Spheroid Culture for Enhanced Differentiation of Human Embryonic Stem Cells to Hepatocyte-Like Cells

Abstract

Stem cell-derived hepatocyte-like cells hold great potential for the treatment of liver disease and for drug toxicity screening. The success of these applications hinges on the generation of differentiated cells with high liver specific activities. Many protocols have been developed to guide human embryonic stem cells (hESCs) to differentiate to the hepatic lineage. Here we report cultivation of hESCs as three-dimensional aggregates that enhances their differentiation to hepatocyte-like cells. Differentiation was first carried out in monolayer culture for 20 days. Subsequently cells were allowed to self-aggregate into spheroids. Significantly higher expression of liver-specific transcripts and proteins, including Albumin, phosphoenolpyruvate carboxykinase, and asialoglycoprotein receptor 1 was observed. The differentiated phenotype was sustained for more than 2 weeks in the three-dimensional spheroid culture system, significantly longer than in monolayer culture. Cells in spheroids exhibit morphological and ultrastructural characteristics of primary hepatocytes by scanning and transmission electron microscopy in addition to mature functions, such as biliary excretion of metabolic products and cytochrome P450 activities. This three-dimensional spheroid culture system may be appropriate for generating high quality, functional hepatocyte-like cells from ESCs.

Hippo Signaling Influences HNF4A and FOXA2 Enhancer Switching during Hepatocyte Differentiation

“…To isolate differentially enriched motifs, we employed the motif-finding algorithm oPOSSUM (http://opossum.cisreg.ca/oPOSSUM3/). We examined embryonic-specific enhancer regions using adult-specific enhancers as a background data set (Subramanian et al, 2014). Reassuringly, our analysis detected TFs such as HNF1a, HNF1b, and CEBPa, which are known to work with HNF4A and FOXA2 during liver development (Figure 5A).…”

Engrafted human stem cell–derived hepatocytes establish an infectious HCV murine model

“…Different strategies have been used to improve the level of differentiation of the HLCs in vitro, such as the use of special matrix, 3D culture, spheroids formation, or coculture with stromal cells (46)(47)(48)(49)(50)(51)(52). However, none of them so far allowed maturation of the HLCs at a level similar to that of PHHs.…”

Human hepatocytes derived from pluripotent stem cells: a promising cell model for drug hepatotoxicity screening

“…However, one of the remaining questions that arises when using HLCs derived from PSCs is the stability of their phenotype and function in long-term cultures, a problem commonly encountered with cultured adult hepatocytes (Schwartz et al 2014). Hence, distinct strategies to improve and stabilize the hepatic phenotype of derived HLCs, such as employing complex 3D culture systems (spheroidal aggregates, perfused bioreactors) (Sivertsson et al 2013;Gieseck et al 2014;Sengupta et al 2014;Subramanian et al 2014;Takayama et al 2014;Yao et al 2014;Kim et al 2015) or using different extracellular matrices (Hay et al 2011), have been promoted.…”