Comparative Analysis of Endoderm Formation Efficiency between Mouse ES Cells and iPS Cells

Abstract

Definitive endoderm (DE) derived from stem cells holds potential to differentiate into hepatocytes. Stem cell therapy using those cells has potential for a treatment of liver disease. To date, various ways of inducing hepatocytes from embryonic stem (ES) cells have been reported by researchers. However, it has not been proved enough that induced pluripotent stem (iPS) cells behave in the same manner as ES cells in endoderm differentiation. The purpose of this study was to establish an efficient method to induce DE from iPS cells, through comparatively analyzing the efficacy of endoderm formation from mouse ES cells. Furthermore, the efficiency of a serum-free medium in the differentiation into DE was investigated. Mouse ES cells and iPS cells were floated in culture medium for 2 or 5 days and embryoid bodies (EB) were formed. Subsequently, DE was induced with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF). RT-PCR and real-time PCR analyses were carried out at each step to determine the gene expression of EB markers. The difference in cellular proliferation between serum-containing and serum-free media was examined by an MTS assay in EB and DE induction. iPS cells showed the paralleled mRNA expression to ES cells in each step of differentiation into EB, but the levels of expression of Sox17 and Foxa2 were relatively higher in ES cell-derived DE, whereas Cxcr4 expression was higher in iPS cell-derived DE. The utilization of serum-free medium for iPS cells showed significantly favorable cellular proliferation during EB formation and subsequent DE induction. Forming EB for 5 days and subsequently DE induction with activin A and bFGF with serum-free medium was an appropriate protocol in iPS cells. This may represent an important step for generating hepatocytes from iPS cells for the development of cell therapy.

Murine embryonic stem cell-derived hepatocytes correct metabolic liver disease after serial liver repopulation

“…We directly compared the frequencies of liver engraftment and liver repopulation from the two populations of ES cell-derived hepatocytes induced by these two methods. In our study, the DE method generated higher quantities of ES-derived hepatic cells by stepwise induction, which was similar to previous studies (Cai et al, 2007; Duan et al, 2010; Gouon-Evans et al, 2006; Iwamuro et al, 2010; Yasunaga et al, 2005). There are probably two reasons for this improvement.…”

“…During DE differentiation, DE cells were induced in stepwise fashion using specific growth factor cocktails and purified by cell sorting for co-expression of Cxcr4 and c-Kit in FACS as described previously (Fig. 1C) (Gouon-Evans et al, 2006; Iwamuro et al, 2010; Li et al, 2011). During hepatic differentiation, Cxcr4 + c-Kit + DE cells cultured at high density had induced alb-GFP expression (Fig.…”

In Vivo Differentiation of Induced Pluripotent Stem Cell-Derived Cardiomyocytes

“…Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) from mouse iPSC-derived EBs on days 6,8,12, and 19; cDNA was synthesized using the Super Script III First-strand Synthesis System (Invitrogen). PCR was performed with KOD-plus (Toyobo, Osaka, Japan).…”

Transplantation of embryonic stem cells improves the regeneration of periodontal furcation defects in a porcine model

“…It is widely accepted that mouse and human iPS cells possess morphological, molecular and developmental features that closely resemble those of blastocyst‐derived ES cells (Amabile & Meissner , Iwamuro et al. ). There are no published studies that report using ES or iPS cells as a cell source to regenerate periodontal defects in animal or human studies.…”