3D Reconstitution of the Patterned Neural Tube from Embryonic Stem Cells

Abstract

SummaryInducing organogenesis in 3D culture is an important aspect of stem cell research. Anterior neural structures have been produced from large embryonic stem cell (ESC) aggregates, but the steps involved in patterning such complex structures have been ill defined, as embryoid bodies typically contained many cell types. Here we show that single mouse ESCs directly embedded in Matrigel or defined synthetic matrices under neural induction conditions can clonally form neuroepithelial cysts containing a single lumen in 3D. Untreated cysts were uniformly dorsal and could be ventralized to floor plate (FP). Retinoic acid posteriorized cysts to cervical levels and induced localize FP formation yielding full patterning along the dorsal/ventral (DV) axis. Correct spatial organization of motor neurons, interneurons, and dorsal interneurons along the DV axis was observed. This system serves as a valuable tool for studying morphogen action in 3D and as a source of patterned spinal cord tissue.

Neural tube morphogenesis in synthetic 3D microenvironments

“…To begin to understand how molecular signals and the extracellular microenvironment coordinate to form early structures of the central nervous system (CNS), we have begun to generate organoids with defining features of developing spinal cord (8). Our previous work with Matrigel has shown that single mouse ESCs could form apical-basal neuroepithelial cysts containing single lumens (8), and that retinoic acid (RA) could induce such cysts to yield patterning along a dorsal-ventral (DV) axis recalling the spatial organization found in the developing neural tube.…”

“…Our previous work with Matrigel has shown that single mouse ESCs could form apical-basal neuroepithelial cysts containing single lumens (8), and that retinoic acid (RA) could induce such cysts to yield patterning along a dorsal-ventral (DV) axis recalling the spatial organization found in the developing neural tube. Although some studies with synthetic PEG matrices have suggested that adhesion ligands play an important role in establishing apical-basal polarity in model systems of epithelial morphogenesis (18), it has remained unresolved which aspects of Matrigel contribute to this spatial organization, and in particular the role of extracellular microenvironment in establishing complex morphogenesis.…”

“…In vitro approaches have attempted to recapitulate key features of these processes, and it has now become possible to generate an increasing variety of self-organizing multicellular tissue constructs, termed organoids, that recreate features of such diverse organs as intestine, liver, brain, and spinal cord (5)(6)(7)(8). Although important aspects of the 3D in vivo organization have been recapitulated in these organoid systems, such studies have been exclusively performed in Matrigel, a poorly defined, largely proteinaceous mixture whose properties cannot be readily modulated.…”

Deriving, regenerating, and engineering CNS tissues using human pluripotent stem cells

“…In theory, platforms that instruct R/C and D/V morphogenetic patterning of developing organoids by exogenous application of morphogen gradients will also be needed. However, recent work describing spontaneous D/V patterning in mouse ESC-derived cysts may indicate that this is not necessary [56]. Regardless, intimate integration of tissue engineering methodologies with organoid derivation protocols will be needed to advance towards instructed organoid morphogenesis and create next generation CNS in vitro models and potentially even organ transplants.…”

Three‐Dimensional Models of the Human Brain Development and Diseases

“…Using this system, neuroepithelial cysts could be reconstituted from mouse embryonic stem cells directly embedded in 3D culture. [199] Three different 3D cell culture systems were compared: (i) Matrigel, (ii) laminin/entactin, and (iii) synthetic PEG hydrogel. ECM proteins improved cyst-forming ability – although they were not required to generate lumen-containing neuroepithelial cysts in the 3D culture system.…”