function and low induction efficiency. In this study, we aimed to establish a three-dimensional (3D) culture condition to mimic the environment in which hepatogenesis occurs in vivo to enhance the differentiation of hiPSCs for large-scale culture and high throughput BAL application.

Methods

We used hydrogel to create hepatocyte-like cell (HLC) spheroids in a 3D culture condition and analyzed the cell-behavior and differentiation properties of hiPSCs in a synthetic nanofiber scaffold.

Results

We found that treating cells with Y-27632 promoted the formation of spheroids, and the cells aggregated more rapidly in a 3D culture condition. The ALB secretion, urea production and glycogen synthesis by HLCs in 3D were significantly higher than those grown in a 2-dimensional culture condition. In addition, the metabolic activities of the CYP450 enzymes were also higher in cells differentiated in the 3D culture condition.

Conclusions

3D hydrogel culture condition can promote differentiation of hiPSCs into hepatocytes. The 3D culture approach could be applied to the differentiation of hiPSCs into hepatocytes for bioartificial liver.

Introduction

Liver failure is associated with high morbidity and mortality and is the seventh leading cause of death worldwide [1]. The bioartificial liver (BAL) system is a cell-based external artificial biological device that has synthetic functions and biotransformation activities that are similar to those of the liver [2]. However, the shortage of primary human hepatocytes, the xenotransplantation-related disadvantages of porcine cells and the limited metabolic function of immortalized hepatic cell lines preclude the widespread acceptance of the BAL system.

Human induced pluripotent stem cells (hiPSCs) that are reprogrammed from a diverse range of cell types, such as hair follicle mesenchymal stem cells,peripheral blood mononuclear cells and skin fibroblasts, possess unlimited self-renewal capability and the potential to differentiate into all three germ layers [3], [4], [5]. The successful generation of functional hepatocytes from hiPSCs could be a potential cell source for BAL devices. Using a step-wise method and cocktails of growth factors/cytokines to promote hiPSCs to hepatocytes is a current protocol. However, lower induction efficiency and significantly lacking liver function are the greatest obstacle to application. To enhance differentiation efficiency, human serum was used to replace fetal bovine serum (FBS) to mimic the environment of hepatogenesis in our previous study. We found human serum, particularly that acquired relatively soon after hepatectomy, can enhance the differentiation efficiency and functionality [6]. These results suggested that conditions that mimic early organogenesis could enhance hepatocyte differentiation and the functionality of hiPSCs.

Recently, several studies have demonstrated that three-dimensional (3D) culture conditions create a pragmatic microenvironment and mimic in vivo development, enhancing hepatocyte differentiation and the functionality of human embryonic stem cells (hESCs) and hiPSCs compared with 2-dimensional (2D) culture conditions [7], [8], [9]. Various 3D culture conditions have been used, including the formation of self-aggregated spheroids on low-attachment surfaces [8], [10], the encapsulation of hepatocytes in alginate and the embedding of cells in synthetic biomaterials [11]. Compared with the other two conditions, synthetic biomaterials provide 3D structures, extracellular matrix (ECM)-mimicking stiffness and an environment that facilitates the diffusion of nutrients and cellular growth factors [12], [13], [14]. Nanofiber hydrogel comprises a animal-free synthetic biomaterial that can mimic native ECM functions and thus support the adhesion and differentiation of hiPSCs. This gel is made by interweaving a self-assembling polypeptide, and the pore size is 50–200 nm. These peptides are completely synthetic, thus avoiding the potential pathogenicity of animal-derived materials. This 3D nanofiber hydrogel is critical for meeting future demands because of the advantages of biocompatibility and retrieval and the lower risk of immunogenic reaction. Thus, embedding cells in a 3D biomaterial construct including a nanofiber scaffold is expected to provide a microenvironment to improve the induction efficiency of hepatocyte differentiation for BAL applications [12], [15], [16].

Recent advances in 3D culture techniques have enabled the development of promising scaffolds for the differentiation of hiPSCs. However, to the best of our knowledge, few studies have applied this nanofiber hydrogel to hiPSC differentiation, and few have tried to determine how this completely synthetic hydrogel influences cell activities and differentiation. In this study, we focused on the cell-behavior and differentiation properties of hiPSCs in a synthetic nanofiber scaffold and designed the fabrication method in 3D culture condition.

Section snippets

Ethics approval and consent to participate

This study was approved by the institutional review board of Tianjin Third Central Hospital, Tianjin, China (file no. 13010). All patients provided written informed consent. The methods were carried out in accordance with the approved guidelines. All experimental protocols were approved by the institutional review board.

Maintenance of hiPSCs cultures

Three human iPSC lines were used for hepatic differentiation (hiPSCs-HF1, hiPSCs-HF2 and hiPSCs-EC1). hiPSCs-HF1 and hiPSCs-HF2 were induced from IMR-90 human fibroblasts using

Differentiation of hiPSCs to definitive endoderm cells with EB formation in monolayer

As a starting point to generate mature hepatocytes, we used monolayer culture strategy for the differentiation of hiPSCs to definitive endoderm (DE) cells with EB formation. As shown in Figure 1B, heterogeneous cells with different morphologies were observed after 1 day, followed by the formation of definitive endoderm after 3 days. These DE cells began to express the definitive endodermal markers FOXA2 and CXCR4 (Figure 1C,D). We assessed DE cell specification by FOXA2 and CXCR4 expression

Discussion

Recent studies have demonstrated that 3D culture conditions with appropriate inducing cytokines could provide hiPSCs with a microenvironment closely mimicking that of hepatogenesis in vivo and enhance the functional of differentiation of hiPSCs [7], [10]. Vallier and colleagues exploited commercial collagen-based scaffolds for the maturation of hiPSC-derived hepatocytes in 3D culture system [19]. Takebe and coworkers succeeded in generating a 3D vascularized organ such as liver [10]. Although

Conclusions

We created a 3D differentiation method consisting of a step-wise monolayer-based differentiation into DE followed by a process of cell self-aggregation into hepatocyte-like cells in 3D spheroids. This culture condition uses a synthetic hydrogel to create the HLC-based spheroids in the 3D culture condition for hepatocyte differentiation from hiPSCs, and the HLC-based spheroids express hepatocyte markers, exhibit functional maturity and have metabolic activity. The generation of HLCs from hiPSCs

Acknowledgments

We thank Dr. Wange Lu (Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research) for editing the manuscript. This study was supported by the Tianjin Health Industry Key Project (No. 16KG150), the Tianjin Health Bureau Funded Project (No. 2012KY02) and Tianjin Science Foundation of China (No. 15JCQNJC45700).

Disclosure of interest: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.

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View full text

Induced Pluripotential CellsThree-dimensional hydrogel culture conditions promote the differentiation of human induced pluripotent stem cells into hepatocytes

Highlights

Abstract

Background aims

Methods

Results

Conclusions

Introduction

Section snippets

Ethics approval and consent to participate

Maintenance of hiPSCs cultures

Differentiation of hiPSCs to definitive endoderm cells with EB formation in monolayer

Discussion

Conclusions

Acknowledgments

Cell

Dig Liver Dis

Biomaterials

Differentiation

Biomaterials

Biomaterials

FEBS Lett

Cell Stem Cell

Cell Stem Cell

J Biotechnol

Acute-on-chronic liver failure: terminology, mechanisms and management

Nat Rev Gastroenterol Hepatol

Bridging the gap: advances in artificial liver support

Liver Transpl

Reprogramming of human somatic cells to pluripotency with defined factors

Nature

Human induced pluripotent stem cells free of vector and transgene sequences

Science

Application of three-dimensional culture conditions to human embryonic stem cell-derived definitive endoderm cells enhances hepatocyte differentiation and functionality

Tissue Eng Part A

Spheroid culture for enhanced differentiation of human embryonic stem cells to hepatocyte-like cells

Stem Cells Dev

Induced Pluripotential Cells
Three-dimensional hydrogel culture conditions promote the differentiation of human induced pluripotent stem cells into hepatocytes