Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/β-catenin, Activin/Nodal and BMP signaling

Abstract

The canonical Wnt/β-catenin signaling has remarkably diverse roles in embryonic development, stem cell self-renewal and cancer progression. Here, we show that stabilized expression of β-catenin perturbed human embryonic stem (hES)-cell self-renewal, such that up to 80% of the hES cells developed into the primitive streak (PS)/mesoderm progenitors, reminiscent of early mammalian embryogenesis. The formation of the PS/mesoderm progenitors essentially depended on the cooperative action of β-catenin together with Activin/Nodal and BMP signaling pathways. Intriguingly, blockade of BMP signaling completely abolished mesoderm generation, and induced a cell fate change towards the anterior PS progenitors. The PI3-kinase/Akt, but not MAPK, signaling pathway had a crucial role in the anterior PS specification, at least in part, by enhancing β-catenin stability. In addition, Activin/Nodal and Wnt/β-catenin signaling synergistically induced the generation and specification of the anterior PS/endoderm. Taken together, our findings clearly demonstrate that the orchestrated balance of Activin/Nodal and BMP signaling defines the cell fate of the nascent PS induced by canonical Wnt/β-catenin signaling in hES cells.KEY WORDS: Primitive streak, Mesoderm, Endoderm, Stem cells, β-Catenin, Wnt Development 135, 2969Development 135, -2979Development 135, (2008 Lindsley et al., 2006), but precise roles of this signaling in human ES (hES) cells remains controversial (Dravid et al., 2005;Sato et al., 2004).We report here that the activation of canonical Wnt/β-catenin signaling in hES cells by conditional activation of stabilized β-catenin disrupted hES-cell self-renewal. Rather, the canonical Wnt/β-catenin and BMP signaling pathway in hES cells has significant roles in establishing the posterior PS/mesoderm progenitors, whereas attenuation of BMP signaling changes the cell fate to the anterior PS/endoderm progenitors. In addition, Activin and Wnt/β-catenin signaling pathways synergistically function in inducing undifferentiated hES cells to differentiate into the anterior PS/endoderm progenitors. This is the first in vitro model system that consistently recapitulates the human early embryogenesis and that enables us to analyze molecular events during the process of early embryogenesis from the epiblast to the PS formation, followed by lineage specification into the mesoderm and endoderm. More importantly, our findings will also be relevant to directed differentiation of specific tissue and cells from hES cells. MATERIALS AND METHODS Activation of β-catenin signaling in hES cellsThe ⌬Nβ-cateninER construct, in which the N-terminal 90 amino acids were deleted, was generated by in-frame insertion into the expression vector containing the hormone-binding domain of a mutant estrogen receptor (Littlewood et al., 1995;Sumi et al., 2007). Cell lines expressing ΔNβ-cateninER were obtained by transfection of hES cell lines KhES-1 and KhES-3 with ΔNβ-cateninER expression plasmids, followed by puromysin selection as described previously (Sumi ...

MSX2 mediates entry of human pluripotent stem cells into mesendoderm by simultaneously suppressing SOX2 and activating NODAL signaling

“…Interestingly, it has been reported that BMP, Wnt and NODAL can regulate each other and form a signaling loop essential for early lineage specification of mouse and human embryonic stem cells [60][61][62]. In this study we have shown that MSX2 acts as a direct target gene of BMP4, while Wnt signaling activates MSX2 expression synergistically with BMP.…”

Both BMP4 and serum have significant roles in differentiation of embryonic stem cells to primitive and definitive endoderm

“…In contrast to these studies, some investigators have demonstrated that BMPs negatively regulates the formation of endodermal progenitors (Poulain et al 2006;Sumi et al 2008). Sumi et al (2008) showed that BMP4 treatment does not induce the differentiation of human ES cells to mesendoderm/mesoderm progenitors. Moreover, Amita et al (2013) demonstrated that trophoblast is the predominant cell type which is derived from human ES cells in response to 10 ng/ml BMP4.…”

“…A combination of BMP4 and activin A induces the differentiation of human ES cells first to definitive endoderm and then to pancreatic cells (Teo et al 2012). In contrast to these studies, some investigators have demonstrated that BMPs negatively regulates the formation of endodermal progenitors (Poulain et al 2006;Sumi et al 2008). Sumi et al (2008) showed that BMP4 treatment does not induce the differentiation of human ES cells to mesendoderm/mesoderm progenitors.…”

Calcium signaling in human pluripotent stem cells

“…Several published papers indicate the role of Wnt in supporting pluripotency [65,66], while other data emphasize Wnt signaling in hPSC differentiation [67]. Recently it has been suggested that different GSK3 pools may play different roles in the control of hPSCs fate determination [56].…”