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

Abstract

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.

In Vitro Modeling of Tissue-Specific 3D Microenvironments and Possibile Application to Pediatric Cancer Research

“…Normal brain cells were isolated from cortices of Wistar rats and allowed to reaggregate and to mature for 20 days, by placing them on a semi-porous membrane at an air-liquid interface, and subsequently generating characteristic brain HiSpots [220,221]. Histological analysis showed that both neurons and astrocytes were present in these 3D cultures, with a preserved physiological architecture.…”

Design and prototyping of a chip-based multi-micro-organoid culture system for substance testing, predictive to human (substance) exposure

“…The use of tissue slices from each cellular layer of the cerebellum, placed horizontally, was proposed for the architectural design of the neuronal segment. Only rare cases of human neuronal tissue culture, containing neurons and all necessary kinds of glia cells, are described (Preynat-Seauve et al, 2009). Channels for the growth and proper connection of the axons of the different layers were provided to restore the destroyed interconnections between the layers caused by this procedure.…”

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology

“…these do not allow the study of drug transport, but may allow the examination of toxicant effects on the blood-brain barrier structure (Al Ahmad et al, 2011). A variant of the membrane technique is the formation of 3D tissue at an air liquid interphase (DuboisDauphin et al, 2010;Preynat-Seauve et al, 2009). Alternatively, cells may be grown in artificial scaffolds to promote threedimensionality (lu et al, 2012a).…”