Short-Term Serum-Free Culture Reveals that Inhibition of Gsk3β Induces the Tumor-Like Growth of Mouse Embryonic Stem Cells

Yanzhen Li,Tamaki Yokohama-Tamaki,Tetsuya S Tanaka,Martin Pera

doi:10.1371/journal.pone.0021355

Yanzhen Li, Tamaki Yokohama-Tamaki + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0021355

Copy DOI

Journal: PloS one	Publication Date: Jun 23, 2011
Citations: 90	License type: CC BY 4.0

Affiliation: University of Illinois Urbana-Champaign

Abstract

Here, we present evidence that the tumor-like growth of mouse embryonic stem cells (mESCs) is suppressed by short-term serum-free culture, which is reversed by pharmacological inhibition of Gsk3β. Mouse ESCs maintained under standard conditions using fetal bovine serum (FBS) were cultured in a uniquely formulated chemically-defined serum-free (CDSF) medium, namely ESF7, for three passages before being subcutaneously transplanted into immunocompromised mice. Surprisingly, the mESCs failed to produce teratomas for up to six months, whereas mESCs maintained under standard conditions generated well-developed teratomas in five weeks. Mouse ESCs cultured under CDSF conditions maintained the expression of Oct3/4, Nanog, Sox2 and SSEA1, and differentiated into germ cells in vivo. In addition, when mESCs were cultured under CDSF conditions supplemented with FBS, or when the cells were cultured under CDSF conditions followed by standard culture conditions, they consistently developed into teratomas. Thus, these results validate that the pluripotency of mESCs was not compromised by CDSF conditions. Mouse ESCs cultured under CDSF conditions proliferated significantly more slowly than mESCs cultured under standard conditions, and were reminiscent of Eras-null mESCs. In fact, their slower proliferation was accompanied by the downregulation of Eras and c-Myc, which regulate the tumor-like growth of mESCs. Remarkably, when mESCs were cultured under CDSF conditions supplemented with a pharmacological inhibitor of Gsk3β, they efficiently proliferated and developed into teratomas without upregulation of Eras and c-Myc, whereas mESCs cultured under standard conditions expressed Eras and c-Myc. Although the role of Gsk3β in the self-renewal of ESCs has been established, it is suggested with these data that Gsk3β governs the tumor-like growth of mESCs by means of a mechanism different from the one to support the pluripotency of ESCs.

Highlights

Embryonic stem cells (ESCs) [1,2,3] and induced pluripotent stem cells [4,5,6,7] are very promising tools for use in drug screening and customized tissue replacement [8] because they are capable of self-renewal that sustains pluripotency
We present experimental evidence that short-term serum-free culture reduces the tumorigenicity of mouse embryonic stem cells (mESCs), which is reversed by pharmacological inhibition of glycogen synthase kinase 3b (Gsk3b)
When mESCs were cultured in chemically-defined serum-free (CDSF) supplemented with 15% fetal bovine serum (FBS), they formed a well-developed teratoma in 5 weeks (Fig. 1G, 2F–2J, and supporting information Fig. S1B)

Summary

Introduction

Embryonic stem cells (ESCs) [1,2,3] and induced pluripotent stem cells (iPSCs) [4,5,6,7] are very promising tools for use in drug screening and customized tissue replacement [8] because they are capable of self-renewal that sustains pluripotency. The self-renewal and pluripotency of mouse stem cells (ESCs and iPSCs) are maintained by extrinsic factors, such as supplementing basal culture medium with leukemia inhibitory factor (LIF) [9,10,11,12,13] and fetal bovine serum (FBS). FBS provides cultures with many other uncharacterized components that may affect the capability of ESCs and iPSCs to self-renew and differentiate. It is firmly established that pharmacological inhibition of glycogen synthase kinase 3b (Gsk3b) promotes the self-renewal of both mouse [22,24] and human ESCs [23], and derivation of mouse ESCs [25]

Methods

Results

Conclusion