(261a) Cell-Cell Contact In Microwell Confinement Primes Wnt/&beta-Catenin Signaling During Differentiation of Human Embryonic Stem Cells

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.