AsianScientist (Apr. 7, 2011) – A new report describes how mouse embryonic stem cells (ESCs) are able to differentiate and assemble into an optic cup, capable of giving rise to a tissue exhibiting the stratified structure characteristic of the retina in vivo.
Published in Nature, the study used a cutting-edge three-dimensional tissue culture system not only to demonstrate this self-organizing capacity of pluripotent stem cells, but the underlying cell dynamics as well.
The mechanistic basis for the formation of the optic cup, with its complex two-walled structure, has been a longstanding question in embryology.
The development of the retina has generally been thought to be triggered by chemical and physical influences from other tissues, such as the lens or cornea, but some, including the father of experimental embryology, Hans Spemann, have suggested that external induction or force is not necessary.
To resolve this question, Mototsugu Eiraku, deputy leader of the Four-dimensional Tissue Analysis Unit, together with colleagues in the Laboratory for Neurogenesis and Organogenesis, RIKEN, Kyoto University and Osaka University, built on a series of techniques and findings emerging from the use of a SFEBq (serum-free culture of embryoid body–like aggregates) ES cell culture system.
By adding extracellular matrix proteins to the SFEBq medium, the group was able to derive epithelially-organized retinal precursors at high efficiencies by day 7 of culture. One day later, optic vesicle-like structure began to form, followed by bi-layered optic cup-like structures by day 10. The pigmented and neuronal character of the outer and inner layers of cells in these spontaneously formed tissues were confirmed by gene expression, and importantly – in the absence of lens or corneal tissue – demonstrating the capacity for self-organization.
They next used multi-photon microscopy to explore the mechanisms behind this process of self-assembly in 3D. They found that after the ES cell-derived retinal precursors differentiated into pigmented epithelial and neuronal layers, the tissue underwent a four step morphological rearrangement on its way to assuming the optic cup structure. When they examined cytoskeletal behaviors in this process, they noted that myosin activity dropped in the region of the epithelium that bend inward to form the cup, giving the flexibility needed to form a pocket driven by expansion of the epithelium through cell division.
Computer simulation of the mechanics behind this revealed that three principal forces can explain the optic cup-forming event. First, the a region of the epithelium must lose rigidity, allowing it to buckle inward, after which cells at the hinge points (defined by the border between presumptive pigment epithelium and neuronal regions) must undergo apical constriction, giving them a wedge-like shape. Once these conditions are met, expansion of the tissue surface by cell division results further involution of the cup, all of which are very much in line with the experimental findings.
As a final test of the in vitro structure’s ability to mirror its embryonic counterpart, Eiraku excised the neuronal layer from the ES cell-derived optic cup and allowed it to develop in 3D cell culture under conditions optimized for spurring neuronal maturation. He found that the retinal neurons underwent active mitosis and ultimately organized into a six-layer stratified and synapse-forming neuronal structure closely resembling that of the post-natal retina.
Potential applications include regenerative medicine approaches to the treatment of retinal degenerative disorders, such as retinitis pigmentosa.
The article can be found at: Eiraku M. et al. (2011) Self-organizing optic-cup morphogenesis in three-dimensional culture.
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Source: RIKEN.
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