The following is a transcript from a discussion with Thomas A. Reh, PhD, Professor of Biological Structure and Principle Investigator examining Retinal Development and Regeneration, joined by Catherine Ray, Reh Lab Project Manager. The objective of the discussion was to learn details about the research the laboratory will be conducting and what resources the WaNPRC would be providing to support it. They were also joined on the phone by WaNPRC administrator of program operations Jesse Day. The project we discussed is pursuing regenerative therapy for glaucoma which is one of the National Eye Institute Audacious Goals.
Catherine Ray: First group of six animals is a cohort to set up the glaucoma induction model.
Tom Reh: There are going to be two phases. There is a guy at Vanderbilt, David Calkins, who’s been working on a glaucoma model in squirrel monkey (Saimiri sciureus). That was the idea was that we would go learn from him how to do it. And then we would bring that surgical technique back here. So that is the first phase.
CR: We’re not quite sure at this point what the training will entail.
TR: Our surgeon, Jen Chao, is in charge of doing that. She’s an MD, PhD in the Department of Ophthalmology here (at the University of Washington). She will be responsible for learning about the actual injection. Basically, the procedure is fairly straightforward. Glaucoma is normally caused by an increase in intraocular pressure. This is basically pressure within your eyeball. And that is partially due to either a congenital or acquired blockage of the aqueous humor’s (the clear fluid filling the space in the front of the eyeball) outflow tract. So your eye’s constantly making new fluid and that fluid is supposed to leave the eye through this outflow tract. When that is blocked or constricted, often people have really strong myopia (nearsightedness). They have problems with that because the eyeball is of a shape that it constricts that.
But anyone can get glaucoma, if you have intraocular pressure increase, and then what happens is the cells in the retina die. A certain class of cells degenerate. These are the ganglion cells. As so what you do when you try to induce this is that you actually inject into the anterior part of the eye, where the aqueous humor is located- little microbeads and then those actually plug up the tract. And so now because the tract is plugged up by these little microbeads, the intraocular pressure goes up, creating glaucoma. It’s actually not that hard to do. Somebody figured out how to do this so it would work and they have done it already in a bunch of different animals, and Dr. Calkins does it in squirrel monkeys and it works. It takes a couple of months to get glaucoma. To get the pressure up and have it elevated over a long enough period of time to create the glaucoma. In mice you can do this and it happens much more quickly, but in monkeys it takes a longer period of time.
Induction of Glaucoma
TR: Project will be in two phases. The first will actually be giving the monkeys glaucoma. And so, the NHPs will be injected with tiny little microbeads into the anterior of the eye which block the output tract. This causes intraocular pressure. A group down in Vanderbilt led by David Calkins has worked this out.
It’s been done in a lot of other animals before but it hadn’t been done in squirrel monkey. He figured out a way to do it. Dr.Chao is a retinal surgeon on humans, and she will do the surgery on the monkeys. It involves injecting these beads into the anterior of the eye. She’s working with Vanderbilt and learn from David Calkins’ surgeon. She’ll then reproduce those injections here. And we we’re thinking that could go on in the first year. That’s what our timeline would be.
CR: Six animals would establish the model here in Seattle after Jen Chao learns from Vanderbilt to see how it’s done. Making sure that we could recapitulate it here.
TR: And so, I think we said six. Six would be ideal. We could do fewer than that. I think it’s gonna be simply reproducing what’s already been done at Vanderbilt. Other monkeys, we would do the next phase of the project, because we’re causing intraocular pressure cause the death of the retinal ganglion cells, which are the cells that convey the message from the eyes to the brain, those cells die from the intraocular pressure increase. And so, what we want to do is see if we can replace them. And so we can get human embryonic stem cells (ES Cells) to make ganglion cells pretty well. We have a human embryonic stem cell line that actually makes these ganglion cells red. Then we can track them with a fluorescent RetCam imaging, and so what we proposed for the next cohort of animals was actually to just try some of these transplants.
CR: Yeah, in the second part of the experiment. The first trial we’re just doing the cells with the glaucoma induction with another cohort. If that works, there are various other experiments that would involve keeping the monkeys for longer a term. We would collect eyes for electrophysiology, etc.
TR: We’ve done injections of human embryonic stem cells into monkeys before, which is one of the reasons why I think that the NIH reviewed our grant positively. I think we’re the only ones in the world who have injected human embryonic stem cells into squirrel monkeys, and one of only three groups to every inject them into any monkey’s eye. So, we reported that in a publication last year. Basically what we did was a sub-retinal injection of embryonic stem cells. It was several years ago when we did this. We don’t think we’re going to immunosuppress this time, but there will be local (intraocular) immunosuppression at the time of the surgery, because they are human cells going into a monkey.
We also proposed, as a worst case scenario, we would derive monkey cells, but we don’t have a squirrel monkey yet. So we’d have to almost make a squirrel monkey IPSL. I know Eliza Curnow here has embryonic stem cells from macaques. That one is closer than human. The other option is that we can engineer the cells to not have T-cell receptors, and they in theory would not get rejected. There is a company in Seattle, Universal Cells that’s doing that for potential human transplants.
So I think those are some options. But I think right now, we’ll probably keep it simple. So we hope the money comes within the next couple months. That’s the idea. There is going to be a regular review by the review panel advisory board for these U grants and we’re meeting with ours on February 5 at the NIH. After that we’ll nail down the timelines and milestones.
Over the next year, it will be six squirrel monkeys.
Nuts and Bolts
CR: We would want to purchase any monkeys until after that takes place. 36 over the course of three years. No more than 6 at a time.
TR: I am pretty sure that we can accomplish what we need to with less than that.
Jesse Day: Please keep me apprised of timelines as you know more.
CR: I had sat down, just really quickly with Animal Trainer Kelly Morrisroe, to talk about behavioral training and she brought this up as well. I don’t think we have a preference as far as sex goes. I think we’re supposed to be keeping it balanced for the NIH.
JD: Evenly distributed as much as possible (for validity).
CR: Based on availability, we don’t care about the sexes of the animals too much.
TR: House females together more so than males?
CR: Males could be fixed if necessary. I think the way the grant is written, these are all three to nine year olds. A little bit more mature age range.
TR: I think in terms of access to the surgery suite and anesthesiology, etc., the way we ran it last time was pretty straight forward. I guess we’ll have to book those sorts of things ahead of time. I remember that Jen was able to bring over a microscope and a RetCam imager from Ophthalmology. Is there an operating microscope available and any kind of device for intraocular imaging?
JD: I would bet that was their equipment that they brought in…. contact the surgery group. That was probably the last study to use it.
TR: I know the Neitz study was doing viral injections. Those are being done here in the I-Wing, correct?
JD: They’re are using the RetCam for that. Talk with the Neitz group about that.
TR: Ok, I think that would be a good idea, we can coordinate and piggy back on top. Again, Jen Chao does the injections for the Neitz group, so she can figure out where everything is. We have an EE protocol? Is that something that the Primate Center takes care of that?
We need to be able to take the intraocular pressure. Once they’re anesthetized, it’s difficult to do because the intraocular pressure declines so you can’t get an accurate reading. So it could be that we would need to train the animals to hold still while we give them a puff of air on the eye.
CR: I sat down and had a meeting with Kelly Morrisroe and she seemed to think that was feasible. According to her, we might even be able to pre-screen some of the monkeys to see if they might be predisposed to receiving behavioral training. That may be a bridge that we need to cross as we get a little bit closer to ordering the monkeys. It seems in terms of our timeline right now, it would be until February that we would even be ordering the animals.
TR: If that is something that your broker can screen animals for. One of the reasons that the Neitzes used squirrel monkeys was that you have this. The males don’t have color vision but the females do, and so by introducing another opsin pigment into the males, they were able to get them to see color.
The other attraction is their eyes have a fovea (a small depression in the retina of the eye where visual acuity is highest. The center of the field of vision is focused in this region, where retinal cones are particularly concentrated). But the animals as a whole aren’t as big as a macaque, which is appealing. The eyes are a good size for surgeons to do their manipulations. Part of why we’re doing this project, there aren’t that many animals that have foveas other than primates, so the work has to be done in nonhuman primates. Other animals can’t focus on the detail like humans and monkeys can. Part of what happens with glaucoma is you lose that sharp vision. So we are trying to restore that ability to see fine detail that is only found in primates.
Stanford is coordinating the project. Johns Hopkins is probably the best in the world at making the stem cell into the ganglion cells. Jeff Goldberg is one of the best people in the world for his studies on regeneration on ganglion cells in mice. Part of this was to also find ways to stimulate the ganglion cells to grow into the brain. It doesn’t do any good if they just stay in the eye. They have to grow accent all the way down the optic nerve, to the brain, which is a long way.
Rigor and Reproducibility
TR: I think the thing about reproducibility, is you gain a lot when three or four labs are involved, because essentially we’re going to be carrying out the same experiments at three different sites. And if we don’t get the same results, then we will have to figure out why that is.
I think part of the problem with reproducibility in science right now is that some of the conditions that make these experiments work are not completely defined because we’re always doing new things. It’s not like we’re doing the same things over and over again. Scientists basically do new things all the time, that’s what they do. So when you do new things, you don’t always know all of the variables that need to be controlled to get it to work. It just happened to work for you in that particular set of conditions and variables, etc. That’s particularly difficult when you talk about the small ends that you have. The small numbers of animals or experiments that you’re able to conduct in primates, and so I actually think in mice, you can do 30 mice and it’s no big deal. If someone does another study in 30 mice and they get a different result, you can figure out pretty quickly what those variables were from site to site. For large animals, you’re not gonna get that chance, so I think doing the experiments at the three sites at the same time, at least we’ll be able to identify the variables between sites as potential sources of irreproducibility. That’s one of the best things about this. It’ll allow for kind of instant reproducibility.
We’ve got some of the best labs in the world that are our collaborators so I figure they have been rigorous up to now.
CR: These are some of the only groups who have ever done this work before. I don’t think anyone else is doing this glaucoma induction model anywhere else.
TR: No, and so in a way, that’s going to validate that model right away. If we can’t reproduce Calkin’s model here, the project can’t move forward. That’s kind of high stakes reproducibility right there. Also remember that we have a scientific review board that’s basically going to look over our work every six months. They’re gonna ask, “How are you folks doing? What’s going wrong? What’s the progress?” t’s still exploratory science in that we really don’t know if these ganglion cells are going to survive the transplantation or not be rejected. Find the right layers. Make the right synaptic connections with their partners. Grow their accents in the brain. I mean, this is a tall order and I doubt we’ll reach all of our goals in five years. I think that the progress that we make will at least define what the things are that we need to work out. I see this as kind of sending us around to the move to see if we get back and what happened along the way before we actually land somebody there. It’s certainly not that historic, but nevertheless, it’s about that complicated.
Audacious Goals Initiative
TR: I think this whole initiative by Paul Sieving of the National Eye Institute has been his big push over the last five years. The point of it is to see if we can, rather than just stopping the eye from degenerating, but to repair it. You know, it is restoring vision back to what it was before someone got a disease or injury. So that’s the whole thing. How can we do better than simply halting the disease, but how can we reverse it. That’s audacious! We don’t do that for very many things.
TR: Sometimes you find that when you do an experiment, you get positive results but it didn’t turn out exactly how you had planned. So for our work that we did in the squirrel monkeys before, we were hoping to transplant rods and cones (Rods are responsible for vision at low light levels (scotopic vision). They do not mediate color vision, and have a low spatial acuity. Cones are active at higher light levels (photopic vision- daylight or other bright light), are capable of color vision and are responsible for high spatial acuity).
We didn’t see any rods and cones in that paper. What we did see were ganglion cells. So the PI of this grant, Jeff Goldberg watched a seminar that I was giving two years ago when I presented some of this. He described it as when you got lemons, make some lemonade. Because you didn’t get photoreceptors integrated into the retina after your transplant, don’t feel so bad. He was interested in glaucoma (a disease that damages your eye’s optic nerve). So that study basically led to this grant. We now have a better idea that we’re more likely to be successful with ganglion cells. So rather than treat macular degeneration, now we’re going to try glaucoma which is a disease of the ganglion cells and not the photoreceptors.
I think in general, we scientists are pretty opportunistic. If we see that the approach that we were taking isn’t really working out as well as we had hoped, then we try something else. In some ways, the science that gets translated into medicine is the science that works over and over again and is successful. This is complicated stuff that we are doing but if it actually works, then I’m sure it will be translated into medicine.
Human Embryonic Stem Cell controversy
TR: One of the postdocs in the lab who will be the primary person growing the stem cells for this project, came to us from Indiana. In Indiana, they’re not allowed to do research on human embryonic stem cells. She did all of her work on induced pluripotent stem cells (Induced Pluripotent Stem Cells (iPS) iPSC are derived from skin or blood cells that have been reprogrammed back into an embryonic-like pluripotent state that enables the development of an unlimited source of any type of human cell needed for therapeutic purposes). So you can create stem cells that are very much like human embryonic stem cells. For her graduate work, she had to work entirely with iPS Cell because they weren’t ES Cells. On the other hand, IPS Cells are a little bit trickier to work with, so it made her a much better scientist actually. Now when she works with ES Cells in Washington, which she can now do, she says, “Well these things are really easy.” In the end, it’s a work around. IPSCs I think provide a very straight forward alternative to ESCs and one that is as successful. In fact, I think in general, the field is moving toward IPSCs because ethically, it is much simpler and they’re not that much different. They’ve been proven to be almost identical.
NHPs in Research
TR: There is controversy around the use of nonhuman primates and some people are putting pressure on lawmakers to reduce it. We’re aware of that. These are really bad diseases for people to get. Glaucoma, macular degeneration, and loss of sight. It’s serious stuff. It justifies to us, our use of the monkey model and stem cells. We feel that these are experiments that can’t be done in any other animal or in any other way. The fovea is real quite unique to primates, and that is kind of where we have to work.