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. 2011;6(6):e21355.
doi: 10.1371/journal.pone.0021355. Epub 2011 Jun 23.

Short-term serum-free culture reveals that inhibition of Gsk3β induces the tumor-like growth of mouse embryonic stem cells

Yanzhen Li  1 , Tamaki Yokohama-Tamaki, Tetsuya S Tanaka
Affiliations

Short-term serum-free culture reveals that inhibition of Gsk3β induces the tumor-like growth of mouse embryonic stem cells

Yanzhen Li et al. PLoS One. 2011.

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The tumorigenicity of mouse embryonic stem cells can be reduced by short-term serum-free culture.
(A): A mouse embryonic stem cell (mESC) line harboring an EGFP reporter driven by the Oct3/4 promoter (Oct3/4::EGFP) was maintained in either standard or chemically-defined serum-free (CDSF) medium as indicated. (B–E): Phase contrast (left) and fluorescence (right) images of the ESC line under the conditions indicated above are shown. Bars, 20 µm. (F–H): After the mESCs were cultured as indicated, they were transplanted into NOD-SCID mice subcutaneously. Teratoma formation was observed by week 11. Bars, 1 cm.
Figure 2
Figure 2. Identification of three germ layers in teratomas.
(A–E): Teratomas developed from mESCs cultured under standard conditions. (F–J): Teratomas developed from mESCs cultured in CDSF supplemented with 15% FBS (CDSF+FBS). (K–O): Teratomas developed from mESCs cultured in CDSF followed by transfer to standard conditions (CDSF to Stand). Ectoderm is represented by neural tissue and keratin pearl, mesoderm is represented by cartilage and striated muscle, and endoderm is represented by ciliated epithelium. Bars, 20 µm.
Figure 3
Figure 3. Mouse embryonic stem cells cultured under CDSF conditions are pluripotent.
(A–D): Mouse ESCs cultured under CDSF conditions were immunostained (right) with either specific antibodies against Nanog (A), Sox2 (B) and SSEA1 (C), or normal mouse serum (D). Also, phase contrast (left), and DAPI-stained (middle) images of these mESCs are shown. Bars, 20 µm. (E): Phase contrast (left) and fluorescence (right) images of a chimeric blastocyst aggregated with the Oct3/4::EGFP mESC line. Bar, 20 µm. (F): A phase contrast image of a male gonad dissected from a chimeric E13.5 embryo. The rectangle indicates the area shown in G. Bar, 100 µm. (G): Enlarged phase contrast (left) and fluorescence (right) images of the male gonad shown in F. Bar, 50 µm.
Figure 4
Figure 4. Phenotypic changes observed in mouse embryonic stem cells cultured under CDSF conditions.
(A): The sizes of the teratomas formed (orange bar, left axis) and the number of days required for the experimental NOD-SCID mice to reach their end points (blue stars, right axis) were compared among mESCs cultured under the conditions indicated (see Fig. 1A). Parentheses indicate the number of biological replicates (i.e., mESCs prepared at different passages) per culture condition. Standard errors of the means are indicated by bars. a: Only one passage in standard conditions followed CDSF culture. b: Two out of seven transplantations showed no sign of teratoma formation when paired with the standard and CDSF-Standard conditions, whereas five out of seven transplantations showed no sign of teratoma formation for 6 months. (B, top): Cell doublings were measured every 48 hours after plating 1×106 cells per well onto 6-well plates (see Materials and Methods for the formula). Only CDSF conditions produced statistically-significant differences compared to standard conditions. Parentheses indicate the number of biological replicates per condition. Standard errors of the means are indicated by bars. (B, bottom): Phase contrast (top) and fluorescence (bottom) images are shown for the Oct3/4::EGFP mESC line (Fig. 1A) grown under the conditions indicated below 1 and 2 days after plating 0.1×106 cells per well in 6-well plates. Bars, 50 µm. (C): Abundance of each transcript indicated above was examined in mESCs cultured under each condition on the right by 25 cycles of PCR. Ef1α was used as a reference.
Figure 5
Figure 5. Screening of factors responsible for the tumor-like growth of mouse embryonic stem cells.
(A): Cumulative numbers of mESCs were compared among ESCs cultured under each condition indicated for 3 passages. Cell counts were normalized to CDSF conditions. A value for CDSF conditions is normalized to 1. Parentheses indicate the number of biological replicates per condition. Standard errors of the means are indicated by bars. +RA, CDSF with retinoic acid; +RL, CDSF with retinol; +RL+RBP, CDSF with retinol and retinol binding protein; +Bmp4, CDSF with Bmp4; +iGsk3β, CDSF with the Gsk3β inhibitor. (B): Total numbers of biological replicates that resulted in formation of teratomas were compared among mESCs cultured under each condition indicated. Orange and yellow boxes indicate the number of biological replicates that developed into teratomas within 6 months and in more than 6 months, respectively. Blue bars indicate the number of biological replicates that failed to form teratomas for more than 6 months. Data for the Gsk3β inhibitor include results obtained with R1 and W4 mESCs. For those biological replicates indicated as (i), (ii) and (iii) in the bar chart, the number of technical replicates, the number of days needed for the experimental animals to reach their end points, and the sizes of the longest axis of the teratomas are shown per technical replicate. Asterisks (*) indicate that W4 mESCs were transplanted. n.d., not determined. (C, top): NOD-SCID mice that received transplantation of mESCs cultured in CDSF supplemented with Bmp4 (right) or the Gsk3β inhibitor (left) are shown. Bars, 1 cm. (C, bottom): Representative histological images are shown. The presence of three germ layers (see the legend for Fig. 2) is evident in teratomas developed from mESCs cultured in CDSF supplemented with the Gsk3β inhibitor. Bars, 20 µm. (D): Abundance of each transcript indicated above was examined in mESCs cultured under each condition on the right by 24 cycles of PCR. Ef1α was used as a reference. White arrowheads indicate the PCR product of c-Myc.
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