Dispatches from ARVO: Day 5

May 6, 2011

By Jennifer Phillips, Ph.D.

The last day of the ARVO meeting was short and sweet, and the very last presentation I saw before heading to the Ft. Lauderdale airport was the one I'm choosing to recap here. The talk was by Hari Jayaram of the University College London's Institute of Ophthalmology , who described a collaborative research project in which cultured human retinal cells were implanted into a rat model of retinal degeneration. At first glance it might sound like the premise of a Mad Scientist thriller, but it was actually quite a well-designed and relevant study. Here's an overview of the experimental rationale, set-up, and outcome:

Main goals of the research: The primary task was to optimize methods of culturing a type o f human retinal cells that had the ability to develop into photoreceptor cells. The subsequent goal was to actually implant these cells into a rat model of retinal degeneration and observe the cellular activity and the effects, if any, on the visual function of the rat.

Methods of achieving the first experiment: Müller Glial Cells (MGCs) were isolated from donor human retinas and cultured with the addition of various growth factors and other molecules. The researchers were seeking to find the best 'cocktail' of ingredients that would stimulate the MGCs to do what they are known to do in intact, living retinas under certain conditions, specifically to de-differentiate and divide to create a population of cells with the potential to become other retinal cells. In this case the goal was to influence these new "cells with potential" to begin developing into rod photoreceptor cells.

The challenge in such an experiment is not only to find the right ingredients to add, but also the right stage of cell specification to achieve. Highly differentiated cells, e.g. fully formed photoreceptors, tend not to be very happy in cell culture, and usually die off pretty quickly. Minimally differentiated cells can live quite happily in cell culture for an amazingly long time, but for the purposes of transplantation, these cells may not have sufficient guidance (by way of internal and external molecular cues) to become the cell type of the researcher's choosing.

Results of the first experiment: In addition to being able to culture MGCs in a relatively undifferentiated (read: having the potential to become a number of different cell types) state, the investigators also hit upon a successful combination of growth factors and molecules known to stimulate rod photoreceptor development. They confirmed their success by analyzing the molecules being produced within the differentiated cultured cells and found that they were churning out molecular markers of rod precursor cells (RPCs)-specialized enough to know to develop into rods, but not so specialized that they would become unhealthy before transplantation time.

Methods of achieving the second experiment: The investigators selected a rat model of retinal degeneration, known as the P23H rat, bearing a mutation in rhodopsin, the light-sensitive protein found in rod outer segments. In both rats and humans this mutation causes RP due to rod photoreceptors death. Unlike the Mertk mouse model described in yesterday's post , in which photoreceptors die because of a protein dysfunction in the neighboring RPE cells, the retinal degeneration in the P23H rat is due to a defect in photoreceptors themselves. This is important because the researchers needed to be sure they were targeting the right population of cells, replacing the particular cell types that were defective, namely rod photoreceptors.

P23H rat retinas received transplants of either the MGC cultures or the RPC cultures. Each rat in the experiment received cells in only one eye, with the other eye remaining untreated for use as an experimental control. The researchers waited 3 weeks, during which time the rats received drugs to suppress their immune response so that they wouldn't reject the donor tissue. After this waiting period, visual function was analyzed by ERG, and retinal tissue was examined to evaluate the growth and development of the implanted cells.

Results of the second experiment: When the researchers observed the retinal tissues of the rats that received the "undifferentiated" MGCs, The researchers observed that these cells had incorporated into the retinal layer usually inhabited by MGCs and had developed an MGC-like morphology. These cells send processes throughout all the other cells layers of the retina, and have a very distinctive shape.

In the retinas of rats receiving "differentiated" RPCs, the investigators reported that these cells incorporated into the photoreceptor cell layer of the retina. No outer segments appeared to form in these cells, suggesting that they did not complete the process of becoming mature rod cells. However, they did form connections with other retinal neurons, which demonstrates the extent to which they were incorporated into the existing retinal structure. The researchers also determined that these cells were producing proteins exclusive to rod cells. So, although the transformation from RPC to fully specialized rod cell was incomplete, the cells did display a "rod-like" molecular profile and cellular behavior.

Visual function was tested by measuring the ERG response to various light intensities. In the eyes treated with RPCs, the investigators detected a greater photoreceptor response to bright light compared to controls. Even without evidence of forming outer segments-the part of the rod cells where rhodopsin functions, the inclusion of RPC-derived cells resulted in a boost in visual function. Interestingly, researchers saw a less dramatic, but still significant increase in photoreceptor response to bright light in the eyes containing undifferentiated MGC transplants. No evidence of additional rod or rod-like cells originating from the transplanted human cells was detected in these eyes by histology, yet the presence of the new Müller-like cells seemed to improve function nonetheless.

Conclusions: These experiments have clear implications for future work toward cell-replacement therapy for photoreceptor degeneration. The experimental design might seem unusual, particularly the idea of putting human cells into a rat retina, but it was important to ascertain how human retinal cells might behave in a complex, living system. Clearly, there were some limitations to this experiment-the cell differentiation was incomplete, in that neither the MGC nor the RPC transplants appeared to mature to 100% fully functional cell types. This may indicate that the cell culture conditions need to be optimized further, but it may also be due to the limitations of cross-species transplants. As you might imagine, human retinal cells are somewhat larger than their rat counterparts, so there may have been some physical limitations to cell growth and differentiation.

Although further optimization and additional testing in animal models will be required before this treatment is ready for clinical trials, this work will likely make a significant contribution to the field of retinal cell replacement.

So, as I wrap up my final report from ARVO, 20,000 feet above Middle America, I can say unreservedly that this is one of the best, most diverse, career enriching meetings around. I'm full of ideas for my own experiments, and I'm already looking forward to the 2012 Annual Meeting.

Jen's final ARVO stats, by the numbers: