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Researches on neural differentiation using embryonic stem cells (ESC) require analysis of neurogenesis in conditions mimicking physiological cellular interactions as closely as possible. In this study, we report an air‐liquid interface‐based culture of human ESC. This culture system allows three‐dimensional cell expansion and neural differentiation in the absence of added growth factors. Over a 3‐month period, a macroscopically visible, compact tissue developed. Histological coloration revealed a dense neural‐like neural tissue including immature tubular structures. Electron microscopy, immunochemistry, and electrophysiological recordings demonstrated a dense network of neurons, astrocytes, and oligodendrocytes able to propagate signals. Within this tissue, tubular structures were niches of cells resembling germinal layers of human fetal brain. Indeed, the tissue contained abundant proliferating cells expressing markers of neural progenitors. Finally, the capacity to generate neural tissues on air‐liquid interface differed for different ESC lines, confirming variations of their neurogenic potential. In conclusion, this study demonstrates in vitro engineering of a human neural‐like tissue with an organization that bears resemblance to early developing brain. As opposed to previously described methods, this differentiation (a) allows three‐dimensional organization, (b) yields dense interconnected neural tissue with structurally and functionally distinct areas, and (c) is spontaneously guided by endogenous developmental cues. S TEM  C ELLS   2009;27:509–520

Development of Human Nervous Tissue upon Differentiation of Embryonic Stem Cells in Three‐Dimensional Culture