Murine pluripotent stem cells that escape differentiation inside teratomas maintain pluripotency

Yangli Pei; Liang Yue; Wei Zhang; Jinzhu Xiang; Zhu Ma; Jianyong Han

doi:10.7717/peerj.4177

Murine pluripotent stem cells that escape differentiation inside teratomas maintain pluripotency

PeerJ. 2018 Jan 4:6:e4177. doi: 10.7717/peerj.4177. eCollection 2018.

Authors

Yangli Pei^#^{1

2}, Liang Yue^#², Wei Zhang², Jinzhu Xiang², Zhu Ma³, Jianyong Han²

Affiliations

¹ State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.
² State Key Laboratories for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.
³ Beijing Dairy Cattle Center, Beijing, China.

^# Contributed equally.

Abstract

Background: Pluripotent stem cells (PSCs) offer immense potential as a source for regenerative therapies. The teratoma assay is widely used in the field of stem cells and regenerative medicine, but the cell composition of teratoma is still elusive.

Methods: We utilized PSCs expressing enhanced green fluorescent protein (EGFP) under the control of the Pou5f1 promoter to study the persistence of potential pluripotent cells during teratoma formation in vivo. OCT4-MES (mouse embryonic stem cells) were isolated from the blastocysts of 3.5-day OCT4-EGFP mice (transgenic mice express EGFP cDNA under the control of the Pou5f1 promoter) embryos, and TG iPS 1-7 (induced pluripotent stem cells) were generated from mouse embryonic fibroblasts (MEFs) from 13.5-day OCT4-EGFP mice embryos by infecting them with a virus carrying OCT4, SOX2, KLF4 and c-MYC. These pluripotent cells were characterized according to their morphology and expression of pluripotency markers. Their differentiation ability was studied with in vivo teratoma formation assays. Further differences between pluripotent cells were examined by real-time quantitative PCR (qPCR).

Results: The results showed that several OCT4-expressing PSCs escaped differentiation inside of teratomas, and these escaped cells (MES-FT, GFP-positive cells separated from OCT4-MES-derived teratomas; and iPS-FT, GFP-positive cells obtained from teratomas formed by TG iPS 1-7) retained their pluripotency. Interestingly, a small number of GFP-positive cells in teratomas formed by MES-FT and iPS-FT (MES-ST, GFP-positive cells isolated from MES-FT-derived teratomas; iPS-ST, GFP-positive cells obtained from teratomas formed by iPS-FT) were still pluripotent, as shown by alkaline phosphatase (AP) staining, immunofluorescent staining and PCR. MES-FT, iPS-FT, MES-ST and iPS-ST cells also expressed several markers associated with germ cell formation, such as Dazl, Stella and Stra8.

Conclusions: In summary, a small number of PSCs escaped differentiation inside of teratomas, and these cells maintained pluripotency and partially developed towards germ cells. Both escaped PSCs and germ cells present a risk of tumor formation. Therefore, medical workers must be careful in preventing tumor formation when stem cells are used to treat specific diseases.

Keywords: Differentiation; Embryonic stem cells; Germ cells; Induced pluripotent stem cells; Pluripotency; Teratoma.

Grants and funding

This study was funded by grants from the National Key Research and Development Program (2016YFA0100202), the China National Basic Research Program (2011CBB01001, 2011CBA01102, 2009CB941003) and the National Thousand Talents Program of China and the Program for New Century Excellent Talents in University (NCET-11-0482). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.