Multiphoton microscopy enables insights that promise regenerative medicine treatment for blindness

Seeking insight into organogenesis, researchers in Japan have demonstrated that mouse embryonic stem cells (ESCs) are able to differentiate and assemble into an optic cup—and then give rise to a tissue exhibiting the stratified structure characteristic of the retina in-vivo. A study published in Nature describes how the scientists used a 3-D tissue culture system to demonstrate not only this self-organizing capacity of pluripotent stem cells, but the underlying cell dynamics as well. Multiphoton microscopy was key to understanding the mechanisms behind this discovery—which is promising for regenerative medicine approaches to treating loss of eyesight.

Mototsugu Eiraku, deputy leader of the Four-Dimensional Tissue Analysis Unit at the Riken Center for Developmental Biology at the Kobe Institute (Kobe, Japan) and colleagues in the Laboratory for Neurogenesis and Organogenesis led by Yoshiki Sasai built on techniques and findings emerging from the use of the serum-free culture of embryoid body-like aggregates (SFEBq) ESC culture system developed by Sasai's lab, which had previously been used to differentiate these pluripotent stem cells into a wide range of neuronal cell types, including structurally organized cerebral cortical neurons. By adding extracellular matrix proteins to the SFEBq medium, the group was able to epithelially organize retinal precursors at high efficiencies by day 7 of culture. One day later, optic vesicle-like structures 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, indicating that optic cup development had been recapitulated in-vitro and in the absence of any external signaling sources, such as from the lens, demonstrating the capacity for self-organization.

They next used multiphoton microscopy to explore the mechanisms behind this process of self-assembly in 3-D. They found that after the ESC-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 in 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 ESC-derived optic cup and allowed it to develop in a 3-D 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.

"What we've been able to do in this study is resolve a nearly century-old problem in embryology, by showing that retinal precursors have the inherent ability to give rise to the complex structure of the optic cup," says Sasai. "It's exciting to think that we are now well on the way to becoming able to generate not only differentiated cell types, but organized tissues from ES and iPS cells, which may open new avenues toward applications in regenerative medicine."

Potential applications include regenerative medicine approaches to the treatment of retinal degenerative disorders, such as retinitis pigmentosa.

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Posted by Lee Mather

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