Embryoid body differentiation. Differentiation of ESCs is commonly performed in monolayer, providing a defined substrate for cell attachment and uniform exposure to exogenously added soluble molecules, such as growth factors or small molecules; however, 3D approaches allow for a biological model of embryonic morphogenesis by facilitating multicellular interactions [34]. The 3D organization and proliferation, migration, and differentiation of an embryo can be modeled by the formation of pluripotent stem cell aggregates, known as embryoid bodies (EBs) [35]. EB formation has traditionally been used to induce spontaneous differentiation of ESCs towards the three germ lineages. Differentiation of EBs is characterized by the initial formation of two epithelial layers, with the specification of exterior cells toward primitive endoderm (PE), mediated by fibroblast growth factor (FGF) signaling via the PI 3-kinase pathway [36,37]. The PE layer eventually differentiates to form visceral and parietal endoderm, while cells at the exterior deposit a basement membrane composed of ECM components including type IV collagen and laminin [37,38]. The basement membrane serves to separate the PE layer from the internal undifferentiated cell population, where cells not in direct contact with the basement membrane may undergo apoptosis to form cystic cavities [39,40]. Culture platforms for EB formation include static suspension, hanging drop and forced aggregation [41]. Spontaneous formation of EBs is initiated by suspension of ESCs in a non-adhesive culture dish in the absence mouse embryonic fibroblasts (MEFs) feeder layers and/or Leukemia inhibitory factor (LIF). Dissociated ESCs are transferred to suspension cultures and allowed to aggregate to form 3-dimensional structures through the homophilic binding of the cell-cell adhesion molecule E-cadherin, which is highly expressed on the cell surface of undifferentiated ESCs [41,42]. However, static suspension of EBs often results in a heterogeneous population due to variable aggregate sizes and agglomeration, ultimately reducing the efficiency of differentiation. More controlled EB formation can be accomplished by the hanging drop method, which involves the suspension of a relatively small number of cells within a single drop (10-20 μL) from the lid of a Petri dish [2]. While allowing for greater control of EB size, the hanging drop technique is labor-intensive and not amenable to scale up for bioprocessing applications. Alternatively, formation of EBs by rot...
