Embryoid Body - Challenges To Directing Differentiation in EBs

Challenges To Directing Differentiation in EBs

In contrast to the differentiation of ESCs in monolayer cultures, whereby the addition of soluble morphogens and the extracellular microenvironment can be precisely and homogeneously controlled, the three-dimensional structure of EBs poses challenges to directed differentiation. For example, the visceral endoderm population which forms the exterior of EBs, creates an exterior “shell” consisting of tightly connected epithelial-like cells, as well as a dense ECM. Due to such physical restrictions, in combination with EB size, transport limitations occur within EBs, creating gradients of morphogens, metabolites, and nutrients. It has been estimated that oxygen transport is limited in cell aggregates larger than approximately 300 µm in diameter; however, the development of such gradients are also impacted by molecule size and cell uptake rates. Therefore, the delivery of morphogens to EBs results in increased heterogeneity and decreased efficiency of differentiated cell populations compared to monolayer cultures. One method of addressing transport limitations within EBs has been through polymeric delivery of morphogens from within the EB structure. Additionally, EBs can be cultured as individual microtissues and subsequently assembled into larger structures for tissue engineering applications. Although the complexity resulting from the three-dimensional adhesions and signaling may recapitulate more native tissue structures, it also creates challenges for understanding the relative contributions of mechanical, chemical, and physical signals to the resulting cell phenotypes and morphogenesis.