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As we mammals age, many of us begin to lose our vision because the neurons in our retinas degenerate. Our retinal ganglion cells can be attacked by glaucoma, or our rods and cones (photoreceptors) can be eroded by macular degeneration or retinitis pigmentosa. Somewhere in the course of evolution we lost the ability to regenerate these types of cells, just as we lost the ability to regenerate limbs. If they’re gone, they’re gone.
But we humans did develop some other things really well: the ability to use reason and the desire to support ourselves. And these qualities have brought us to the brink of compensating for some of our evolutionary shortcomings.
That’s amazing enough we can now grow human stem cells into retinal “organoids” — tiny balls that contain all the different types of cells that are needed to make a functioning retina even organized into the correct layers.
But now we have learned that if we divide the organelle into individual cells, these cells are able to spontaneously form connections to transmit a signal (synapses) with other retinal cells. This means that a patient can have their own stem cells grown into retinal cells and applied to their own retina, these new cells can functionally replace the old ones and vision can be restored. No gene therapy needed, thank you very much.
You can read all about this final hurdle overcome in the laboratories of the University of Wisconsin Drs. David Gam and Xinyu Zhao c the January 4 edition of Proceedings of the National Academy of Sciences.
Just last year, Gamm’s lab had shown that rods and cones (photoreceptors) made from stem cells can respond to light just like healthy ones. This is a great development for creating individual cells for therapy, but to be part of a functional retina, these rods and cones must be able to transmit their signals to the rest of the retina. This happens through synapses, ultrathin connections between neurons through which signaling molecules (mainly glutamate) are transmitted:
Retinal organoids (ROs) gave Gamm and Zhao hope that defective parts of the retina could be restored for real by stem cells, because not only did all RO cells form the layers they were supposed to, but also make connections to each other inside the RO with synapses. You can see how similar the RO structure is to a real retina in terms of cell types and synapses (colored in green):
So the question is, if we break up these RO cells and apply the appropriate ones to the patient’s retina, will they be able to rewire these synaptic connections? That’s what the Gamm and Zhao labs set out to test here.
They spiked some ROs with papain, which is an enzyme from papaya used as a meat tenderizer and digestive aid, but also famous to destroy synapses. (So no injecting papain directly into the eyeballs, right?)
After the papain treatment, they saw that the proteins that are important for the function of the synapse were fortunately still there, but had somehow moved back into the cells. So it seemed that the cells would have a good chance of re-establishing synapses with each other if they could reorient themselves.
They cultured these RO cells together as individuals for 20 days on a dish, in a situation similar to what they would encounter when applied to a real retina. But how can you tell if neurons have formed these tiny synapses and that these synapses are functioning?
Fortunately, there’s a clever way to do this called “synaptic tracking.” It turns out that the rabies virus can be transmitted between neurons only through functioning synapses, so we can use it to understand not only whether there are synapses, but how well they are working. (Feels like a good time to add the rabies virus to the very long and still growing list of things you shouldn’t inject into your eyeballs.)
The way this is done is very cool and stick with me here because you’ll end up with some colorful pictures that will make it pretty obvious what happened.
We first need to get the rabies virus to infect only a small percentage of our cells without scouring the entire culture, and we also need to mark these cells as “starters” in some way. So first we need to do a little setup.
We’ll start with a different virus—a lentivirus—into which we’ve inserted a gene for green fluorescent protein (GFP) that we’ve targeted to the nucleus. Then we will be able to spot all the cells that are infected with our lentivirus because they will have a big green dot in the center. We can do some trial and error with the amount of lentivirus we use so that we end up with about 5% of our cells infected.
We’re going to put two other genes in our lentivirus, called TVA and Rgp, and we’ll see why they’re both important in just a second.
Then we’ll go ahead and infect our cells with the rabies virus, but change the gene for its coat protein. Normally this is Rgp, but we will replace it with another one called Env. Viruses that use Env as their envelope proteins can only infect cells that have TVA, and that’s exactly why we just put TVA in our cells with a green dot. Now we can drop the rabies virus onto the culture and it will only infect the cells with green dots.
We will insert a gene for mCherry (a red fluorescent protein) into our rabies virus so that all cells infected with it will have a red color throughout the cell and it will be easy to spot rabies infected cells. So all of our “start” cells with a green dot will get rabies because they all have TVA and that will turn our “start” cells into festive red and green.
Recall that we had also placed the Rgp gene in our lentivirus, so our cells with green dots also produced Rgp protein. Once the rabies virus infects our green dot cells, they will regain their original coat protein, revert to their old selves, and… ohhhhhh.
So now about 5% of our cells are red-green “starter” cells and they can infect other cells in the culture with rabies (and turn them red) only if they are connected to other cells by working synapses! If this happens, we should see red cells without a green dot – ie. rabies-infected cells that are not starter cells. Bam! Here’s the preview, now let’s get down to it…
A nice control to start with is the whole system we just talked about, but without Rgp in the lentivirus. This means that the starter cells should not be able to infect other cells because the rabies will not have its normal coat protein. All we need to see are starter cells colored red and green.
So the small graph on the left below shows red and green starter cells that cannot infect other cells even if there are active synapses. The bluer pictures on the left have an extra stain called DAPI that detects DNA in blue, so each cell will show up in blue. This way you can visualize the percentage of cells that were infected as starter cells. Then on the right side we get rid of the blue DAPI so you only see red and green. Notice that everyone who is red also has a green dot.
OK, now let’s do the real test where Rgp is incorporated into the lentivirus so now the rabies virus can infect other cells but only through active synapses. Same deal for colors, and now we hope to see some red-only neurons:
We see a lot of rabies infection on non-starter cells, which means we have active synapses! And that means we are to clinical trials!
“We developed this story together in the lab, piece by piece, to build confidence that we were headed in the right direction,” says Gamm, who patented the organoids and co-founded Madison-based Opsis Therapeutics, which is adapting the technology for human treatments eye diseases based on the UW-Madison findings. “Ultimately, all of this leads to human clinical trials, which are the clear next step.”
After confirming the presence of synaptic connections, the researchers analyzed the cells involved and found that the most common retinal cell types forming synapses are photoreceptors—rods and cones—which are lost in diseases such as retinitis pigmentosa and age-related macular degeneration. as well as some eye injuries. The next most common cell type, retinal ganglion cells, degenerate in optic nerve diseases such as glaucoma.
“This was a major revelation for us,” Gam says. “This really shows the potentially broad impact that these retinal organelles can have.”