Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation

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Abstract
To realize the therapeutic potential of human embryonic stem cells (hESCs), it is necessary to regulate their differentiation in a uniform and reproducible manner. We have developed a method in which known numbers of hESCs in serum-free medium were aggregated by centrifugation to foster the formation of embryoid bodies (EBs) of uniform size (spin EBs). These spin EBs differentiated efficiently and synchronously, as evidenced by the sequential expression of molecular markers representing stem cells, primitive streak, and mesoderm. In the presence of hematopoietic growth factors, reproducible differentiation was achieved with blood cells formed in more than 90% of EBs. Using chimeric EBs generated from mixtures of green fluorescence protein–positive (GFP+) and GFP– hESCs in a clonogenic assay, hematopoietic precursor frequency was estimated to be approximately 1:500 input cells. This method of EB formation provides a generally applicable means for modulating and objectively monitoring the directed differentiation of hESCs.