WNT/β-Catenin signaling pathway regulates non-tumorigenesis of human embryonic stem cells co-cultured with human umbilical cord mesenchymal stem cells

Abstract

Human pluripotent stem cells harbor hope in regenerative medicine, but have limited application in treating clinical diseases due to teratoma formation. Our previous study has indicated that human umbilical cord mesenchymal stem cells (HUCMSC) can be adopted as non-teratogenenic feeders for human embryonic stem cells (hESC). This work describes the mechanism of non-tumorigenesis of that feeder system. In contrast with the mouse embryonic fibroblast (MEF) feeder, HUCMSC down-regulates the WNT/β-catenin/c-myc signaling in hESC. Thus, adding β-catenin antagonist (FH535 or DKK1) down-regulates β-catenin and c-myc expressions, and suppresses tumorigenesis (3/14 vs. 4/4, p = 0.01) in hESC fed with MEF, while adding the β-catenin enhancer (LiCl or 6-bromoindirubin-3'-oxime) up-regulates the expressions, and has a trend (p = 0.056) to promote tumorigenesis (2/7 vs. 0/21) in hESC fed with HUCMSC. Furthermore, FH535 supplement does not alter the pluripotency of hESC when fed with MEF, as indicated by the differentiation capabilities of the three germ layers. Taken together, this investigation concludes that WNT/β-catenin/c-myc pathway causes the tumorigenesis of hESC on MEF feeder, and β-catenin antagonist may be adopted as a tumor suppressor.

Figure 1. Down-regulation of β-catenin and CMYC in hESC co-cultured with HUCMSC.

(a) qRT-PCR of c-myc of hESC/MEF, hESC/HUCMSC and hESC/MHM. (b) Western blotting analysis of β-catenin and c-myc of hESC/MEF, hESC/HUCMSC and hESC/MHM. Further shifting to MEF co-culture (hESC/MHM) reversed these expressional changes (a,b). (c) TCF/LEF activity of hESC cultured on different condition was measured by luciferase assay, and Firefly luciferase activity was normalized to Renilla luciferase activity, which was adopted as internal control. Values are shown as the mean of three replicates ± standard deviations. (d) Real-time PCR analysis of DNA fragments precipitated in a ChIP assay by using a β-catenin antibody. Primers designed for the 5′ promoter of c-myc were adopted to detect specific β-catenin binding. Data are represented as percentage input. Error bars represent SEM. (e) Three germ-layer differentiation gene expressions of embryoid body (EB) derived from hESC cultured on MEF and HUCMSC were compared by qRT-PCR (ectoderm: β-3-tubulin, MAP2, GFAP; endoderm: GATA4; mesoderm: GATA6, Hand1). *p < 0.05, **p < 0.01, ***p < 0.001. All cropped blots were run under the same experimental conditions in (b).

Figure 2. The β-catenin signaling activator LiCl and BIO up-regulates β-catenin and CMYC in hESC/HUCMSC.

After 10 mM LiCl treatment for 24 hours, hESC/HUCMSC maintained a normal morphology for embryonic stem cells (a). Expressions of CMYC after either LiCl (10 mM) or BIO (5 μM) treatment were up-regulated at both mRNA (b) and protein (c,e) levels. (d) Western blotting analysis of nuclear translocation of active β-catenin in response to BIO 5 μM treatment for 24 hours. Scale bar = 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001. All cropped blots were run under the same experimental conditions in (c–e).

Figure 3. LiCl-treatment promotes teratoma formation in hESC/HUCMSC xenograft.

Tumor formation was observed 12 wks after sc injection of 1 × 10⁵ hESC cells on HUCMSC feeder into NOD/SCID mice. Two of 7 mice grew tumors when injected with LiCl-treated hESC/HUCMSC (a,b). Hematoxylin and eosin stain of the re-sected tumor indicated a histology mature teratoma with evident endoderm (c,d), ectoderm (e) and mesoderm (f) components.

Figure 4. β-catenin transactivation antagonist FH535 and DKK1 inhibits β-catenin and CMYC expression in hESC co-cultured with MEF.

(a) The typical morphology of hESC remained unchanged after treating hESC/MEF with 10 μM FH535 for 8 hours. Scale bar = 1000 μm. (b) qRT-PCR analysis CMYC mRNA expression of hES/MEF treated by FH535 (10 μM) and DKK1 (250 ng/ml) for 24 hours. (c) Western blotting analysis of Myc protein was down-regulated to a level equivalent to that in hES/HUCMSC. Quantitative expression of β-catenin and c-myc protein in three independent experiments is shown in the two right-hand panels. (d) Western blotting analysis of nuclear translocation of active β-catenin in response to DKK1 (250 ng/ml) treatment of hES/MEF for 24 hours. Quantification of nuclear fraction of β-catenin (in triplicate) is shown in the right panel. (e) Western blotting analysis of c-myc in hESC/MEF after treating DKK1 for 24 hours. *p < 0.05, **p < 0.01, ***p < 0.001. All cropped blots were run under the same experimental conditions.

Figure 5. hESC/MEF following β-catenin antagonist (FH535) treatment maintained pluripotency.

(a) Immunocytochemistry of hESC/MEF after 10 μM FH535 for 8 hours with pluripotency markers. (b) Immunocytochemistry of EB derived from hESC/MEF with three germ layers markers. Three germ layers markers: ectoderm (MAP2, tuj1), endoderm (ATBF1) and mesoderm (brachyury). (c) RT-PCR analysis of pluripotency genes (OCT4, sox2, nanog) and differentiation genes specific for germ cell (GDF9), endoderm (GATA4), mesoderm (HAND1, GATA6) and ectoderm (β-III-tubulin, MAP2, GFAP) were observed. GAPDH was adopted as a control. (d) Hematoxylin and eosin staining of teratoma formed by hESC/MEF treated with FH535 (1: ectoderm; 2: mesoderm; 3: endoderm). ES: human embryonic stem cell, F: FH535, EB: embryoid body. Scale bar = 50 μm. All cropped gels were run under the same experimental conditions in (c).