Applications of differentiated progeny generated from human embryonic stem cells (hESCs) broadly span cell replacement therapies and screening studies (toxicology, disease-drug modeling). These applications require differentiation into lineage-specific cell types from hESCs that are largely dependent on several reported embryoid body (EB) formation methods. However, methodologies for in vitro EB differentiation have not been quantitatively evaluated and compared. Using the hematopoietic lineage as a test for differentiation competency, we performed multiparameter comparisons of three prevalent EB methods: (1) suspension (SP), (2) hanging drop (HD), and (3) forced aggregation (FA). Although FA improved the homogeneity between hEBs, the highest hematopoietic induction efficiencies were observed in EBs formed in SP culture independent of the presence or absence of serum. Despite the EB formation method used, EB-based hematopoietic differentiation could be potently influenced by EB size and was augmented by paracrine signaling between cocultured EBs. Our study identifies physical and physiological parameters contributing to the efficiency of hESC differentiation in EB formats and reveals that EB methods are best tailored to specific applications unique to cell replacement vs small molecule screening or early human development.
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