My name is Deepak Srivastava, and I direct the Gladstone
Institute of Cardiovascular Disease and the Roddenberry
Stem Cell Center at Gladstone, and I'm a professor
at the University of California in San Francisco.
This is the second part of my talk, and in this part, I'll share with you
how we can use the developmental biology gene networks
that we've discovered to impact not just our understanding
of birth defects, but also how we can use that for regenerative
medicine approaches through cellular reprogramming.
Can we use these developmental pathways we've discovered
to somehow take an adult heart and create new muscle in a
regenerative approach? And it turns out that the heart, like
most other organs, has not only muscle cells but a number of
support cells. In the heart, we call them cardiac fibroblasts,
but most other organs in your body have support cells around
the functioning cells that are there to form the architecture
and support the cells that are necessary for that organ to function.
The heart, it turns out, is quite special in this in that fully
half of the cells in the organ are of this type of cardiac fibroblast.
And those are the same ones that are activated in the
site of injury and make scars. So what you see here is a
heart, this is in the case of a mouse, but a human heart is very similar.
If it's been damaged in say a heart attack, where in red you see
normal muscle, and here in the left ventricle, you see this
broad purple area which is scar tissue that is present
after an injury to the heart. Now if we wanted to repair
this heart, you can tell that the fundamental problem is that we lost
muscle. We'd want to create new muscle there somehow.
And we and others are trying approaches to take the beating
heart cells that I've shown you before from stem cells and
somehow inject them into this heart to create new muscle.
It's turned out that we've had a lot of trouble there in terms of
getting those heart cells to stick and persist. And even if we
do that, to get them to electrically connect with their neighbors
so that they can beat in synchrony. So then years ago, we
asked could we take a different approach? Could we somehow
harness the fact that we have this abundant pool of
fibroblasts that are already in the organ. Could we somehow
trick those cells into not being fibroblasts, but somehow be
turning into new myocytes or reprogram them into new
myocytes. And this is really inspired by Shinya Yamanaka's
discovery that you could take a skin fibroblast and turn it
into a stem cell. So we thought maybe with the right cues we could
take this cardiac fibroblast and right there within the organ,
convert its fate into a new cardiomyocyte, if we knew the right
factors to induce that cell-fate switch. So, the question as illustrated
here. Can we take a cardiac fibroblast and convert it into
what we call an induced cardiomyocyte-like cell?
And to do this, we really relied upon all of these developmental
biology networks. Because we thought we'd learn from nature
-- how nature normally does it, maybe we could redeploy
the key factors in a dish. And that turned out to be the case.
When we screened a large number of transcription factors and
microRNAs that we knew to be critical for development of the heart,
and asked what was the minimal essential cocktail, it turned out
to be the three factors that I'm showing you here. Two of which we've
talked about extensively already, Gata4 and Tbx5. And maybe that
makes sense now, because of what I've showed you. That these
two proteins sit together to drive cardiac cell fate during
embryogenesis. And a third really critical factor, Mef2c.
That's also essential for normal cardiac formation in an embryo.
So it turned out that these three factors in a dish could push cells
towards this cardiac fate, somewhat inefficiently, but broadly
reprogram these cells. But what we really wanted to do
is know if these three factors could do the trick in the cells that are
already in the heart. And it turns out that they can.
So, if we take a mouse heart and tie off the coronary artery
and induce a heart attack, and do so in a mouse where we can
genetically label with a fluorescent protein the fibroblast
pool and its progeny, we could then track whether or not
we've really converted a resident fibroblast into a new myocyte.
And it turns out that we can not only do this, but we can do it in a way that
new myocytes that are formed electrically couple with their neighbors,
they're most similar to an adult ventricular cardiomyocyte.
And most importantly, we were able to show that these new
myocytes are sufficient to actually improve the cardiac output
in these mice after damage by MRI. And if you look at these
hearts, histologically, where we've done the experiment
that you see here and then wait about 3 months, and take these
hearts out and section them.. Normally, after injury, if we just
introduce with the virus these control genes a dsRed
marker, we see a ventricle like what you see here, with
a big large scar on the free wall of the ventricle, that's indicated
in blue here. In contrast, when we put these three genes
in, we still see a thin-walled ventricle, there's scar tissue.
But now you can begin to see these areas of red, threads of
myocardium in the scar area. And if you look by this yellow
fluorescent protein, these all used to be non-myocytes that have converted
into new myocytes. So this was encouraging, but we knew
that this process with just these three genes was relatively
inefficient in vitro. And we knew that we had improvement even
in vivo. And so we embarked upon a chemical screen, where we
-- Tamer Mohamed, who has a pharmacist background, is a postdoc
in the lab who screened over 5,000 small molecule compounds
to ask could any of them make this process more efficient?
And it turns out that he identified about 20 different chemical
compounds that could. If we measured the number of cells that
were turning on a cardiac specific reporter, in this case alpha-myosin heavy chain.
At about two weeks after introducing these genes. And you can
see here that the number of hits range here from fold
increase from 2 to 7 fold. And what we're encouraged by is
about four of these 19 hits all affected the TgfB signaling
pathway. And about four of them affected the Wnt signaling pathway.
Both of them are very important signaling pathways in various
embryological pathways. So the fact that we're hitting the same
pathways multiple times encouraged us that these might be
true hits. And in fact, we showed that these small molecules
were working specifically through these pathways.
So it turns out that these small molecules not only increased
the number of cells that could convert, they also accelerated
the pace of reprogramming. So normally, in a dish,
it takes about 6 weeks for these cells to convert their fate all
the way to a beating cardiomyocyte, and with the TgfB
inhibitor, this process was accelerated so that it happened within
about 3 weeks. And most remarkably, when we added both
inhibitors, we could see beating cells in a dish as early as
just one week after introducing the genes. So this is really quite
remarkable, if you think about the wholesale epigenetic
shift that has to occur for a cell to go from one cell type
to another, just within a week and induce this process. And
we have interrogated that process very carefully. And we're beginning
to see how the epigenome changes, and it all happens
within the first few days in this setting, which is really quite remarkable.
So, we wanted to really know can these chemicals
improve the ability of these genes to reprogram in vivo. And
improve the function. And it turns out, if we repeat the experiment
that I showed you earlier, and now do it by injecting not only the
genes with the virus into the heart, but also treat the animals
with these chemical inhibitors of Wnt and TgfB
for a couple of weeks, now we see much more robust
formation of new muscle. So what you see here is the
control setting, where at the apex of the heart down at the bottom
of the heart, if you section you see a lot of scar tissue.
And that gets a little bit less as you get higher up in the ventricle.
In comparison, in this setting where we've put both genes and compounds,
even at the apex, all of this region right here that you see is all
muscle. IR reporter are all cells that used to be non-muscle, so we're getting
a lot of more muscle formation, about 5 times as much
as without the compounds. And if we look at the function
of the heart by measuring what we call the ejection fraction, which
is the fraction of blood squeezed out of the heart with every beat.
And we measure the decline in the ejection fraction over time,
by doing ultrasounds on a weekly basis, what you can appreciate
in the red and green lines is the decline that we saw in either just
a control virus or control virus plus the chemicals alone,
which didn't make a lot of difference. The purple line shows you the
change in ejection fraction with just the genes, which is better.
And then the blue line is the change with the genes plus these
chemical inhibitors, which you can see is significantly better
even with the genes alone. And it happens very quickly,
as early as a week, consistent with what we found
-- the rapidity of reprogramming. So we're hopeful that now
we've got a better combination that can actually improve the
heart function even more significantly in this setting.
But what I've showed you so far is just the mouse, and
obviously we'd like to translate this toward the human
condition. And so, it turned out that the three genes I mentioned to you in mouse
actually did nothing in a human cardiac fibroblast. So we had
to go back and ask, what are we missing? And it turns out,
that the addition of two more genes were enough, and again these
are transcription factors also, to reprogram a human cardiac
fibroblast to be more like what we saw in the mouse, but in a human
induced cardiomyocyte. But in mouse, we had see that the
efficiency in vivo was a lot better than in vitro, and so
we pushed forward with trying to test this combination
in a heart that's larger like the human, namely the pig.
And a pig's heart is physiologically more similar to the human.
And the three mouse genes alone didn't do the trick in
human cells either, so we thought this was a good system to
know if this might work in human hearts. And so, so far what
we've done is used this retrovirus, which only infects dividing cells, and
we haven't done it yet with the chemicals. But what we have
done is the following. We've taken pigs and blown up a balloon
in their left anterior descending artery, which is the one that's commonly
occluded in humans in an infarct. We've created a big
infarct and waited three days, put these pigs into an MRI machine,
to determine the degree of cardiac dysfunction that we
were left with. And then two days later, we've treated these
pigs with gene therapy using a retrovirus that infects dividing
cells, the fibroblasts namely and not the myocytes. And then waited two
months, put them back in the machine, measured if there's any
improvement in their cardiac function. So this we did a bit to mimic
the human condition, and what you're seeing here are the results
of that experiment in a group of about 5 pigs treated with
the factors, and the four pigs with the controls. And we haven't
done it yet with the most robust effort with the chemicals,
or with the really proper gene therapy vector. But despite
that, what we're seeing is if you compare the change in
ejection fraction from the point where there's been damage,
and compare every pig to itself every time. What we can see
is that there's a significant difference between the treated pigs
and the control pigs over that two month period.
So this type of difference of about 7% in the absolute
ejection fraction is significant, it's about a 25% relative
improvement from where they started. And we think that we can actually
even do much better than this by testing with these chemical inhibitors
that I mentioned. And with the proper vector that can get into
more of the cardiac fibroblasts. So, we've tested whether or not
these chemical inhibitors I mentioned actually help the human
cardiac fibroblasts reprogramming, and it turns out they
do. And not only do they help the reprogramming like they
did in mouse, it turns out it's simplifies the gene cocktail
that's required to only the three genes you see here
that can give us pretty good reprogramming with these
human cardiac fibroblasts that you see have these nice
patterns that we call sarcomeres in these cells.
So, the reason this is important is that with this combination
we're getting down to a small enough number of genes
that you could imagine packaging those genes in a gene
therapy vector that's non-integrating in the genome, which
would be the most clinically viable approach. And that approach would involve
an adeno-associated vector. Now, the problem with adeno
associated vectors turned out to be that if you wanted to infect a
cardiac myocyte, we actually have great AAVs to do that.
But if you want to infect a cardiac fibroblast, which would be
the target in this therapeutic approach, none of the currently
available AAVs do that very well. And so to overcome this
obstacle, we took advantage of the fact that our colleague, David Schaffer at
Berkeley, had developed this error prone AAV library, where
there are millions of random mutations that are going on
in these AAVs. So you can choose from those millions
and try to identify one that have tropism for a human, and in
our case pig, fibroblast over a human cardiac myocyte. So the way
we did that is we did rounds of selection, asking are there any
AAVs in this pool of millions that can efficiently infect human
cardiac fibroblasts? And then we take those and we ask, to remove
those that could efficiently infect a human embryonic stem cell
derived cardiac myocyte. And we did this round of selection
about seven times. And finally came up with an AAV that
had in fact greater tropism for a human cardiac fibroblast.
So then the question is, can this vector actually express
high enough levels of these reprogramming factors to
actually convert the cell. And so we cloned in the genetic
material into this vector, put them into a human cardiac fibroblast.
And what you see here is a cell that used to be a fibroblast, and I think
you can appreciate is that it's vigorously beating just one week
after introducing these genes. So we feel like we've now
got a good cocktail that can be used to induce cell fate conversion.
We've got a good vector to deliver these, and we're ready
now to do the final trials in large animals before moving
this technology towards a human clinical trial. And there are many hurdles that
have to be overcome still, in terms of safety issues, and delivery.
But we can begin to see a line of sight towards the clinical
application of this reprogramming technology. And this notion
of harnessing cells in your body to reprogram them to the type
that you want is really not unique to the heart, but in fact,
could be utilized for many different organs in the body, because all
we have to do is be able to crack the code, if you will, for
what are the minimal essential genes that will dictate a
cell fate. And then introduce that in the proper organ, in
that setting. And others have now begun to show this for a
variety of different organs, including cells in the brain, the liver,
pancreas, spinal cord, etc ... And so we believe this paradigm
of in vivo cellular reprogramming could be used for many
different organs that might benefit from a regenerative approach.
And I think we'll see that in the coming years. So, what I've shown
you today is how we can utilize the knowledge of the key
developmental biology networks that are involved in cell fate
determination and organogenesis. I've shown it to you in the heart, but the same
is going on for many different organs in the body, and how we can
use that information to both understand the basis on a deep mechanistic
level of human birth defects, but also how we can apply that
knowledge to regenerative medicine in the adult.
So, with that I'd just like to thank the members of the
laboratory that did the work that I shared with you.
Those individuals are shown here and range from very
talented graduate students and postdocs to very dedicated
research assistants. And we've had a number of collaborators
throughout our scientific community that have greatly
contributed to the work. And thank you for your attention,
and I hope this has been informative.