Synergistic action of Wnt and LIF in maintaining pluripotency of mouse ES cells

doi:10.1016/j.bbrc.2006.02.127

Biochemical and Biophysical Research Communications

Volume 343, Issue 1, 28 April 2006, Pages 159-166

https://doi.org/10.1016/j.bbrc.2006.02.127 Get rights and content

Abstract

Leukaemia inhibitory factor (LIF) was the first soluble factor identified as having potential to maintain the pluripotency of mouse embryonic stem (ES) cells. Recently, a second factor, Wnt, with similar activity was found. However, the relationship between these completely different signals mediating the overlapping functions is still unclear. Here, we report that the conditioned medium of L cells expressing Wnt3a maintains ES cells in the undifferentiated state in feeder-free culture, followed by expression of stem cell markers and their ability to generate germline chimaeras. However, although the activity of this conditioned medium is dependent on Wnt3a, recombinant Wnt3a protein cannot maintain ES cells in the undifferentiated state. As supplementation with Wnt3a to the sub-threshold level of LIF alone was not sufficient to maintain ES self-renewal, the results of maintenance of the undifferentiated state indicated the synergistic action of Wnt and LIF. Induction of constitutively activated β-catenin alone is unable to maintain ES self-renewal but shows a synergistic effect with LIF. These observations indicate that the Wnt signal mediated by the canonical pathway is not sufficient but enhances the effect of LIF to maintain self-renewal of mouse ES cells.

Section snippets

Materials and methods

ES cell culture. ES cells were maintained on feeder-free gelatine-coated plates in FCS-containing medium supplemented with LIF: Glasgow minimal essential medium (GMEM; Sigma–Aldrich) supplemented with 10% FCS (selected batches, Sigma–Aldrich), 100 μM 2-mercaptoethanol (Nacalai Tesque), 1× non-essential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen), and 1000 U/ml (U) LIF (ESGRO; Invitrogen). EB3 and OLG2-3 ES cells were generated by introduction of Oct3/4 knockout vector carrying

Results and Discussion

To determine whether activation of the Wnt signal is absolutely sufficient to maintain pluripotency of mES cells, we first examined the effects of conditioned medium from mouse fibroblast L cells expressing Wnt3a (L-Wnt3a CM) as a source of Wnt activity on mES self-renewal using a feeder-free culture system [11], [13]. To determine the actual effect of Wnt3a in the conditioned medium on mES cells, we used two different negative controls: conditioned medium from wild-type L cells (L CM) and

Acknowledgments

We thank Dr. Shinji Masui (RIKEN CDB) for technical suggestions regarding the ROSA-TET expression system. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture of Japan (to H.N.), the Ministry of Health, Labor and Welfare of Japan (to R.N.), and Leading Project (to H.N.).

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Culturing pluripotent stem cells effectively requires substrates coated with feeder cell layers or cell-adhesive matrices. It is difficult to employ pluripotent stem cells as resources for regenerative medicine due to risks of culture system contamination by animal-derived factors, or the large costs associated with the use of adhesive matrices. To enable a coating-free culture system, we focused on UV/ozone surface modification and atmospheric pressure plasma treatment for polystyrene substrates, to improve adhesion and proliferation of pluripotent stem cells. In this study, to develop a feeder- and matrix coating-free culture system for embryonic stem cells (ESCs), mouse ESCs were cultured on polystyrene substrates that were surface-modified using UV/ozone-plasma combined treatment. mESCs could be successfully cultured under feeder-free conditions upon UV/ozone-plasma combined treatment of culture substrates, without any further chemical treatments, and showed similar proliferation rates to those of cells grown on the feeder cell layer or matrix-coated substrates.
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As previously reported, both ΔC β-cat and wt β-cat isoforms restored self-renewal and AP staining intensity defects (Figure S4C, bottom panel), suggesting that canonical Wnt pathway activity is dispensable for self-renewal in these culture conditions. However, nuclear β-catenin activity appears to be required for inhibition of differentiation in serum and in absence of LIF (Ogawa et al., 2006; Sato et al., 2004). Therefore, we performed clonal assay and AP staining of Eβ47 rescued cells in serum without LIF.
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The Dishevelled proteins transduce both canonical Wnt/β-catenin and non-canonical Wnt/planar cell polarity (PCP) signaling pathways to regulate many key developmental processes during embryogenesis. Here, we disrupt both canonical and non-canonical Wnt pathways by targeting the entire Dishevelled family of genes (Dvl1, Dvl2, and Dvl3) to investigate their functional roles in the early embryo. We identified several defects in anterior-posterior axis specification and mesoderm patterning in Dvl1^+/−; Dvl2^−/−; Dvl3^−/− embryos. Homozygous deletions in all three Dvl genes (Dvl TKO) resulted in defects in distal visceral endoderm migration and a complete failure to induce mesoderm formation. To identify potential mechanisms that lead to the defects in the developmental processes preceding gastrulation, we generated Dvl TKO mouse embryonic stem cells (mESCs) and compared the transcriptional profile of these cells with wild-type (WT) mESCs during germ lineage differentiation into 3D embryoid bodies (EBs). While the Dvl TKO mESCs displayed similar morphology, self-renewal properties, and minor transcriptional variation from WT mESCs, we identified major transcriptional dysregulation in the Dvl TKO EBs during differentiation in a number of genes involved in anterior-posterior pattern specification, gastrulation induction, mesenchyme morphogenesis, and mesoderm-derived tissue development. The absence of the Dvls leads to specific down-regulation of BMP signaling genes. Furthermore, exogenous activation of canonical Wnt, BMP, and Nodal signaling all fail to rescue the mesodermal defects in the Dvl TKO EBs. Moreover, endoderm differentiation was promoted in the absence of mesoderm in the Dvl TKO EBs, while the suppression of ectoderm differentiation was delayed. Overall, we demonstrate that the Dvls are dispensable for maintaining self-renewal in mESCs but are critical during differentiation to regulate key developmental signaling pathways to promote proper axis specification and mesoderm formation.

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Synergistic action of Wnt and LIF in maintaining pluripotency of mouse ES cells

Abstract

Section snippets

Materials and methods

Results and Discussion

Acknowledgments

Dev. Biol.

Chem. Biol.

Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides

Nature

Molecular mechanism to maintain stem cell renewal of ES cells

Cell Struct. Funct.

Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3

Genes Dev.

STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells

EMBO J.

A novel mechanism for regulating clonal propagation of mouse ES cells

Genes Cells

Embryonic stem cell lines derived from human blastocysts

Science

Establishment of embryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI

Dev. Dyn.

Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor

Nat. Med.

Defining the role of Wnt/beta-catenin signaling in the survival, proliferation, and self-renewal of human embryonic stem cells

Stem Cells