Three blind mice no longer

Have you ever seen such a thing in your life as three blind mice having cells transplanted into their eyes, reforming and helping them regain their sight? Researchers from the University of Oxford have seen exactly that.

The study was led by Professor Robert MacLaren in the Nuffield Department of Clinical Neurosciences at the University of Oxford, together with Dr Mandeep Singh, an eye surgeon from the National University Hospital of Singapore. The findings have been published online in Proceedings of the National Academy of Science.

Retinal degenerations, such as retinitis pigmentosa (RP), involve a progressive loss of photoreceptor cells from the outer nuclear layer (ONL). The researchers already knew that the downstream circuitry of the human inner retina could be reactivated long after photoreceptors were lost, as has been demonstrated through sub-retinal stimulation in blind patients. So a new treatment was sought - photoreceptor transplantation via cell therapy.

The cells to be transplanted were rod precursor cells - that is, cells destined to become rod photoreceptors. Such cells have, in previous studies, been generated through embryonic stem cell culture and implanted into the retina - however, these experiments took place during early degeneration, when the ONL was still relatively well formed. The Oxford study took place with almost no ONL left, and would require the precursors to, according to the researchers:

“… resume their developmental program to form a polarised ONL after transplantation, with the formation of light-sensitive outer segments (OS) containing enzymes critical for light sensitivity. Furthermore, the cells would need to reconnect synaptically with residual downstream retinal neurons for light-evoked signals to be relayed to central targets.”

To test whether a functional ONL could be reconstructed, the researchers used a group of mice, aged 10-12 weeks, whose rods had been dead since they were three weeks old. Another group of mice would donate their rod precursors, which had been filled with green fluorescent protein (GFP) expression so the researchers could track them after transplantation into the host. There was also a ‘sham’ group of mice who received retinal cells harvested from rod-free donors in order to test for the potential effects of other neuronal and non-neuronal cells present in the precursors.

“Two weeks after transplantation,” the researchers stated, “we found numerous GFP-positive donor cells appropriately interposed between the host inner nuclear layer (INL) and retinal pigment epithelium (RPE), recreating a new ONL up to 10 cells thick in places.”

After confirming that the precursors had not only survived transplantation but also followed normal development, elaborated OS and connected with the host, the researchers tested their effect on the mice. First they used pupil light response (PLR), ie, a stimulus of light in the pupil which would prove “not only the existence of light-sensing OS but also that efferent connections existed to central targets.” Before treatment the mice had measured a pupil size of 66.3% after the stimulus; after treatment this went down to 30.9% (in comparison, the sham group’s pupils decreased by around 12.8%). Furthermore, laser speckle imaging showed blood-flow changes in the visual cortices of precursor-transplanted mice that were not seen in the sham group.

Finally, the two groups of mice were placed in a dimly lit chamber which was connected to a dark chamber. Since mice are nocturnal, they were expected to make their way into the darkness. One mouse from the sham group simply moved around in circles, not recognising one area from the next and spending 87.3% of the 10 minute trial in the light. The test mouse, on the other hand, quickly fled to the dark and spent only 46.3% of its time in the light. Videos of the tests can be viewed here and here.

With the mouse model standing in for humans suffering from complete outer retinal degeneration, the researchers hope that their technique can be replicated in human patients using induced pluripotent stem (iPS) cells. These are stem cells that have been generated from the patient’s own cells, such as skin or blood cells, which can then be directed to form precursors of the retina cells. Professor MacLaren says that this has been achieved by others; the next step is to find a reliable source of cells in patients that can provide the stem cells for use in such transplants.

“Our study shows what we could achieve with a cell-based approach,” said Professor MacLaren. “We have shown the transplanted cells survive, they become light sensitive, and they connect and reform the wiring to the rest of the retina to restore vision.

“The ability to reconstruct the entire light-sensitive layer of the retina using cell transplantation is the ultimate goal of the stem cell treatments for blindness we are all working towards.”