Enrichment of Pluripotent Stem Cell-Derived Hepatocyte-Like Cells by Ammonia Treatment.

Daihachiro Tomotsune,Masako Fujii,Sakiko Takizawa-Shirasawa,Ken Matsumoto,Tadayuki Yokoyama,Katsunori Sasaki,Fengming Yue,Kanji Hirashima,Shree Ram Singh

doi:10.1371/journal.pone.0162693

Abstract

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are potential resources for the regeneration of defective organs, including the liver. However, some obstacles must be overcome before this becomes reality. Undifferentiated cells that remain following differentiation have teratoma-forming potential. Additionally, practical applications require a large quantity of differentiated cells, so the differentiation process must be economical. Here we describe a DNA microarray-based global analysis of the gene expression profiles of differentiating human pluripotent stem cells. We identified differences and commonalities among six human pluripotent stem cell lines: the hESCs KhES1, KhES2, KhES3, and H1, and the iPSCs 201B7 and 243G1. Embryoid bodies (EBs) formed without requiring supplementation with inducing factors. EBs also expressed some liver-specific metabolic genes including the ammonia-metabolizing enzymes glutamine synthetase and carbamoyl-phosphate synthase 1. Real-time PCR analysis revealed hepatocyte-like differentiation of EBs treated with ammonia in Lanford medium. Analysis of DNA microarray data suggested that hepatocyte-like cells were the most abundant population in ammonia-treated cells. Furthermore, expression levels of undifferentiated pluripotent stem cell markers were drastically reduced, suggesting a reduced teratoma-forming capacity. These results indicate that treatment of EBs with ammonia in Lanford medium may be an effective inducer of hepatic differentiation in absence of expensive inducing factors.

Highlights

Inclusion of microarray data from pluripotent stem cell lines grown in a Matrigel-coated dish with mouse embryonic fibroblasts (MEFs)-conditioned medium (MCM) and under Embryoid bodies (EBs)-forming conditions in the analysis revealed that differences in gene expression profiles arose during EB formation (Fig 1C and 1D)
Pluripotent stem cells cultured in MCM maintained an undifferentiated state than those grown on a MEF layer (ML) (Fig 1E)
Our findings reveal that the six undifferentiated pluripotent stem cell lines have a varied differentiation tendency

Summary

Introduction

We performed a global gene expression analysis of six differentiating pluripotent stem cell lines and identified several hepatocyte-specific genes that are expressed at the early induction stage of hepatic differentiation in the absence of expensive inducing factors. This suggests that ammonia could select for and enrich populations of pluripotent stem cell-derived hepatocytes. We included ammonia in our hepatocyte induction protocol and again analyzed global gene expression using DNA microarray.

Results

Conclusion