Though many advances have been made in controlling human embryonic stem cell (hESC) behavior, large-scale stem cell tissue engineering applications still face two main obstacles: spontaneous differentiation during expansion in the undifferentiated state and low yields for many differentiation protocols. We have developed a 3-D microwell array that presents a tunable, homogeneous system for studying the effects of various microenvironmental cues on stem cell processes. Previous work has shown that this microwell system promotes long-term self-renewal of hESCs and can generate uniform embryoid bodies (EBs) with enhanced cardiogenic potential. Elucidating the mechanisms underlying these effects can provide effective targets for controlling self-renewal and differentiation. Here we used the microwell system to evaluate the effects of modulating cell-cell contact on Wnt/β-catenin signaling in hESCs.
Wnt signaling has been implicated in both self-renewal and cardiogenesis of hESCs, and competition for β-catenin between the Wnt pathway and cadherin-mediated cell-cell interactions can impact cell behavior. Flow cytometry results showed that hESCs cultured in 3-D microwells had higher expression of E-cadherin per cell than hESCs grown on 2-D controls. Since β-catenin must be present to stabilize cadherin-mediated contacts, the increase in E-cadherin expression in microwells limited the amount of β-catenin available to translocate to the nucleus and participate in Wnt signaling, as demonstrated by immunofluorescence data showing a lack of nuclear β-catenin localization in hESCs cultured in microwells. These results were confirmed via Western blot analysis of nuclear and cytoplasmic protein extracts from microwells and 2-D controls. Additionally, the lack of nuclear β-catenin in microwells corresponded to a downregulation of Wnt target genes FST, AXIN2, CCND1, FZD7, and SLUG. Although there was reduced Wnt signaling in microwells, data from a Wnt reporter line showed that EBs formed from microwells contained higher levels of Wnt signaling than EBs from 2-D controls, and gene expression analysis of the Wnt-positive cells within EBs showed an upregulation of genes associated with cardiogenesis as compared to the Wnt-negative cells. These differences in Wnt/β-catenin signaling suggest a potential mechanism by which microwell confinement affects hESC self-renewal and cardiac differentiation that could be translated into development of scalable, efficient processes for tissue engineering applications.