Differentiation of Human Pluripotent Stem Cells Into Definitive Endoderm Cells in Various Flexible Three-Dimensional Cell Culture Systems: Possibilities and Limitations.

Margarita A Bystriakova,Marjo Yliperttula,Ara Taalas,Mariia S Bogacheva,Xiaoqiang Xiang,Alan W Leung,Yan-Ru Lou,Emilia Flander,Riina Harjumäki

doi:10.3389/fcell.2021.726499

Abstract

The generation of human stem cell-derived spheroids and organoids represents a major step in solving numerous medical, pharmacological, and biological challenges. Due to the advantages of three-dimensional (3D) cell culture systems and the diverse applications of human pluripotent stem cell (iPSC)-derived definitive endoderm (DE), we studied the influence of spheroid size and 3D cell culture systems on spheroid morphology and the effectiveness of DE differentiation as assessed by quantitative PCR (qPCR), flow cytometry, immunofluorescence, and computational modeling. Among the tested hydrogel-based 3D systems, we found that basement membrane extract (BME) hydrogel could not retain spheroid morphology due to dominant cell–matrix interactions. On the other hand, we found that nanofibrillar cellulose (NFC) hydrogel could maintain spheroid morphology but impeded growth factor diffusion, thereby negatively affecting cell differentiation. In contrast, suspension culture provided sufficient mass transfer and was demonstrated by protein expression assays, morphological analyses, and mathematical modeling to be superior to the hydrogel-based systems. In addition, we found that spheroid size was reversely correlated with the effectiveness of DE formation. However, spheroids of insufficient sizes failed to retain 3D morphology during differentiation in all the studied culture conditions. We hereby demonstrate how the properties of a chosen biomaterial influence the differentiation process and the importance of spheroid size control for successful human iPSC differentiation. Our study provides critical parametric information for the generation of human DE-derived, tissue-specific organoids in future studies.

Highlights

Spheroids and organoids are three-dimensional (3D) clusters of cells
In 3D cell culture, only cells in the outermost layer of spheroids/organoids are directly accessible by media components, but cells apart from the outermost layer must rely on other means such as diffusion or active transport in order to gain access to the media
We show how the size of the human induced pluripotent stem cells (iPSCs) spheroids and 3D culture conditions influence definitive endoderm (DE) differentiation

Summary

Introduction

Spheroids and organoids are three-dimensional (3D) clusters of cells. Spheroids can be made from a variety of cells including stem cells, tumor cells, and organ-specific cells. Organoids are made from stem cells or progenitors that can self-organize into organ-specific structures (Fatehullah et al, 2016; Lou and Leung, 2018) Both spheroids and organoids can produce in vivo–like structures and hold great potential in human development research, disease modeling, drug research, and tissue replacement via transplantation. For potential applications of spheroids/organoids in regenerative medicine, several xenofree and chemically defined hydrogels have been developed (Gjorevski et al, 2016; Nowak et al, 2017; Broguiere et al, 2018; Candiello et al, 2018). Suspension culture has been reported to be effective in the formation of cell aggregates and organoids (Kim et al, 2016; Bergmann et al, 2018; Kumar et al, 2019; Wimmer et al, 2019)

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