Recently, various approaches for controlling the embryonic stem (ES) cell microenvironment have been developed for regulating cellular fate decisions. It has been reported that the lineage specific differentiation could be affected by the size of ES cell colonies and embryoid bodies (EBs). However, much of the underlying biology has not been well elucidated. In this study, we used microengineered hydrogel microwells to direct ES cell differentiation and determined the role of WNT signaling pathway in directing the differentiation. This was accomplished by forming ES cell aggregates within microwells to form different size EBs. We determined that cardiogenesis was enhanced in larger EBs (450 m in diameter), and in contrast, endothelial cell differentiation was increased in smaller EBs (150 m in diameter). Furthermore, we demonstrated that the EB-size mediated differentiation was driven by differential expression of WNTs, particularly noncanonical WNT pathway, according to EB size. The higher expression of WNT5a in smaller EBs enhanced endothelial cell differentiation. In contrast, the increased expression of WNT11 enhanced cardiogenesis. This was further validated by WNT5a-siRNA transfection assay and the addition of recombinant WNT5a. Our data suggest that EB size could be an important parameter in ES cell fate specification via differential gene expression of members of the noncanonical WNT pathway. Given the size-dependent response of EBs to differentiate to endothelial and cardiac lineages, hydrogel microwell arrays could be useful for directing stem cell fates and studying ES cell differentiation in a controlled manner.hydrogel microwells ͉ stem cell differentiation ͉ WNT signal pathway T he developmental versatility of embryonic stem (ES) cells offers a powerful approach for directing cell fate and is a promising source of progenitors for cell replacement therapy and tissue regeneration (1). ES cells can differentiate into a wide spectrum of cell types, such as cardiomyocytes and endothelial cells, by forming embryoid bodies (EBs) (2). Those lineages arise from distinct mesoderm subpopulations that develop sequentially from premesoderm cells (3). Such lineage specification is highly coordinated with differential changes in gene expression (4-8).Despite the therapeutic potential of ES cells, one of the significant challenges to their widespread clinical use, is the inability to homogeneously direct ES cell differentiation into specific lineages. One reason for the heterogeneity in EB differentiation is caused from variations in EB size (9, 10). To address this challenge for controlling the differentiation of ES cells, various microscale technologies (i.e., surface patterning, hydrogel microwells, and microfluidic systems) have been developed for directing the stem cell fate (11-15). Micropatterning techniques have been used to evaluate the effect of EB size on ES cell differentiation. For instance, microfabricated adhesive stencils were used to pattern ES cells for controlling initial ES cell aggregate sizes...

Directed embryonic stem (ES) cell differentiation is a potentially powerful approach for generating a renewable source of cells for regenerative medicine. Typical in vitro ES cell differentiation protocols involve the formation of ES cell aggregate intermediates called embryoid bodies (EBs). Recently, we demonstrated the use of poly(ethylene glycol) (PEG) microwells as templates for directing the formation of these aggregates, offering control over parameters such as size, shape, and homogeneity. Despite these promising results, the previously developed technology was limited as it was difficult to reproducibly obtain cultures of homogeneous EBs with high efficiency and retrievability. In this study, we improve the platform by optimizing a number of features: material composition of the microwells, cell seeding procedures, and aggregate retrieval methods. Adopting these modifications, we demonstrate an improved degree of homogeneity of the resulting aggregate populations and establish a robust protocol for eliciting high EB formation efficiencies. The optimized microwell array system is a potentially versatile tool for ES cell differentiation studies and high-throughput stem cell experimentation.