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

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 3␤) in multiple mouse and human ESCs results in meso/endodermspecific 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.The unique property of pluripotent ESCs 2 to differentiate into three germ layers derivatives makes ESCs an ideal source of cells for regenerative therapy (1). One of the most pressing problems in developing ESC-based applications has been the inefficient differentiation of ESCs into the specific therapeutic cell type of choice and the presence of unwanted differentiated cells of other germ layers. A strategy to overcome this problem is to derive lineage-specific progenitor stem cells from ESCs.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, therefore, the differentiation will be robust and more efficient. In addition, they can self-renew, and thus, can be maintained as a renewable source of cells.ESCs can aggregate to form embryoid bodies (EBs) (2, 3), which resemble an intact embryo, and thus, many protocols for in vitro differentiation of the ESCs utilize formation of EB as the first step. However, there can be a mixture of differentiating cells in using an EB approach, whereas differentiation using monolayer culture can yield more uniform and homogenous results. A successful strategy for the use of ESCs in regenerative medicine could involve formation of lineage-restricted progenitor cell using a monolayer culture system as a first step.Gene expression analysis and fate maps together indicate that endoderm and mesoderm are derived, at least in part, from bipotent mesendodermal cells that separate during gastrulation (4, 5) Active canonical Wnt signaling is detected in pregastrulating embryo, through primitive streak (PS) formation, and during gastrulation (6). The evolutionary conserved Wnt signaling pathway is absolutely essential for...

Smek promotes histone deacetylation to suppress transcription of Wnt target gene brachyury in pluripotent embryonic stem cells

“…On the other hand, sustained Wnt-signaling activation can induce differentiation of ESC. A recent study showed that long-term treatment of ESCs with Wnt3a protein in the presence of LIF induces differentiation into bipotent mesendodermal cells that produce mesoderm [33]. Similarly to this, we show that depletion of Smek proteins induces mesoderm differentiation.…”

Maintaining embryonic stem cell pluripotency with Wnt signaling

“…By contrast, other studies of modulated Wnt pathway activity in Xenopus and mouse embryos in vivo and in mouse ES cells in vitro have concluded that Wnt signaling promotes mesendodermal differentiation (Bakre et al, 2007;Gadue et al, 2006;Lindsley et al, 2006;Sokol, 1993;Takada et al, 1994). Adding to the confusion, TCF3, a binding partner of -catenin that represses cell differentiation in multiple tissues (Nguyen et al, 2006), has also been proposed to restrict mouse ES cell self-renewal (Cole et al, 2008;Tam et al, 2008;Yi et al, 2008).…”

“…Not only does Wnt signaling specify the anteroposterior (AP) body axis in most metazoan animals, but it has also been reported to promote ES cell pluripotency (Nusse et al, 2008;Wend et al, 2010), to specify the mesendodermal lineage and to inhibit neuroectodermal differentiation in mouse ES cells (Aubert et al, 2002;Bakre et al, 2007;Haegele et al, 2003;Lindsley et al, 2006;Sato et al, 2004) and in vertebrate embryos (Yoshikawa et al, 1997;Itoh and Sokol, 1999). Strikingly, whether Wnt ligands and receptors themselves have a proven role in pluripotency continues to be the subject of ongoing debate (Nusse et al, 2008;Wend et al, 2010).…”

A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates

“…BMP2 together with Wnt3a constitutes a potent combination of factors to induce the mesoderm (16,17). We thus used the cardiogenic morphogen BMP2 in a defined serum-free medium and in the presence of the FGF receptor inhibitor SU5402 (18).…”