Cell-cell contact in microwell confinement primes Wnt/β-catenin signaling during differentiation of human embryonic stem cells
The cellular response, and thus cell fate decisions, of human embryonic stem cells (hESCs) can be dramatically influenced by culturing hESCs in a 3D microenvironment as opposed to the traditional monolayer of cells on plates. However, the underpinning molecular events are seldom explored and have yet to be fully understood. Here, we investigate how 3D culture increases adherens junctions, which influence the activation of the Wnt/β-catenin signaling pathway that is important during early differentiation. In order to assess effects of 3D culture on hESC developmental pathways, we devised a microwell culture system that permits precise control of colony size and shape. To manufacture microwells, wells are etched in silicon wafers to form reusable PDMS stamps, which in turn serve to mold polyurethane substrates that are used to culture hESCs. Cell adhesion and growth are confined to the insides of the wells by depositing gold and then a protein-resistant, alkanethiol self-assembling monolayer on all but the bottom of the wells. Using this microwell system, we determined molecular differences of hESCs cultured in 3D versus 2D and then the effects on cell signaling and differentiation. First, E-cadherin expression is greater per hESC cultured in microwells compared to hESCs from traditional 2D culture. Next, during the initial stages of embryoid body (EB)-based differentiation, EBs generated from microwells activate Wnt/β-catenin signaling earlier than those made from traditional 2D culture. Subsequently, EBs from microwells favor differentiating toward mes-endoderm lineages rather than the neuroectoderm. Furthermore, microwell confinement alone encourages development into cardiomyocytes. Therefore, to link the amount of E-cadherin prior differentiation to the earlier activation of Wnt/β-catenin signaling immediately following differentiation, we established inducible E-cadherin knock-down and knock-in hESC lines. Thus, by modulating the levels of E-cadherin of hESCs cultured both within microwells and on traditional 2D plates, we can delineate E-cadherin's roles in Wnt/β-catenin signaling during differentiation. In conclusion, we have established that by culturing hESCs in a 3D configuration, we “prime” the cells for an earlier up-regulation of Wnt/β-catenin signaling and mes-endoderm development.