Human cerebral organoids (hCOs) provide an excellent model for the study of human brain development and disease. Here, we present a protocol to obtain hCOs directly from two-dimensional (2D) pluripotent stem cell (PSC) cultures, avoiding cell dissociation and posterior embryoid body (EB) aggregation. We describe steps for subjecting 2D cultures to a neural fate and subsequently developing hCOs. We then detail the evaluation of different cellular types. For complete details on the use and execution of this protocol, please refer to González-Sastre et al..
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