they form synapses themselves
As we mammals age, many of us begin to lose our sight as the neurons in our retinas degenerate. Our retinal ganglion cells can be attacked by glaucoma or our rods and cones (photoreceptors) can be damaged by macular degeneration or retinitis pigmentosa. Somewhere during evolution we lost the ability to regenerate such cells, just as we lost the ability to regenerate limbs. Once they’re gone, they’re gone.
But we humans have developed some other things very well: the ability to use reason and the desire to sustain ourselves. And these traits brought us to the brink of compensating for some of our evolutionary deficiencies.
It’s amazing enough that we can now grow human stem cells into retinal “organoids” — small balls that contain all the different types of cells that are needed to create a functional retina, even organized into the right layers.
But now we have learned that if we break the organoid into individual cells, these cells are able to spontaneously form connections to communicate signals (synapses) with other retinal cells. This means that a patient’s own stem cells could be grown into retinal cells and applied to their own retina, and the new cells could functionally replace the old ones, and vision could be restored. No gene therapy needed, thank you very much.
You can read all about this last hurdle smitten at the University of Wisconsin in the laboratories of Dr. David Gamm and Xinyu Zhao in magazine issue of January 4 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 react to light just like healthy ones. It’s a great development for creating individual cells for therapy, but to be part of a functioning retina, those rods and cones need to be able to transmit their signals to the rest of the retina. This happens through synapses, ultra-thin connections between neurons through which signal molecules pass (mainly glutamate) pass:
Retinal organoids (ROs) gave Gamma and Zhao hope that defective parts of the retina could be reconstituted as real ones from stem cells, because not only do all RO cells form the layers they should, but also make connections mutually within the RO with synapses. You can see how similar the RO structure is to the real retina in terms of cell types and synapses (colored green):
So the question is, if we break down these RO cells and apply the appropriate ones to the patient’s retina, will they be able to make these synapse connections again? That’s what the Gamm and Zhao labs set out to test here.
They broke some ROs with papain, an enzyme from papaya that is used as a meat tenderizer and aid in digestion, but also known destroy synapses. (So no injecting papain directly into the eyeballs, OK?)
After the papain treatment, they saw that the proteins that are important for the functioning of the synapses were fortunately still there, but they 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 just reorient themselves.
They grew these RO cells together as individuals for 20 days on a plate, in a situation similar to what they would encounter when applied to a real retina. But how can you tell if neurons have formed those tiny synapses and if those 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 functional synapses, so we can use it to find out not only whether synapses are present, but how well they work. (This seems like a good time to add the rabies virus to the very long, but still growing, list of things you shouldn’t inject your eyeballs with.)
The way this is done is really cool, and stick with me here because you’ll end up with some colorful photos that will make it pretty obvious what happened.
First we have to get the rabies virus to infect only a small percentage of our cells without searching the whole culture, and we also have to somehow label those cells as “starters”. So, first we have to set it up a bit.
We’ll start with another virus—a lentivirus—into which we’ve inserted a green fluorescent protein (GFP) gene that we’ve targeted to the nucleus. Then we will be able to see 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 into our lentivirus called TVA and Rgp, and in a moment we’ll see why they’re both important.
Then we’ll go ahead and infect our cells with the rabies virus, but we’ll change the gene for its coat protein. Normally it 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 this is exactly why we put TVA in our green dot cells. Now we can release the rabies virus into the culture and it will only infect the green dot cells.
We will put a mCherry (red fluorescent protein) gene into our rabies virus, so any cells infected with it will have a red color throughout the cell and it will be easy to spot the rabies infected cells. So all of our “starting” cells with a green dot will get rabies because they all have TVA, and that will turn our “starting” cells into festive red and green.
Recall that we also put the Rgp gene into our lentivirus, so our green dot cells also produce the Rgp protein. Once the rabies virus infects our green dot cells, they will regain their original hair protein, go back to their old self and… ohhhhhhh.
Now about 5% of our cells are red-green “starter” cells and they can infect other cells in 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—that is, rabies-infected cells that were not the original cells. bam! Here is your visualization, 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 initial cells should not be able to infect any other cells, because rabies will not have its normal coat protein. All we should see are the starting cells, colored red and green.
So the small graphic on the left below shows red-green seed cells that cannot infect other cells, even if there are active synapses. The bluer images on the left have an additional stain called DAPI, which detects DNA in blue, so every cell will be blue. In this way, you can visualize the percentage of cells that are infected as initial cells. Then on the right 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, let’s do a real test now, where Rgp is included in the lentivirus, so now the rabies virus can infect other cells, but only through active synapses. Same deal with colors, and now we hope to see only red neurons:
We see a lot of rabies infections of non-starting cells, which means we have active synapses! And that means we are to clinical trials!
“We’ve been piecing this story together in the lab, piece by piece, to build confidence that we’re going in the right direction,” says Gamm, who patented the organoids and co-founded Madison-based Opsis Therapeutics. which is adapting technology to treat human eye disorders based on UW-Madison discoveries. “All of this leads, ultimately, 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 types of retinal cells forming synapses were photoreceptors—rods and cones—which are lost in diseases such as retinitis pigmentosa and age-related macular degeneration. as in certain eye injuries. The next most common cell type, retinal ganglion cells, degenerate in optic nerve disorders such as glaucoma.
“That was an important discovery for us,” says Gamm. “It really shows the potentially broad impact that these retinal organoids could have.”