Speaker 1: Welcome to the Salk Institute’s Where Cures Begin podcast, where scientists talk about breakthrough discoveries with your hosts Allie Akmal and Brittany Fair.
Allie Akmal: Core facilities are resources shared by all Salk labs so that each lab doesn’t have to develop its own expertise in, say, gene sequencing or specialized microscopy. Ken Diffenderfer directs a stem cell core facility, and we got a chance to talk with him about what exactly that involves. What does a stem cell core do?
Ken Diffenderfer: Yeah, so our lab focuses on providing human stem cell models to researchers at the institute for the purposes of studying aging, development and disease in human cell systems.
Allie Akmal: Tell me a little bit about what stem cells are for people who don’t know.
Ken Diffenderfer: Yeah, absolutely. Great question. So stem cells are a type of cell that has an incredibly unique ability to turn into a different cell type.
Allie Akmal: And what do you mean by a different cell type?
Ken Diffenderfer: Right. We are very complex multicellular organisms that cells are integrating together and organs and organ systems are integrating to form us, right? All of that complexity comes from ultimately a single cell, right? So the power of a STEM cell to develop into all of these unique constituent of parts of organs of organ systems is really what makes them unique. This property that they have that allows them to do this is what we call differentiation. And so at the most basic level, a stem cell is a cell that can differentiate into something else.
Allie Akmal: Is that kind of like specializing?
Ken Diffenderfer: Yeah, absolutely. You can think of it that way. Yeah. So the cell has kind of no defined purpose and then it makes life decisions, and over time it becomes an incredibly specialized unit that has a very unique purpose inside of whatever part of the body it is potentially existing.
Allie Akmal: Wow. That’s super cool. You sound pretty excited when you talk about stem cells. How did you get into doing this?
Ken Diffenderfer: Yeah. So my first exposure to stem cells was as an undergraduate at Cal State Channel Islands and had the opportunity to work with a lab there that was studying a neural stem cell line and really using this as a kind of platform to see how some drugs that a chemist on campus was developing would kind of cause that cell and to react, right? Would it cause it to divide more, to divide less? Would it cause that neural stem cell to make specific types of neurons at higher frequency than others? But it really struck a nerve with me that these cells are incredibly powerful.
Allie Akmal: So you studied them as an undergrad.
Ken Diffenderfer: Yep.
Allie Akmal: And then you went to grad school?
Ken Diffenderfer: Yeah. Yeah. So I went to graduate school also at Cal State Channel Islands. I did a master’s program there, which had a stem cell technology as component to it. And a part of that was doing a year long internship, which I did locally in the San Diego area, at the Scripps Institute. That kind of led me into a position here at Salk.
Allie Akmal: And what are some of the exciting ways that stem cells can be used in research?
Ken Diffenderfer: Oh yeah, so I mean they are abundant and diverse. A few of the really exciting ways we use them here at the Salk Institute is through deriving patient-specific stem cell lines for the purpose of studying disease, and this is a technology that was developed by a Japanese scientist by the name of Shinya Yamanaka back in 2006 where we can effectively take a permanently differentiated cell, a cell that has made all of its life choices. It’s no longer a stem cell, and we could access that material through a biopsy, through a blood draw and take those cells and revert them all the way back to the early stages in human development.
Allie Akmal: How would you move that backwards in time?
Ken Diffenderfer: Most often times when we are doing this, the skin biopsy comes to us in what we call a punch biopsy, but it’s just a little kind of core of skin, roughly three millimeters in diameter. We’ll take that little chunk of skin and we will treat it with enzymes overnight. And those enzymes are just breaking apart all of the individual cells that are kind of inside that biopsy. And then we can essentially put those cells out onto a plastic dish, grow them out, expand them as they start to cover the entire surface of the dish. And then we can introduce four key genes to those cells. And these are the four key genes that Dr. Yamanaka discovered back in 2006. And so we use highly engineered viruses that have had all the disease causing elements of them removed. And we essentially just use them as shuttles for genetic material. And so we will infect the cells with these viruses and then allow them to make this transition from a permanently differentiated, permanently developed cell all the way back to the earliest stages of human development, which we described as pluripotency.
Ken Diffenderfer: And so that process takes roughly three to four weeks. And so as essentially as skin cells hanging out in the dish, and then over time they start to slightly change. And we see the skin cells change from these very elongated cells that cluster very tightly together into these very, very small, tightly compacted groups of cells that we call colonies. And these colonies are the induced pluripotent stem cells starting to form.
Allie Akmal: So at that point, those cells are capable of becoming a heart cell or a lung cell or a brain cell?
Ken Diffenderfer: Absolutely. At that point, those cells are pluripotent, which is the unique ability of a stem cell to turn into absolutely any cell type in the human body.
Allie Akmal: Wow.
Ken Diffenderfer: And if we think about doing this in a patient specific manner like we can with the induced pluripotent stem cell technologies, most people get very, very excited. I have a relative who has Parkinson’s. You can study that, right? I have a relative who has cystic fibrosis. You can study that. And so there is an immediate connection people can start to make these technologies and see not only the value to kind of the academic community and the medical community as we kind of probe these diseases and better understand them, but also how these understandings can lead to better treatment, better therapies.
Allie Akmal: So help me get a picture of what the stem cell core looks like. Do you have big tubs of liquid that have undifferentiated stem cells just floating in them?
Ken Diffenderfer: Yeah. That is a great question. We are literally right around the corner. Do you want to actually head over and take a tour?
Allie Akmal: You mean now?
Ken Diffenderfer: Yeah, absolutely.
Allie Akmal: Sure.
Ken Diffenderfer: I feel like we’re on an episode of “Radio Lab”. Okay. So let’s enter the Core.
Allie Akmal: Okay.
Ken Diffenderfer: If the door unlocks for us. There we go. Okay, Allie, well welcome to the Salk Stem Cell Core.
Allie Akmal: Thanks, Ken.
Ken Diffenderfer: We have a few specialized pieces of equipment that allow us to actually image the cell models that we work with. Some are just kind of standard microscopes that allow us to take a cell culture out of an incubator, put it on the stage of the microscope and visualize it, and some are actually a little bit more exciting. So I want to show you right now.
Allie Akmal: The Yokogawa Cell Voyager 1000?
Ken Diffenderfer: We have yes that, so this is a cool system that allows us to do very high resolution imaging, confocal style imaging. But this chamber is actually designed to keep the cells at 37 degrees Celsius, which is body temperature, and also injects a small amount of CO2 inside of them, and it mimics the exact same conditions that the cells grow in when we store them in incubators. And so we can do imaging experiments over a very long course of time, program the software that runs the microscope to image every five minutes an hour over the course of a day, two weeks, a month.
Allie Akmal: Wow.
Ken Diffenderfer: And really track how cells are behaving over that time and monitor those changes visually. And this is the one we use most frequently. This is our Incucyte. So this one is actually a microscope that lives inside of an incubator. But what makes this one very cool is that it can actually image multiple plates at the exact same time. It’s actually imaging right now. So I can’t open it and show you.
Allie Akmal: Kind of looks like a wine fridge, but for cells.
Ken Diffenderfer: It does look like a wine fridge for cellS. Yeah, a little bit warmer than your wine fridge. Yeah.
Allie Akmal: Okay.
Ken Diffenderfer: Yeah.
Allie Akmal: Cool.
Ken Diffenderfer: So that’s what we have in this bit. If we move down the hallway here and see another one of our bays, and this bay is a bay we have dedicated to media prep. This is a little bit of a long corridor where we have some benches along one side and then some shelving with various glassware that we use for production and media.
Allie Akmal: And when you say media, you’re not talking about CNN or NPR?
Ken Diffenderfer: No, I am not talking about those fantastic news outlets. I’m talking about the liquid that the cells grow in. Right. And so this is our cell culture media. And a lot of the cells models used here in the stem cell core have incredibly specialized criteria for that liquid media that they grow in. And so we have the luxury of having some expertise in what those requirements are here. And we can actually make these complicated medias in house. And so we do a lot of that here in this bay. It’s not super busy now, but about every other week we do one of these very high volume preps of this specialized media. And it’s all done by hand. There’s not robots or automation doing this. It is our small team of technicians and myself that are physically mixing these various components together and sterile filtering it and then providing it to our user base afterwards. Yeah.
Allie Akmal: That’s sort of like making custom cocktails for cells.
Ken Diffenderfer: Exactly. Yeah. But we don’t drink this one unfortunately. Yeah. Cool. So moving down, if we turn to our right, we have one of our tissue culture bays, and these bays are absolutely the heart of the facility. This is where all the cell culture activity happens.
Allie Akmal: So what kind of cells do you have for us today?
Ken Diffenderfer: Oh my gosh, we have so many things. The first thing I want to show you are some fibroblasts.
Allie Akmal: Fibroblasts.
Ken Diffenderfer: Fibroblasts. So these are cells that we actually can derive from a small biopsy of human skin. These fibroblasts are one of the very, very common starting materials for doing that IPSC or induced pluripotent stem cell derivation process. Right. And the fibroblasts we’re actually looking at today are from a collaboration with a large group of researchers at UCSD that study Alzheimer’s disease. And so these fibroblasts are actually from an individual with Alzheimer’s disease, and we are growing them out. So this is the starting material right here. And before we actually get under the microscope, I just want to show you, if you look into the well here, and this was one of our well plates. We have 24 little wells on this plate. And one of these wells has a very, very small amount of media in it and has a little disk of tissue that you might be able to see there. And that’s actually-
Allie Akmal: Oh yeah.
Ken Diffenderfer: The skin, the skin punch biopsy just sitting on the very, very bottom of the plate. And what we do is when these biopsies come in, we treat them overnight with enzymes. And then the next day we’ll come in and rinse off the enzymes and then put that little small disc of tissue onto the bottom of the plate that’s been coded with gelatin, the exact same gelatin you would use at home to make jello with. It’s just kind of a biological kind of cell culture grade-
Allie Akmal: Wow.
Ken Diffenderfer: Of it. And then we let it attach to that gelatin and put the tiniest, tiniest amount of media on it. Just enough that it stays hydrated, but not so much that that media would go over the top of the tissue. Because if that happens, the tissue will start to float. And we want it to sit on that gelatin. And as it sits on that gelatin, little cells are going to start to crawl out of that biopsy. And that’s actually how we derive the cells from this tissue.
Allie Akmal: Wow. And you’re controlling the microscope with the computers that-
Ken Diffenderfer: So this microscope does have a small little camera on it, and this really allows us all to look at the exact same thing at the exact same time instead of everyone trying to squeeze onto the two little eyepieces. And so we have a small little camera, and that’s just going to broadcast this image onto the computer screen here. Let me focus this in appropriately. Here’s the edge of the tissue. It’s a little small circular disc. Let’s scan around and see if we can see any cells. I think I see some. Get this focus appropriately. So if you look right at the edge here, it’s a bit hard to see on the screen. You can see we have these little cells, these little circular cells right at the edge of this tissue and that in there, they’re kind of crawling out of the tissue there.
Allie Akmal: So the little translucent circles, is that what you mean?
Ken Diffenderfer: Yeah, yeah. The ones attached to the bottom of the plate here.
Allie Akmal: Okay.
Ken Diffenderfer: Oh yes. Here’s much more dynamic area. So this is really a greater, better example of that, and so you can see here I’m on the top part of the biopsy that area is very active with cells that are crawling out of it.
Allie Akmal: To be clear, we’re not seeing any motion in real time, but there is a greater concentration of cells at the edge of the biopsy tissue whose movement outward would be obvious if we were to look at an image taken earlier in time. It almost looks like a reef in an ocean.
Ken Diffenderfer: Yeah.
Allie Akmal: That you’re seeing below the surface.
Ken Diffenderfer: Yeah. Absolutely. You could think of it that like that. Yeah, yeah, yeah. So the next thing, so you guys are induced pluripotent stem cells. They’re looking like a very, very compacted tight group of cells that we would typically see in very early human development.
Allie Akmal: Like soon after a sperm has fertilized an egg?
Ken Diffenderfer: Yeah, like five days after that.
Allie Akmal: Wow.
Ken Diffenderfer: Yeah. Yeah. But these cells are not from that process. Right? These are induced pluripotent stem cells that have come from human skin that we’ve reprogrammed to the induced pluripotent stem cell state, but they still kind of mimic a lot of those qualities, a lot of those behaviors that we would see with an embryonic stem cell.
Allie Akmal: That’s amazing.
Ken Diffenderfer: So what I would show you next is one of the kind of exciting endpoints we can generate. And these are cardiomyocytes or heart muscle cells. Okay. And the very, very cool thing about these cells is that as we go through the process of differentiating them from pluripotent stem cells and they mature, the mature cells will do something very, very cool. Hopefully you guys can see.
Allie Akmal: Oh my gosh.
Ken Diffenderfer: That these cells are actually rhythmically pulsating.
Allie Akmal: Yes.
Ken Diffenderfer: And this is no trickery. I’m not shaking the plate. This is what these cells do as they mature.
Allie Akmal: That’s crazy. They’re all synced.
Ken Diffenderfer: They are somewhat synced, right? Still not the dramatic organization of a four chamber heart that we would expect to see. But it’s not a kind of random event. Right? There is some organization even just in a monolayer, single layer of cells that is making-
Allie Akmal: Yeah, it’s wild. At the earliest stages, they kind of know what they’re meant to do.
Ken Diffenderfer: Yep. Yep. So that’s what we’re looking at here.
Allie Akmal: That is really amazing.
Ken Diffenderfer: Beating heart cells. Yeah.
Allie Akmal: That is so…
Ken Diffenderfer: So it’s hard to describe, right?
Allie Akmal: It’s hard to stop looking.
Ken Diffenderfer: It is very hard to stop looking. We get very geeky about this and love watching this. When we have a bad day, we’ll just come and look at cardiomyocytes.
Allie Akmal: It’s really calming.
Ken Diffenderfer: Fawn over them. Yeah. We just need some like Zen type music in here. We can all get relaxed. Bust out the yoga mats.
Allie Akmal: Very cool.
Ken Diffenderfer: Yeah.
Allie Akmal: And how long can these stay out like this?
Ken Diffenderfer: Yeah, so the cardiomyocytes in our hands anyway are very sensitive to temperature change. So as these stay outside of the incubator, they’re going to start to slow down and eventually they’ll stop beating. We throw them back in the incubator, they’ll start beating again within a few hours. Okay. Also, when we change media on these cells, we see the same thing happens that they slow down or stopped beating. And then if we throw them back incubator within a few hours, they’re beating again. So you guys, actually I rescued these for you when we were coming through earlier.
Allie Akmal: How’s that?
Ken Diffenderfer: One of our technicians here was about to change media in all these. So I just caught him right before.
Allie Akmal: Oh wow.
Ken Diffenderfer: Otherwise we probably wouldn’t see beating cells right now.
Allie Akmal: Wow.
Ken Diffenderfer: Yeah.
Allie Akmal: They would have just been resting and getting used to the new media.
Ken Diffenderfer: They would have been resting and getting used to the new media. Exactly.
Allie Akmal: Oh my gosh, you’ve given us the grand tour.
Ken Diffenderfer: Thanks.
Allie Akmal: You hit all the highlights.
Ken Diffenderfer: Thanks so much for coming by. I’m so glad we can share a little bit of the cells with you today and show you this.
Speaker 1: Join us next time for more cutting edge Salk science. At Salk, world-renowned scientists work together to explore big, bold ideas from cancer to Alzheimer’s, aging to climate change. Where Cures Begin is a production of the Salk Institute’s Office of Communications. To learn more about the research discussed today, visit salk.edu/podcast.