How Circulating Stem Cells Exit Blood Vessels

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00:00:13.08 Did you know that heart attacks are one of the leading causes of mortality worldwide,
00:00:18.23 affecting millions of people every year?
00:00:21.19 One of the most dangerous and frightening things about a heart attack is that, after
00:00:26.14 the heart has been damaged, there's very limited capability to heal itself and regenerate.
00:00:32.05 And there are actually very few treatment options for patients who have had heart attacks.
00:00:37.00 So, in the past few years and decades, stem cells have emerged as a new therapy to treat
00:00:43.07 these damaged hearts.
00:00:44.16 But the thing is, to treat it, you have to get the stem cells to the area of damage.
00:00:50.05 So, one way to do this would be to undergo open-heart surgery and directly inject the
00:00:54.23 cells into to the heart where the damaged tissue is.
00:00:58.08 However, open-heart surgery is a very complicated and dangerous procedure.
00:01:02.08 So, in a new emerging therapy, you're actually able to inject cells directly into the bloodstream,
00:01:09.09 using a catheter at a distant part of the circulation, and then use that catheter to
00:01:13.21 find and actually inject cells into the blood vessels of the heart tissue.
00:01:18.05 And so, once inside the blood vessels, these cells have the ability to find damaged tissue,
00:01:23.09 cross the blood vessel wall, enter where this damaged tissue is, and help with regeneration.
00:01:29.11 The only thing is, only a small fraction or portion of these cells actually exit the blood vessel
00:01:35.15 and reach the damaged tissue.
00:01:37.18 And how they actually exit is unknown and it's a mystery.
00:01:41.19 And so this is a huge mystery that no one seems to know, and it was something that
00:01:46.18 I was interested in uncovering.
00:01:49.06 I'm Tyler Allen, a graduate student at North Carolina State University,
00:01:52.24 and I am a stem cell investigator.
00:01:55.08 So, let's find out how these cells are getting out.
00:01:57.11 So, like I said previously, it's been known for some time that cells, or the stem cells,
00:02:02.12 actually do exit blood vessels and reach damaged tissue.
00:02:05.22 So, this was found out by researchers and scientists who did experiments where they
00:02:10.12 injected cells into the blood stream or into blood vessels at one time point, and then
00:02:15.13 they waited and they came back and they imaged the same blood vessel at another time point,
00:02:20.03 and saw that the cell was, strikingly, outside of the blood vessel.
00:02:24.08 And so they knew that, somehow, they were exiting, but how they were actually getting
00:02:28.17 out was still unknown and was an elusive mystery that I was interested in solving.
00:02:34.16 So, one way that we kind of... or one question that we had was, do they exit similar to other
00:02:41.07 blood vessels or other blood cells in the... the vessel or in the circulation?
00:02:45.11 So, there's a very common or well-known blood cell that actually exits, and this is known
00:02:51.11 as the white blood cell.
00:02:52.15 So, the white blood cell, it actually travels around through the circulation or through
00:02:56.20 the blood flow and the blood vessels, and, usually as an immune or inflammatory response,
00:03:01.22 it will exit the blood vessel.
00:03:03.15 And so it does so in a very characteristic manner, where it squeezes through, kinda like
00:03:07.14 this video that just showed the white blood exiting the blood vessel.
00:03:11.24 So, it was a question as to whether stem cells use this method.
00:03:16.15 So, we know that they exit but, due to the limitations of the imaging technology we have,
00:03:22.15 we don't really know how stem cells exit.
00:03:24.14 So, a lot of people just assumed that it was similar to white blood cells, where they just
00:03:28.21 squeeze through.
00:03:30.14 But I was actually curious to understand and to know if they did indeed use the same method.
00:03:35.10 So, it was a big question for me, did they exit the same way as white blood cells?
00:03:40.01 And the reason that I really questioned this is because stem cells and white blood cells
00:03:44.10 are extremely different from one another.
00:03:46.22 So, maybe they do undergo a different way to get out, because white blood cells, they've
00:03:51.17 got specific membrane proteins and cytoskeletal properties that most other cells, including
00:03:57.24 stem cells, simply just don't have.
00:04:00.02 So, it was my thought that, well, maybe the stem cells are using a different method that's
00:04:06.13 completely distinct from white blood cells, because they don't have the components needed
00:04:10.09 to squeeze through and exit like white blood cells.
00:04:13.14 But, like I said before, it's very hard to see this in humans and it's very hard to see
00:04:17.21 this in most mammals, so I turned to the zebrafish.
00:04:21.16 So, here's an image of a zebrafish and they're called that because they've got these
00:04:25.05 nice stripes, similar to zebras.
00:04:27.13 But zebrafish are actually more related to you, and humans, than you might think.
00:04:32.13 So, zebrafish share about 70% of the protein coding region of the genome as humans, so
00:04:39.05 they are actually excellent models to study biological processes that occur in humans.
00:04:43.21 So, I specifically use them at the embryonic stage.
00:04:47.03 And so this is really cool because I can see completely through them and they're
00:04:51.04 completely transparent at this stage.
00:04:53.00 And also, at this stage in life and in development, they haven't developed their adaptive immune
00:04:58.13 system, so if you introduce cells that are foreign to them, then they won't reject them.
00:05:03.02 So, what I'm able to do is I use a specific type of embryo where all of the blood vessels
00:05:08.18 are fluorescent with green fluorescent protein.
00:05:11.08 So, they... they're green, like this one down here.
00:05:14.02 So, what I'm able to do is actually take cells and then directly inject them.
00:05:19.01 So, how I do this... or, the... the first thing that I wanted to do is actually see
00:05:23.17 if these were even good models.
00:05:25.06 So, I wanted to see if mammalian cells that were introduced into the zebrafish embryo
00:05:30.19 could exit.
00:05:32.06 So, remember before when I said they're... the white blood cell normally exits and it
00:05:36.21 exits all the time.
00:05:37.21 So, I wanted to see if white blood cells from a mammalian model would actually behave the
00:05:42.15 same way and exit and squeeze through vessels... or, squeeze through blood vessels in the zebrafish.
00:05:47.21 So, what we did is we took whole blood from a mammal and there are... a rat, specifically,
00:05:52.18 in this case, and then we isolated the white blood cells.
00:05:55.18 And then what I did is I labeled these white blood cells and then I was able to inject
00:06:00.13 them into the zebrafish.
00:06:02.06 So, let's take a look.
00:06:03.20 So, here is the setup that I use for injecting cells.
00:06:07.09 Here's me at a microinjector and then the red box is the actual setup that I use and
00:06:11.14 the zebrafish are in there, but let's take a closer look.
00:06:14.06 So, here's the zebrafish.
00:06:16.10 And here it is -- the microinjector and then you've got the... the area that comes down
00:06:21.12 into where the zebrafish's heart is and leads directly into the circulation.
00:06:26.05 So, I'm able to inject cells directly into the circulation.
00:06:30.17 And then what they can do is they'll kind of travel around the circulation and through
00:06:34.13 the body of the zebrafish, into the tail or
00:06:37.08 to several other regions or areas of the zebrafish embryo.
00:06:41.06 And then, this is where it gets really cool.
00:06:43.14 So, I'm able to get these nice, beautiful images.
00:06:46.17 So, in this case, this is the area or a region of the zebrafish, and you can see the blood
00:06:51.21 vessel is in green here, and then the injected cells are actually in red.
00:06:56.07 And then, what I'm able to do is take slices of images at different points and then using
00:07:01.11 a software I'm able to actually do 3D reconstructions of these images.
00:07:06.06 And then, this, this is where it gets cool, because it allows me to take journeys through
00:07:10.14 with the blood vessel -- we're actually able to dive in and swim through the blood vessel
00:07:15.10 and actually see what's going on inside and see where the cells are in relation to the
00:07:20.06 blood vessel.
00:07:21.06 So, with this, let's take a look at what happens when we inject white blood cells, mammalian
00:07:26.16 white blood cells, into the zebrafish model.
00:07:30.05 So, here, we've got a picture or we've got a video of a white blood cell in red and then
00:07:35.21 the blood vessel, here, in green.
00:07:37.11 So, right now, it's... or, at the beginning of this video, it's inside the blood vessel,
00:07:41.18 and then around here is the outside of the blood vessel and you can kind of see the
00:07:45.22 blood vessel wall right here in green.
00:07:48.13 So, let's take a look and see if it undergoes the characteristic manner where it kind of
00:07:52.16 squeezes through.
00:07:53.21 So, you see... oh!... and there it is -- it's out.
00:07:58.04 So, it actually... interesting... or, interestingly enough, it does squeeze out similar to how
00:08:02.22 they natively squeeze out when they're in their own environment.
00:08:05.14 So, even in this zebrafish model, mammalian cells, or mammalian white blood cells, can
00:08:10.19 squeeze and exit out just fine.
00:08:12.18 So, this was actually really exciting because this showed that the zebrafish embryo model
00:08:17.13 was an excellent model to study how mammalian cells exit blood vessels.
00:08:22.00 So, with this, we want to move on to what we were really interested in and this was
00:08:26.13 the stem cell.
00:08:27.13 So, what we did is we labeled, or we took stem cells, adult cardiac and other types
00:08:32.11 of mammalian stem cells,
00:08:33.22 and we labeled them and we injected them into the same zebrafish model.
00:08:38.06 And so, in this case, or in this video, we've got the red... these are the stem cells, this
00:08:43.00 time, and the green are also, like in the previous one, the blood vessel... so, except
00:08:47.12 in this one, we've got both the 3D reconstruction and the 2D video, which are going to play
00:08:52.19 in tandem.
00:08:53.19 So, let's take a look and see what happens with these stem cells when they're inside
00:08:57.24 of the blood vessel, and let's see if they exit.
00:09:00.14 Maybe they'll exit like the white blood cells or maybe not -- let's take a look.
00:09:05.10 So, here we have the cells and they're leaving, and then they're actually
00:09:11.15 almost being pushed out, it seems.
00:09:14.06 Now, they're out.
00:09:17.01 That was... that was really cool, because that was... that was a lot different from
00:09:21.00 the white blood cell.
00:09:22.06 We saw the white blood cell was squeezing out, but this... they actually maintained
00:09:26.09 their form and it was almost as if they were being pushed out of the blood vessel.
00:09:31.04 And so this was really interesting and exciting when we saw this, because this was the first
00:09:35.15 time that anyone had ever seen or visualized
00:09:38.23 the method that stem cells used to exit blood vessels.
00:09:42.06 This was exciting.
00:09:43.06 I mean, this has never been seen before and so we were actually really, really interested
00:09:47.19 in what was going on here and what was going on with this process.
00:09:50.24 So, let's take another look at another cell.
00:09:53.21 So, here, we've got another type of stem cell, and it's the same setup, so the red is the
00:09:59.00 stem cell, the green is the blood vessel, and we've got the 3D reconstruction and the
00:10:02.18 2D playing in tandem.
00:10:03.23 So, let's take another look at this.
00:10:05.24 So, right now it's inside the blood vessel and then...
00:10:09.07 BAM -- it's just pushed out.
00:10:11.06 So, again, it doesn't squeeze out.
00:10:14.00 Even this one, which is a different type of stem cell, is still actually being pushed
00:10:18.04 out by the blood vessel in a very characteristic and interesting manner.
00:10:22.20 So, this... this showed us that the stem cells, regardless of the type of stem cell it was,
00:10:28.18 undergo this kind of pushing method and not the squeezing method that was thought, like
00:10:33.01 the white blood cells.
00:10:34.03 So, this was actually really interesting because it showed that we had discovered or observed
00:10:39.09 an entirely new phenomenon, or way for cells to exit blood vessels.
00:10:44.08 So, what we did is we actually named this process and so it's called angiopellosis -- so,
00:10:50.18 angio relating to blood vessels and then pello is Greek for to drive or push out.
00:10:55.03 And so, we saw that this process was a multi-step process that began with the cell traveling
00:11:00.19 through the blood vessel or through the bloodstream, and then would attach or become lodged in
00:11:05.19 the blood vessel wall.
00:11:07.08 And then the vessel wall would then start to wrap around and kind of hug the cell and
00:11:12.15 then actively push it from the inside into the outer region, where it could then exert
00:11:17.03 its beneficial or therapeutic effects.
00:11:20.00 So, we wanted to see next is, was this specific to stem cells?
00:11:26.18 So, in science, since the process of biology and the biological processes are so dynamic,
00:11:33.02 you really want to make sure that what you're seeing is not just an artifact and that this
00:11:37.08 is specific to the type of cell or whatever model or system you're looking at.
00:11:41.23 So, we had all these questions when we found out or we discovered this method for cells
00:11:46.21 to exit.
00:11:47.21 We were interested in, was this specific to cells?
00:11:51.01 Was anything that was around or... around the same size -- would it also exit?
00:11:56.09 Or was it something that was on the cells or a cellular component that was causing this?
00:12:01.00 So, we needed a good control to make sure that it wasn't just anything around the same
00:12:05.22 size in the same shape that was undergoing this method, or if it was.
00:12:10.06 So, what we did is we took microspheres.
00:12:13.14 And so, microspheres are actually these polymer, kind of round balls that are around the same
00:12:19.01 size and shape as the stem cells.
00:12:21.15 And the only difference is that they're... or, one of the only differences is that they're
00:12:25.10 not biological -- they have no biological components, they're completely synthetic.
00:12:30.09 So, we wanted a control to see, well, if we inject something that's around the same size
00:12:36.10 and the same shape as these stem cells, how would it react?
00:12:39.10 Or would it react in a similar method?
00:12:41.05 A different method?
00:12:42.14 Or would nothing or something happen at all?
00:12:45.03 So, what we did is we took those microspheres and then we labeled them with a fluorescent
00:12:50.06 marker and then we actually did the same thing -- we injected them into the zebrafish embryo
00:12:54.12 and we saw what would happen.
00:12:57.02 And so, here, in this example, the green is the blood vessel and then the yellow, round
00:13:02.03 ball that you see is the microsphere.
00:13:04.07 And so, we imaged and we inject... or, we injected them and then we imaged and then
00:13:08.09 we looked for hours and hours and hours... and then we patiently waited.
00:13:14.21 And then nothing happened.
00:13:16.06 They would circle around, they would travel through the circulation, some would clump
00:13:20.19 together, some would become lodged in the vessels in different places, but none of them
00:13:25.21 would ever exit, none of them would escape.
00:13:29.21 They wouldn't exit through angiopellosis or through the method like white blood cells.
00:13:34.04 They would just stay inside the blood vessel.
00:13:36.11 So, this was... this was really interesting because then we had another question.
00:13:41.09 It was, is there something about the cell that is prompting the blood vessel or is prompting
00:13:47.13 them to exit or leave out of the bloodstream or the circulation?
00:13:51.10 Because, if you have these inert non-biological microspheres that are around the same size
00:13:57.17 and the same shape, they don't do anything, they don't do anything at all.
00:14:01.18 So, we thought, or we devised this plan to kind of tease out what was going on in this
00:14:07.21 method and what was going on.
00:14:09.14 So, what we did is we took these same microspheres in these same stem cells, and what we did
00:14:16.03 is we kind of... we gutted out the stem cells so that only their shell or their membrane
00:14:20.24 was present, and then we... we dissolved or we disassociated the cell's membrane and then
00:14:26.17 reformed them around the microspheres.
00:14:29.20 So, think of like a costume party.
00:14:32.07 So, these microspheres, they're going to a costume party and, for this situation, this
00:14:38.11 example, the costume is the membrane or the skin of the stem cell.
00:14:43.13 So now, we've got these microspheres that have the coated membranes of the stem cells
00:14:49.18 and we're going to use these to see what happens when they're introduced into the system.
00:14:53.16 So, what we did is we took these coated microspheres and we injected them again into the zebrafish
00:14:59.22 embryos and then we saw what would happen.
00:15:02.07 So, in here, you've got two microspheres that are kind of close to each other inside the
00:15:06.23 blood vessel.
00:15:07.23 And let's... let's take a look and let's see what happens.
00:15:11.06 So, see right now they're inside, and then now they're being pushed and you see they're
00:15:18.24 actually outside at this point.
00:15:20.22 So, here, we've got the blood vessel and see, now, they've actually exited to this
00:15:25.13 extravascular cavity surrounding or outside of the blood vessel.
00:15:30.01 And so, this was... this was really interesting, because this showed that the cells... or,
00:15:36.04 there was a component of the cell on the membrane that, even when on a non-biological substance,
00:15:42.12 could prompt them to exit the blood vessel.
00:15:45.14 So, this was... this was exciting because it showed that, even if a polymer microsphere
00:15:51.23 was coated with something biological, it could escape.
00:15:54.21 So, we were interested to find out, well, okay...
00:15:57.24 so, we know it's something on the membrane.
00:16:00.09 We know that there's some component that's causing this.
00:16:04.10 What could it be?
00:16:05.10 And so we thought back and, well, we thought and we thought... and we're like... we remembered
00:16:10.06 the white blood cell.
00:16:11.12 So, remember, I talked about the white blood cell and how it regularly escapes from the
00:16:15.12 bloodstream all the time.
00:16:17.06 So, we thought, okay, the white blood cell has lots and lots of membrane proteins that
00:16:22.05 help it with this process of squeezing through
00:16:24.19 and interacting with the blood vessel to actually escape.
00:16:28.16 And so we... we searched around and we found that there was a membrane protein that was
00:16:33.01 actually found on both white blood cells and stem cells, and this type of stem cells that
00:16:37.20 we were using, and it was present on both.
00:16:40.01 So, we... we devised of a way to kind of see if they were the same or if these two processes
00:16:45.17 -- stem cells and white blood cells exiting -- relied on the same molecular pathway or
00:16:50.08 the molecular system.
00:16:51.18 So, what we did is we blocked the antibody... or, using an anybody, we blocked this protein
00:16:57.22 on white blood cells.
00:16:59.15 And so, we took these white blood cells that had been blocked, and then some that were
00:17:03.13 just normal white blood cells the hadn't been blocked, and we introduced both into the zebrafish
00:17:08.14 model, and we wanted to see if, in this model, that blocking this protein, or this membrane
00:17:14.09 protein, could inhibit the white blood cells from exiting, and then we could see if this
00:17:18.06 was a good model to test this.
00:17:19.23 So, on the far left, we have some white blood cells
00:17:22.17 that have been injected that are not blocked.
00:17:25.16 And then, closest to me, we have white blood cells that have been injected that this membrane
00:17:30.05 protein has been blocked.
00:17:31.16 So, right now, the red is the white blood cell and the green is the blood vessel.
00:17:35.21 So, let's take a look and let's see what happens.
00:17:38.10 So, in here, in this video, you see it's going and going and, then, on the very far left,
00:17:45.10 the ones that have not been blocked, they're actually able to exit just fine and kind of
00:17:50.15 come out of the blood vessel characteristically, and squeeze out just fine.
00:17:54.06 But, here, in the one closest to me, the white blood cells are actually trapped inside the
00:17:58.22 blood vessel and they're not able to exit.
00:18:00.24 It's almost as if they've been blocked or inhibited from squeezing through.
00:18:05.08 So, what we wanted to do next we'll see if this held true for the stem cells.
00:18:10.06 So, we actually took that same membrane protein, using the same method, we blocked it on the
00:18:15.24 stem cells.
00:18:17.04 And then we injected these stem cells into the zebrafish embryo, and wanted to see what
00:18:22.02 would happen.
00:18:23.07 And we saw that, actually, even when this protein was blocked, they could still exit
00:18:28.24 or they could have skill... still escape from the blood vessel in the same characteristic
00:18:33.12 angiopellosis method, where they're being pushed out from the inside.
00:18:37.03 So, this was... this was really interesting because, now, this showed that white blood
00:18:41.19 cells and stem cells, they both require these membrane components and these biological components
00:18:47.17 to exit, but they're two completely different components that are allowing them to achieve
00:18:53.06 these things.
00:18:54.06 So, this further established that angiopellosis was a completely different method than the
00:18:59.19 one used by white blood cells to exit the blood stream, or to exit blood vessels.
00:19:03.22 So, this was... this was really interesting and this showed that we had pretty much defined
00:19:08.12 or discovered a completely new method for cells to exit blood vessels.
00:19:13.16 And it represented a method that stem cells used to exit blood vessels to
00:19:18.06 reach damaged tissue.
00:19:20.00 And so this is really important, one, because if we understand this method then this is
00:19:26.09 understanding a unique and basic biological process that's going on.
00:19:30.20 But, also, it's interesting because, remember at the beginning when I talked about heart
00:19:35.03 attacks, and how dangerous they are and how there are very few, limited treatments...
00:19:39.10 well, remember the treatment where we inject the stem cells and they're able to cross the
00:19:43.03 blood vessel wall and reach the damaged tissue.
00:19:45.19 So, this is a promising treatment or promising therapy but, like I said before, only a small
00:19:51.02 fraction of the stem cells actually exit and reach the damaged tissue, which is a problem.
00:19:57.00 But, if we can understand how they're doing so, if we can understand angiopellosis, and
00:20:02.08 all the mechanisms and the molecules involved, then we can manipulate these and augment,
00:20:08.11 and increase the amount of stem cells that are exiting the blood vessel and reaching
00:20:12.16 the damaged tissue, potentially increasing the amount of regeneration happening and saving
00:20:17.20 countless or improving lives as well.
00:20:20.22 So, I want to thank you all for listening to my talk.
00:20:23.22 And I especially want to thank my advisor, Dr. Ke Cheng, as well as my lab members, my
00:20:28.14 collaborators, and funding,
00:20:30.10 as well as iBiology for giving me the opportunity to give this talk.
00:20:34.21 And thank you and have a great time.

This Talk

Speaker: Tyler Allen

Audience:

Educators of H. School / Intro Undergrad
Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: May 2017

Talk Overview

Injecting adult stem cells into the bloodstream could help regenerate tissue damaged by heart attacks. For this to happen, circulating stem cells need to exit blood vessels and reach the damaged tissue. While scientists knew that circulating stem cells are able to exit the bloodstream, how they did it was a mystery. In this lecture, Tyler Allen describes angiopellosis, a new way for circulating stem cells exit blood vessels when injected into the blood for therapies.

This talk is part of the Young Scientist Seminars, a video series produced that features young scientists giving talks about their research and discoveries.

Speaker Bio

Tyler Allen

Tyler Allen received his bachelor’s degrees in Biology and Plant Biology from North Carolina (NC) State University. Upon graduation, Allen joined the laboratory of regenerative medicine expert, Dr. Ke Cheng, as a PhD student at NC State’s College of Veterinary Medicine. Allen’s research involves understanding how adult stem cells exit blood vessels, when injected into… Continue Reading