In vitro factories for producing replacement photoreceptors in eyes with retinal degenerative disorders are becoming an increasingly realistic possibility

Roibeard O’hEineachain

Posted: Wednesday, July 19, 2017

In vitro factories for producing replacement photoreceptors in eyes with retinal degenerative disorders are becoming an increasingly realistic possibility as stem cell technology continues to advance, according to presentations at the Retina 2016 meeting held in Dublin, Ireland.
The potential of the new technologies extends to several aspects of retinal disease. Apart from a cell replacement therapy in the more distant future, in the shorter-term stem cells and stem cell-derived cultures may serve as a means of testing drugs and gene therapy in vitro, said David Gamm MD, PhD, Director of the McPherson Eye Research Institute at the University of Wisconsin, Madison, Wisconsin, USA.
“Ultimately, all the applications of stem cell technology we can conceive of are predicated upon our ability to understand and perhaps even manipulate the developmental processes that occur in a dish. But what must first be ensured is the authenticity of the differentiated cell or tissue products,” he added.
Already retinal pigment epithelium (RPE) stem cell cultures are being used in clinical trials. However, compared to photoreceptors, RPE stem cells may be cultured, purified and manipulated with relative ease. In addition, unlike cultured RPE stem cells, photoreceptors mature in an environment that also contains other neuroretinal cell types, Dr Gamm said.
Originally, many centres used two-dimensional plated protocols with either embryonic or human induced pluripotent stem cells (hiPSCs). However, cells cultured in this way failed to form distinct layers and also often contained undesired cell types.

Subsequently, Dr Gamm and others developed three-dimensional (3D) protocols where aggregates of embryonic cells or hiPSCs were started on a culture plate and then transferred to a suspension, or kept suspended throughout. That eliminated many unwanted cells, and research has shown that neuronal tissues grow better in 3D culture medium.
Using this approach, and other additional steps, they found that they could induce hiPSCs to differentiate into neuroretinal cells in a spherical layered configuration, resembling the natural retina. These layered structures are sometimes referred to as optic vesicle structures, or retinal organoids.
“These structures not only produce all major neuroretinal cell classes, but do so in a conserved spatio-temporal manner with the capacity to generate laminated neuroretinal tissue,” Dr Gamm explained.
He noted that the advantages of the 3D hiPSC approach for neural retinal culture include greater neural retinal cell enrichment and the generation of tissue-like structures with advanced cell morphology and function.
However, the 3D approach at present is a cumbersome process requiring significant culture manipulation. In addition, scaling up the process remains a challenge to be met if the technology is to be used on a wide scale in the clinic or clinical laboratory, he said.

Daytime vision is highly dependent on cone photoreceptors. Retinal degeneration resulting in their loss is a leading cause of blindness, said Robin Ali PhD, FMedSci, University College London, UK.
A potential regenerative strategy for many forms of retinal diseases is the replacement of lost cone receptors by cell transplantation.
He noted that the subretinal space is very conducive to such an approach because it is a natural anatomical space and is accessible to surgery. In addition, the blood-brain barrier provides it with some protection from the host immune system.
Preliminary clinical trials involving transplanted human embryonic stem cell-derived RPE have so far shown safety and limited efficacy. Successful RPE stem cell transplantation requires only the formation of a functional monolayer of cells. Photoreceptor transplantation has the additional requirement of functional connectivity. Research is showing that this is feasible, Dr Ali said.
For several years, Dr Ali and his associates have been characterising and optimising cone differentiation in cells from mouse and human embryonic stem cells (hESCs). They have been able to culture photoreceptors exhibiting cone-specific phototransduction-related proteins. In 2006 they demonstrated that effective photoreceptor transplantation is possible in adult mice, provided the photoreceptor precursors are at a very specific stage of development.
“Although the effect was slight, it was an important proof-of concept. This knowledge might be used to generate appropriate cells for transplantation from stem cells,” he said.

An additional discovery they have made is that much of the improvement in vision reported so far in laboratory animals has not been primarily due to integration of the cone photoreceptors, but instead appears to result from a transfer of RNA and/or protein material between the implanted cells and the remaining host cone photoreceptors.
However, a percentage of transplanted cells did integrate with the surrounding cells and expressed biomarkers for phototransduction. Dr Ali and his associates are investigating whether implantation at later precursor stages of cone cell development might yield a higher rate of integration.
“The exciting thing is that we are now able to do a lot of experiments with a purified population of human cones. There is still a lot of work before we can go to a clinical trial but we are now defining a protocol to generate clinical grade cell lines and we are starting to work with larger animals. We expect clinical testing to begin in around five years,” Dr Ali added.

David Gamm:
Robin Ali:

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