Embryoid Body - Formation of EBs

Formation of EBs

EBs are formed by the homophilic binding of the Ca2+ dependent adhesion molecule E-cadherin, which is highly expressed on undifferentiated ESCs. When cultured as single cells in the absence of anti-differentiation factors, ESCs spontaneously aggregate to form EBs. Such spontaneous formation is often accomplished in bulk suspension cultures whereby the dish is coated with non-adhesive materials, such as agar or hydrophilic polymers, to promote the preferential adhesion between single cells, rather than to the culture substrate. As hESC undergo apoptosis when cultured as single cells, EB formation often necessitates the use of inhibitors of the rho associated kinase (ROCK) pathway, including the small molecules Y-27632 and 2,4 disubstituted thiazole (Thiazovivin/Tzv). Alternatively, to avoid dissociation into single cells, EBs can be formed from hESCs by manual separation of adherent colonies (or regions of colonies) and subsequently cultured in suspension. Formation of EBs in suspension is amenable to the formation of large quantities of EBs, but provides little control over the size of the resulting aggregates, often leading to large, irregularly shaped EBs. As an alternative, the hydrodynamic forces imparted in mixed culture platforms increase the homogeneity of EB sizes when ESCs are inoculated within bulk suspensions.

Formation of EBs can also be more precisely controlled by the inoculation of known cell densities within single drops (10-20 µL) suspended from the lid of a Petri dish, known as hanging drops. While this method enables control of EB size by altering the number of cells per drop, the formation of hanging drops is labor intensive and not easily amenable to scalable cultures. Additionally, the media can not be easily exchanged within the traditional hanging drop format, necessitating the transfer of hanging drops into bulk suspension cultures after 2–3 days of formation, whereby individual EBs tend to agglomerate. Recently, new technologies have been developed to enable media exchange within a modified hanging drop format. In addition, technologies have also been developed to physically separate cells by forced aggregation of ESCs within individual wells or confined on adhesive substrates, which enables increased throughput, controlled formation of EBs. Ultimately, the methods used for EB formation may impact the heterogeneity of EB populations, in terms of aggregation kinetics, EB size and yield, as well as differentiation trajectories.