Generation of Multipotential Mesendodermal Progenitors from Mouse Embryonic Stem Cells via Sustained Wnt Pathway Activation

Yvonne Yiling Koh,Jamie Chen Yee Mong,Lawrence W. Stanton,Kee Yew Wong,Aina Hoi,Manjiri Manohar Bakre

doi:10.1074/jbc.m704287200

Abstract

Pluripotent embryonic stem cells (ESCs) are capable of differentiating into cell types belonging to all three germ layers within the body, which makes them an interesting and intense field of research. Inefficient specific differentiation and contamination with unwanted cell types are the major issues in the use of ESCs in regenerative medicine. Lineage-specific progenitors generated from ESCs could be utilized to circumvent the issue. We demonstrate here that sustained activation of the Wnt pathway (using Wnt3A or an inhibitor of glycogen synthase kinase 3beta) in multiple mouse and human ESCs results in meso/endoderm-specific differentiation. Using monolayer culture conditions, we have generated multipotential "mesendodermal progenitor clones" (MPC) from mouse ESCs by sustained Wnt pathway activation. MPCs express increased levels of meso/endodermal and mesendodermal markers and exhibit a stable phenotype in culture over a year. The MPCs have enhanced potential to differentiate along endothelial, cardiac, vascular smooth muscle, and skeletal lineages than undifferentiated ESCs. In conclusion, we demonstrate that the Wnt pathway activation can be utilized to generate lineage-specific progenitors from ESCs, which can be further differentiated into desired organ-specific cells.

Highlights

The advantages of lineage-specific progenitor cells over embryonic stem cells (ESCs) are that they differentiate into a limited number of cell types of a particular lineage and, the differentiation will be robust and more efficient
Wnt Pathway Activation and Isolation of mesendodermal progenitor clones” (MPC)—Mouse ESCs were cultured continuously for 3 weeks in the presence of leukemia inhibitory factor as described above either in growth medium supplemented with 1 ␮M GSK-3␤ inhibitor (Eli Lilly) or in 50% growth medium and 50% Wnt3A conditioned medium (CM) to activate the Wnt pathway
Activation of Wnt Pathway Induces Differentiation of ESCs along Meso/Endoderm—Wnt signaling was induced in two ways: by the addition of active Wnt3A CM and a selective inhibitor of GSK-3␤

Summary

Introduction

The advantages of lineage-specific progenitor cells over ESCs are that they differentiate into a limited number of cell types of a particular lineage and, the differentiation will be robust and more efficient. The molecular analysis indicated that Wnt pathway activation led to induction of meso/endodermal markers (T-brachyury, Gata2, Nkx2.5, Hand1, Foxa2, AFP, Gata4, and Sox17) in E14 cells by 7–10 days, and the induction was stronger at day 21 (Fig. 1, D and E).

Objectives

Results

Conclusion