Stem Cell Advances in Heart Disease: Generating Muscle within an Existing Heart | Deepak Srivastava

Stem Cell Advances in Heart Disease: Generating Muscle within an Existing Heart | Deepak Srivastava


While we ultimately want to build
whole parts of the heart, The more immediate goal
that’s more– is reachable
not in five to ten years, but maybe in the more near future, is to be able
to regenerate new muscle in an existing heart. In existing parts of the heart. And this could be
tremendously advantageous in numerous forms
of both pediatric and adult forms of heart failure– one of which, as shown here, is caused by heart attacks,
largely in adults, where a coronary vessel
is occluded. The blood flow to that part
of the heart is lost, and those muscle cells die. And the result is the scar
that you see on your right, here, that’s indicated in blue where the wall of the ventricle
becomes very thin and is–this is really
fibrotic tissue. The heart has little or no
capacity to regenerate itself, and so one is left
with this scar permanently, and over time the heart gets–
deteriorates and gets worse and worse. And this is what five million
people in America suffer from. Ultimately, the only course
right now is a heart transplant for any of these types
of heart failures at the end stage. But only about 2,000
heart transplants are done in the United States
every year because of a clear
shortage of donors. Now, what we know is that the heart is actually made up
not just of muscle. In fact, only less than half
of the cells in the heart are muscle. The other half of the cells
in the heart are what we call “fibroblasts,” which are really just support cells
for the architecture of the heart and secrete important signals
to the neighboring muscle. But it’s also those fibroblasts that get activated after injury
and migrate to the site of damage and are the ones
that lay down this scar tissue. And so we asked a few years ago–
began to ask is, “If the heart has this great
reservoir of cells “already there in the organ, “then might we be able to
not reprogram fibroblasts to stem cells”– as Shinya Yamanaka did– “but rather, directly
convert those fibroblast cells “that are already in the organ into newly born
cardiomyocytes?” If we could do that,
then we could harness this vast potential of cells
already in the organ and turn them into new muscle cells
for regenerative medicine without having to put new cells
into the organ– and I’m sure you’ve heard and seen
through many–so many grants– all the hurdles that we
are trying to overcome with cell-based therapies
of introducing cells. I think we can
overcome those with time, but there are clearly
many hurdles we face there. And this might
be a little bit easier. And so, the approach we took
was to go back to how nature normally
builds a heart– which we had
invested the last 15 years in trying to figure out
those networks– and we asked, “If we essentially
leveraged that information “and threw the kitchen sink,
if you will, “of those networks
at a fibroblast cell, “could we get a cell to become
more like a cardiomyocyte in a dish?” And, in fact, we were able to. And it turns out that a combination of just three key
master regulatory genes, some of which
I told you about earlier, are enough to turn a… cardiac fibroblast
into a muscle-like cell. And those were Gata4, which I mentioned
causes human disease, Tbx5, which
causes a similar disease, and a third factor, Mef2c. Now, in a dish, these three factors
could push cells to a cardiomyocyte-like state, but most cells were
partially reprogrammed, and a few went all the way
to become beating heart cells, even on plastic. But the question
we really wanted to know is not, “Could we do this on plastic?” but, “Could we do this
in a living organism, “in an organ– specifically in the heart
in an animal?” And so we did that experiment
more recently. We tied off a coronary artery
in a mouse, created–mimicked–
to mimic a heart attack. We injected the three factors with the virus–the sort of
a gene-therapy approach, waited four weeks
and then took those hearts out and dispersed the cells in the– from the heart into a dish so we could
examine each individual cell and see how well we were able to reprogram the cells
into new muscle cells. And about 50% of the cells
looked something like this. We labeled–genetically
traced those cells that would– started off as a fibroblast
and become a muscle cell with a red marker
that you see here. And about 50% of the cells
looked like this, where they
looked essentially just like a real cardiac muscle cell,
which is shown here. So that looked pretty good. And if we stained those
with markers of the– of the what we call “sarcomeres,” or the beating units in the muscle, it looked something like this. The green cell here is a real,
if you will– or a native cardiac muscle cell. And the yellow cell
is a newly born cardiac muscle cell that used to be a fibroblast cell. And you can see that these also have these
beautiful striations indicative of a cardiac
muscle cell. But the real proof in the pudding
in this situation is, “Can these cells beat?” because for heart cells,
we have a very rigorous test of whether they’re
fully reprogrammed or not. And so, it turns out
that when we reprogram cells in the “in vivo,”
native environment with all the right signals
around them and the stretching–the mechanical
forces they might experience– over 50% of the cells that are reprogramming go all the way to beating. And that’s what you see here
if I play this movie for you. Here this cell that’s
florescent green is genetically labeled to indicate that it used to be a fibroblast,
or a non-muscle cell. And this is a real muscle cell that was already in the heart that we’ve taken out. And so now you can
see when we electrically stimulate, both of these cells
are able to contract– and as I mentioned, about 50%
of the florescent green cells could be fully
reprogrammed like this. And so we were very
encouraged by this and proceeded to see if they
could make a difference in vivo. But before that,
we had to confirm one thing that’s very important. It’s not enough to make a cell– a muscle cell that beats
in the heart if it doesn’t electrically couple
with its neighbors. It would beat by itself,
would not generate force, but moreover be a potential source
for rhythm disturbances. And so this experiment that I’ll
show you here was critical. And that is to determine
if these cells can actually couple with one another–
both the newly born ones, but also the previously
existing one. So here what you see
is a cluster of cells that we’ve isolated now
from a reprogrammed heart in vivo. And now it’s not a single cell,
but there are three cells here. One, two, and three. Only the red cell is newly born,
and these two are old cells. And when I play
this movie down here, we were
looking at the wave of calcium that goes through these cells. And what you’ll see
is that when we stimulate, all three cells have the wave
in synchrony, which means that they
are electrically coupled with one another, and therefore,
they beat at the same time. And so with this knowledge in hand, then we proceeded to do the–
the trial. Okay, but I forgot
I was gonna show you this movie. So just to illustrate for you what we’ve done here
in a diagrammatic form, many of you, I suppose,
have seen this Waddington diagram of this hill of a ball
rolling down, and a pluripotent cell
becoming a specific cell type. And you’re probably
with Shinya Yamanaka’s work where he took these balls,
essentially–if you will, and rolled them up the hill
back to a pluripotent state where they could go back
into any one of these states. What we’ve done here
is something different, where we’ve essentially
taken these cells and jumped them over the hill,
if you will, from one cell type
in the adult directly into another
without having to go back up the hill
and then come back down. So this is something we
refer to as “direct reprogramming” because it’s going directly
from one adult somatic cell type to another. Okay, so here’s
the experiment, then– the sort of pre-clinical
experiment, if you will. We took a large cohort of mice, did these infarctions, and then injected
the reprogramming factors, and then a serial time-points
did echocardiography on the mice to see even with this
crude injection in to the muscle, could we make a difference
in their function, and at the end did MRIs to look to see– because that’s really the best test
to look at cardiac function. And so I’m showing you here
a representative MRI on your left, and then the quantification across tens of mice
in each category. And the dashed line here indicates the cardiac function
by MRI of a normal mouse that hasn’t had an infarct. This blue bar
represents control mice that had an infarct, but had a virus injected
that was just a control virus encoding a control gene. And you can see
that it’s decreased. And then the green bar indicates
the experimental mice that had the three
reprogramming factors. It’s clearly not normal, but it’s much better
than without treatment. And the MRIs–
representative MRIs are on the left– and we’ll go through right now. If we section these mice
after sacrificing them, the controls
look something like this, where you see now
in a cross-section where the left ventricle
is here, that this is the thin
left ventricle wall with the scar tissue. And the treated mice
looked something like this, where there’s still a thinning
of the left ventricle, there’s scar, but you can begin to see muscle
interspersed now through the scarred area. And we could genetically
label those cells and ask if those red cells
that you see in this scarred area, thinned area, were just leftover myocytes, or newly born ones– and we have
genetically labeled these, and I’ll just tell you
that these, in fact, are all newly born
and have the markers that tell us that these
used to be non-myocyte. So they’re actually
newly reprogrammed cells. So we’re very encouraged by this because we think that this
represents a way to potentially regenerate heart muscle
without putting cells in– and all the associated hurdles, but rather harnessing the cells
that are already in the organ and tricking them to do something
that they would not normally do. Now, there’s a lot of work
to be done going forward to really understand this biology. And so, with the help of a CIRM
Basic Biology grant, we’re now aggressively
trying to understand the mechanism by which this occurs– meaning, where do these three
transcription factors that are controlling thousands
of other genes– where do they sit on the DNA, and what are they
turning on and off that actually makes this happen? And what I’ll just tell you
from our early results is that it happens within hours. It’s phenomenal
how quickly the genome is being reprogrammed
by these factors. And within three days, most of the transcriptional changes
across the genome have already occurred. And then over the next
several weeks, There are fine tuning
of gene changes that ultimately result
in the cardiomyocyte phenotype. But the bulk of the activity
is very, very early. We did not anticipate
or expect that to be the case. And we have the tools now to actually
understand the epigenetics and the transcriptional changes that occur at hourly intervals, and we’re doing exactly that
with your help. Now even while we
push to understand this and refine the technology, we’re aggressively pushing forward
with pre-clinical studies in larger animals,
specifically the pig to begin to see if this strategy
could work in a larger heart that’s more similar
both in size and physiology to the human’s. And again, this is with the help of the CIRM
Early Translational Award that we’re now
able to do our studies in pigs to test both the efficacy
of this approach as well as the safety. And so we’ve already initiated
our first sets of pigs to begin to test
how the delivery is occurring and what levels we’re getting. And I’m very hopeful that in the next year
to year-and-a-half, we’ll begin to have the results to see if this really–
this approach really can work in a larger heart. And we’re very, very
excited about this, and are grateful, again. This is not the kind of work
we could do any other way. It’s expensive, and NIH just doesn’t
support this sort of thing that’s no longer
in the discovery phase, but really is translational to move this
into a pre-clinical state. Now I’ll just close
by saying that this approach we’re very excited about
because we don’t think it’s unique to the heart. We–there’s evidence from–
at least in a dish, now, for many other cell types
that one can directly reprogram adult somatic cell into not just heart cells,
but brain cells by folks at Gladstone, as well as Marius Wernig
at Stanford, to blood cells, to liver cells, pancreatic cells, and it’s our hope
that others will now do these types of studies in vivo. And just last week,
a group reported that that they were
able to convert, in vivo, cells in the brain–neurons, into spinal-motor-neuron-like cells that are lost in ALS. And so I think you can
think of this strategy for multiple, different
regenerative approaches because the developmental biology of each of these cell types
is now fairly well understood, and it’s that developmental biology
that then allows us to switch the–
flip the switches, if you will, and tell a cell to become
what we want it to become. And so, I’ll close there by just saying that I think the combination
of the powers of human genetics along with the stem cell biology, particularly
using reprogramming technology, puts us in a situation where
we have unprecedented opportunity to both understand human disease
in ways we couldn’t before through disease modeling, and leverage our knowledge to begin to think of new ways to regenerate organs going forward. And so I just want to thank
members of my laboratory, that are shown here, that are just a brilliant
group of people. Many CIRM scholars
are in this picture that have been supported
by our training grant at Gladstone, and all of them
utilize our shared facility that you all have generously
supported at Gladstone. And we’ve had great collaborators including Shinya and his lab
at Gladstone, Benoit Bruneau, who I mentioned, Bruce Conklin, and Shen Ding, who moved to join us last year from the Scripps Institute. Thank you very much.