To investigate the utility of such a system, we created scaffold free three-dimensional (3D) human neural organoids, from human iPSCs derived from AD patients and healthy controls and test each line for pluripotency (S1 Fig). Several protocols have been developed to create neural organoids from human pluripotent stem cells [53,64–67]. We followed the protocol published by Kadoshima et al. [64] with minor modifications (see Methods) and successfully created complex dense 3D neural tissues from a number of human iPSC lines from AD patients and healthy control (S2A Fig and S1 Table). We subjected the 3D cultures, heretofore referred to as organoids, to immunohistochemistry with antibodies against a neuronal protein (MAP2) as well as SOX2, a marker for neural progenitor cells, to determine the presence of neural cell types. After one month of culture, we observed the emergence of translucent regions of neuroectoderm and immunolabeling for neuronal MAP2 as well as SOX2-positive neural progenitor cells, as previously described [64] (S2 Fig). At 60 days (60d) and 90–100 days (90d) in culture, the organoids demonstrated formation of a rolling morphology structure with
