Modulation of Wnt/β-catenin signaling in human embryonic stem cells using a 3-D microwell array

Abstract

Intercellular interactions in the cell microenvironment play a critical role in determining cell fate, but the effects of these interactions on pathways governing human embryonic stem cell (hESC) behavior have not been fully elucidated. We and others have previously reported that 3-D culture of hESCs affects cell fates, including self-renewal and differentiation to a variety of lineages. Here we have used a microwell culture system that produces 3-D colonies of uniform size and shape to provide insight into the effect of modulating cell-cell contact on canonical Wnt/β-catenin signaling in hESCs. Canonical Wnt signaling has been implicated in both self-renewal and differentiation of hESCs, and competition for β-catenin between the Wnt pathway and cadherin-mediated cell-cell interactions impacts various developmental processes, including the epithelial-mesenchymal transition. Our results showed that hESCs cultured in 3-D microwells exhibited higher E-cadherin expression than cells on 2-D substrates. The increase in E-cadherin expression in microwells was accompanied by a downregulation of Wnt signaling, as evidenced by the lack of nuclear β-catenin and downregulation of Wnt target genes. Despite this reduction in Wnt signaling in microwell cultures, embryoid bodies (EBs) formed from hESCs cultured in microwells exhibited higher levels of Wnt signaling than EBs from hESCs cultured on 2-D substrates. Furthermore, the Wnt-positive cells within EBs showed upregulation of genes associated with cardiogenesis. These results demonstrate that modulation of intercellular interactions impacts Wnt/β-catenin signaling in hESCs.

Extracellular Matrices Decellularized from Embryonic Stem Cells Maintained Their Structure and Signaling Specificity

“…[22][23][24][25] These organizations have been shown to affect the secretion of ECM proteins and autocrine factors, producing an ECM microenvironment of distinct characteristics. 20,26,27 For example, deposition of endogenous transforming growth factor-beta inhibitor, Lefty, into ECMs has been reported for undifferentiated ESCs but not EBs, whereas the EBs displayed a unique ECM-shell structure and different cytokine secretion profiles compared to undifferentiated ESCs.…”

Wnt-YAP interactions in the neural fate of human pluripotent stem cells and the implications for neural organoid formation

“…However, the EBs formed from 3-D microwell culture contain more canonical Wnt signaling activity at early stage of differentiation and promote cardiac differentiation of hESCs. 15 These studies indicate that modulation of culture systems of PSC aggregates affects Wnt signaling.…”

“…15,25 Slower rotary speed (25 rpm) was shown to increase the nuclear accumulation of b-catenin at early time point and the expression of cardiac genes MESP-1 and MEF-2C. 25 3-D microwell culture was shown to promote the membrane localization of b-catenin and thus downregulate Wnt signaling.…”

“…Increased Ecadherin expression can sequester b-catenin at the adherent junctions, which results in less b-catenin available for translocation to the nucleus and the down-regulation of Wnt signaling. 15 The loss of E-cadherin expression enables b-catenin translocation into nucleus to activate Wnt signaling. During differentiation, the influence of Wnt/b-catenin has the context-dependent effects on hPSC fate decisions.…”

The Pharmacology of Regenerative Medicine

“…Pharmacological Tools for Differentiation from Pluripotent Stem Cells. Further application of 3D culture technologies and tissue-specific matrix components, in addition to soluble growth factors and cytokines, have the potential to improve in vitro differentiation from both pluripotent stem cells and lineage-restricted stem/progenitor cells present in fetal and adult tissues (Ott et al, 2008;Baptista et al, 2011;Bratt-Leal et al, 2011;Kraehenbuehl et al, 2011;Leipzig et al, 2011;Mari-Buye and Semino, 2011;Spence et al, 2011;Wang et al, 2011c;Azarin et al, 2012;Cardinale et al, 2012;Purpura et al, 2012). In this arena there exists great potential for regenerative pharmacology to explore synergies among peptidic signaling molecules (growth factors, cytokines), ECM components and synthetic biomaterials, and small molecule pharmaceutical compounds.…”