Silence can be a pleasant experience. Imagine
standing in the middle of the Utah desert
on a warm summer day. It's quiet, it's warm,
and it's relaxing. Now imagine you're born
deaf, you cannot hear, you cannot communicate
by spoken words to your friends and family.
Life has only recently got better for hard
of hearing patients with the development of
modern hearing aids and cochlear implants.
But with new technology new challenges arose.
Our ears have only evolved to respond to sound
with very high sensitivity and high selectivity.
They have not evolved to tolerate some of
the man made sounds that have very unusual
and unnatural physical characteristics. These
sounds are for example explosives, industrial
noise, musical instruments, power tools, and
headphones. The rise time and the peak energy
and the sustained duration of some of these
sounds are highly detrimental to our hearing.
In addition, some modern drugs, chemotherapy
drugs, as well as certain antibiotics, are
hazardous to our hearing. This in combination
to the effects of aging and genetic disposition
in some instances, can cause a worldwide pandemic,
and this is what we're currently experiencing.
There are about 350 million people all over
the world that suffer from disabling hearing
loss. At the center of our sense of hearing
is a very interesting cell. It is called the
hair cell. They sit deep within our brain
and are enclosed by a dense bone. They have
nothing to do with the cells that are on your
head, the hairs on your head. These are modified
neurons. The name giving part for the hair
cell is a hair bundle. It is a protrusion
of the cell sticking out of the epical surface.
And bathing inside the inner ear fluids where
they become stimulated by sound waves and
acceleration forces. This conversion from
a mechanical energy into a chemical energy
can be nicely visualized when one fills a
hair cell with a sensitive die that responds
to the calcium ions that influx into the cell.
This happens at the top of the cell where
there is a probe attached and this moves the
hair bundle to the right side. And whenever
it pulls on it and moves it to the right side
you can actually see the flash of the ions
entering the cell. This is visualizing the
process of the conversion of mechanical energy
into chemical and electrical energy very nicely.
Many labs around the world are interested
in finding the molecules and the molecular
components of this transduction machinery.
And as I will explain in a second there are
many reasons why this sense hasn't really
been elucidated at the molecular. And I want
to mention one other thing. I mentioned deafness.
And the issues with deafness. Hair cells that
are within your brain are very scarce. There
are only 12,000-15,000 in the human cochlea.
And they do not regenerate. Once they are
lost in response to a toxic sound or to an
antibiotic it cannot be regenerated by natural
means. Therefore overtime you constantly and
gradually lose your hearing and it cannot
come back. It is uncurable. That is one of
the major points as to why deafness has not
gotten a cure yet. So the two points of interest
of research are how does it work this conversion
of mechanical energy into electrical energy
and secondly how are we able to bring back
lost hair cells to cure deafness. I want to
illustrate one of the reasons why it has been
so difficult to get a grasp of the hair cells
in the inner ear. Our brain has 100 billion
neurons. This was a huge number for me up
until 2 weeks ago. (laughing) Besides the
100 billion neurons in the brain there are
12-15 thousand cochlear hair cells. If you
transfer this ratio to a city like New York
City, with 8 million people. If these 8 million
people would correspond to the number of neurons
in the brain, the equivalent of the hair cells
that you could get out of a single cochlea
would be equivalent to one person walking
down 42nd street. Another comparison for a
meaningful drug screening essay, I would assume
you would need 1 million cells that you could
grow easily in a standard cell culture dish.
To fill this culture dish with inner ear cells
you would need the equivalent of 40 experimental
animals. I think this is not only impractical
but not ethical and I would not be able to
look into the eyes of my dogs when I go home
at night. So we needed to come up with another
source, a renewable source, so we turned our
attention to embryonic stem cells. Embryonic
stem cells can give rise to every cell type
in the body. And we thought maybe theres a
way of suppressing all the other organs that
are developing in the body and enhancing the
formation of ear cells. So how do we do that?
We learn from development. The body, the mouse,
the human, any organism already shows us how
to make an ear. We just need to look carefully.
We see that the early signs of ear development
is the thickening of the outer layer of the
embryo, this layer is called the ectoderm.
This surface layer thickens at an early stage
in a mouse after 8 days of development. And
shortly there after this thickening of the
outer surface is dimpling in, its invaginating.
And this dimple is later forming a vesicle,
a round ball, that goes on and forms the ear.
So there are 3 things we can learn from this.
First is that the ear develops from the surface
layer of the embryo. So we need to get embryonic
stem cells to form surface cells. Second there
are many genes already present that are expressed
and present in this dimpling and thickening
of the outer layer. One of them is shown here
in red. Its a gene called PAX2. And PAX2 is
very important for ear development. If you
lose it your ear doesn't form correctly. Many
of the cell types are missing. Therefore many
of the cell types are missing. Therefore we
think that PAX2 is a very important marker
that we can use for our studies. A third finding
comes from experiments done from a colleague.
Who took a ball, the vesicle, and he transplanted
from the embryo, the ear region, to a limb.
And what he found is that all the major cell
types of the ear develop no matter the environment
that it was transplanted in. This means the
cells that are red here have all the information
to form the major cell types of the inner
ear. So we strategized and we thought maybe
theres a way of just generating these PAX2
expressing cells. The way we did this was
to coax embryonic stem cells first to develop
into the surface layer. Then we looked at
what other people have found about ear induction
and how the ear forms. There are certain proteins
a group of growth factors called the FGFs.
And when we add these growth factors to the
surface layers of cells we can see that they
upregulate in a dramatic fashion this PAX2
transcription factor. So what can we do with
these cells? One experiment a post doc of
mine did was he took these cells and injected
them back in to the ear into the otic vesicle
in the stage where we think the cells are
in development. Namely in the stage where
all the cells are PAX2 positive. ANd he used
a chicken embryo. And I mentioned that the
ear develops from a little ball. And then
we let the ball develop into a fully grown
inner ear. And after that happened we just
took a slice. We were looking for the mouse
cells the mouse embryonic stem cells that
we had coaxed into early ear cells that they
were able to form ear cells. And what you
can see here is the in green are the mouse
cells. All the other cells surrounding the
mouse cells that are not green are chicken
cells. And in red is a hair cell marker that
labels sensory hair cells and the yellow color
is the composite color of the green and the
red. And this means that the yellow cells
on the right side are mouse hair cells in
a chicken inner ear. So what have we learned?
I think that one thing we have learned is
that we can generate from embryonic stem cells
which is a renewable source and we can generate
as many cells as we like as long as we have
a large enough incubator, We can generate
hair cell like cells, cells that look like
hair cells and express genes that hair cells
have, they also have some functional characteristics
of these cells. They do not look perfect.
Especially if you compare them with hair cells
that are present in the mammalian and the
human and mouse cochlea. They are little away
from perfect. This is an example on the bottom
of a native cochlear hair cell. Another interesting
thing that we learned is that combining other
technologies with this technology. We are
now able to find proteins and genes that are
at the correct location for the transduction
machinery. I mentioned previously that we
do not know a lot about how the mechanical
stimulation is being converted into chemical
and electrical energy. And this is one of
the new findings that we have is the protein
that is at the tip of the hair bundles right
at the correct place. I think that the future
will tell us a lot. I think that in the next
decade we will probably know how these cells
actually work and the ear catches up to smell
and taste in that regard. And being in a medical
center, we always wonder where does this research
lead us. And i'd like to illustrate this here.
There are two pathways that we are going right
now. On the top you see where the hair cells
are located in the human cochlea. And the
hair cells are the dark blurbs in there. And
after a toxic insult, they are gone. This
is what the inner ear of a person that is
deaf looks like. Theres a single cell layer
of a damage cochlea. Now with stem cell technology
there are two pathways that are imaginable.
One that is shown on the left is cell transplantation.
And we have shown that at a very rudimentary
stage in the chicken embryo we transplant
cells that have the ability to repopulate
these single cell epithelia, and then hopefully
lead to a epithelium that has reseeded hair
cells that at some point for a patient will
hopefully hook up to the nervous system and
work properly as the original cells were.
The other pathway that I think is very promising
is to use stem cell generated cells in culture
dishes to use them for high throughput drug
screening without killing so many animals.
In fact you do not have to kill any animals
for this kind of research because we do everything
in a test tube. And once we have positive
candidates we can apply them to the same pathway
seen on the right and hopefully get regeneration.
And i think this is the goal of my lifetime
to at some point be able to reach the bottom.
So very egoistically that I'm not turning
deaf at some point. Taking me out of play,
there are other people out there that this
technology can really help. So this is quite
motivating and I'm glad to be at a place like
Stanford where there is a lot of collaboration
going on where we can really achieve this
type of goal.
