cc >> Today's speaker for DART
talks will be Dr. Sean Palacek, who works with the Chemical and
Biological Engineering Department.
He'll be speaking on tissue engineering, specifically skin
for regenerative medicine. ( no audio )
As much of a discussion as possible to keep me from just
talking and you from sleeping. So please feel free to jump in
with questions, comments, your thoughts at any particular time.
Before I get started, though, I would like to ask how many
people here are engineers. Just a couple of us.
So one of the goals of what I want to do this evening is give
you a little bit of an insight into how engineers think about
stem cells and regenerative medicine.
I'm going to talk a bit about some of the work in our lab
that's trying to generate skin from embryonic stem cells.
These approaches really can be generalized to a large number of
cell types that are of therapeutic interest.
The topics we're going to discuss, first a little bit of
an introduction into what is an embryonic stem cell; why are
these of interest to regenerative medicine; and
really, why have these cells rejuvenated the field of tissue
engineering. Tissue engineering went through
a big downturn in the 1990s. A lot of the money that went
into the field didn't end up in clinical products.
And investment declined dramatically.
This money has started to flow back in the field because of the
potential use of stem cells. Then we're going to talk about
embryonic stem cell self-renewal and differentiation, ways that
we can design culture systems that let us grow up the large
numbers of cells that we would need for regenerative medicine
applications. And finally, we'll discuss how
we can use stem cells in therapeutic applications,
specifically, skin tissue engineering.
The first human embryonic stem cells were derived on campus
here. I think most of us know this, in
Jamie Thomson's lab in 1998. I'd like to go through this
process so we can better understand what these cells are
capable of. Most of the current embryonic
stem cell lines are generated by the process of in vitro
fertilization. So you mix a sperm and an egg in
a dish. You grow it for about five days.
It grows to the blastocyst stage.
Here we have on the order of a hundred cells.
There's an inner cell mass surrounded by a trifectaderm.
This is before implantation of blastocysts into the uterine
wall would occur in vitro. The inner cell mass is isolated
and the cells are then cultured in a dish on fibroblast feeders.
Most of the cells die, but occasionally you'll get a cell
that survives and proliferates. These form the embryonic stem
cell line. These embryonic stem cells are
not quite identical to the inner cell mass.
There are some differences in function and gene transcription.
But what's important to note is that the embryonic stem cells
are really the earliest cell type that we can easily study.
Here's a picture of the first embryonic stem cell line.
These cells grow in tightly packed clusters.
They really like to have cell-cell contact.
So if you isolate a single cell, they don't survive very well.
Although the power of these cells comes from the fact that
they can differentiate to any cell type in the adult.
So things like pancreatic cells are of interest for treating
diabetes. Cardiac cells for heart disease.
And neural cells for a variety of nerve disorders.
A way we can get cells to differentiate is just by pulling
them off a plate and culturing them in suspension, if we remove
of the self-renewal factors. So you get these spheres of
cells called embryoid bodies. You can think of this as
recapitulating some of the very early developmental stages, but
it's in a very uncontrolled manner.
We don't see the structure form that gives rise to the human
body. You do see clumps of different
cell types arise in a seemingly random pattern.
So what we'd like to be able to do is control this
differentiation. Before we talk about that, I
want to address this idea of the difference between embryonic and
adult stem cells. So embryonic stem cells have
certain advantages over adult stem cells.
Adult stem cells are found in a lot of tissue.
They can be harvested and used in therapeutic applications.
And adult stem cells are currently being used for
therapeutics. I think embryonics have certain
advantages that may give them the leg up in the long run.
So embryonic stem cells have an enhanced proliferative capacity.
We can take a single cell line and theoretically culture it an
infinite number of times. They do change upon passaging,
however, so there is some effective limit.
Whereas adult stem cells, like the cells in our body, have a
limited number of divisions. There's a difference in
differentiation, as well. Embryonic stem cells can form
any cell type found in the body, whereas adult stem cells form
cell types related to the tissue which they're identified with.
So from an engineering point of view, the embryonic stem cells
give us a much more scalable processing uniform starting
material. We can, in theory, generate an
unlimited supply of any tissue type found in the body from a
single cell source, whereas adult stem cells, there's a
limited availability, not very many people want to donate their
brains to harvest the stem cell. And because of the expansion
potential here, you can only generate a limited amount of
product. We've touched on these
properties that give embryonic stem cells a lot of hope, and
perhaps some of the hype that's been associated with them.
The three main applications for these cells, and one is
biological, gives us a window into some of the early
developmental processes in humans.
We can study this in a dish, whereas it's not ethical and
feasible to study it in the body.
These cells can be a source of cells and tissues for screening
effects of drugs and other compounds on human tissue.
Again, it's hard to come up with human heart and brain tissue,
but there's a tremendous interest in pharmaceutical and
biotech industry in learning how different compounds will affect
these tissues. So if we can grow them in a
dish, they give us a source of cells for the screening.
And then there's the regenerative medicine aspect.
Embryonic stem cells give us a source of tissues that may be
able to be implanted in humans to repair diseased or damaged
tissue. There's also a downside to
these cells. Because of where they're
generated from. Until recently, it required
destruction of human embryos to generate these cells.
Some exciting work from Jamie Thomson's work here on campus,
and a lab in Kyoto, last November, has potentially given
us a way to address this problem.
What they've been able to do is take adult cells, in both cases,
skin fibroblasts, and express four genes in them that are
highly expressed in embryonic stem cells.
The set of four genes is different in the two studies.
But the results are the same. These cells return to an
embryonic stem cell-like state. So we go from these very flat,
large fibroblasts to a small, tightly packed embryonic stem
cell. In addition to preventing the
destruction of embryos, what this may mean, is that people
can generate embryonic stem cell lines from their own cells and
tissue. These can then be used for
therapeutic applications. So this is really a tremendous
breakthrough that the field has been waiting for.
There's still a lot of challenges in actually using
embryonic stem cells in therapeutics, though.
And today, we're going to hit on a couple of these, and not
discuss some of the other ones. So one of the problems is that
we really don't have very good tools to culture these cells,
either at the research scale, or to scale up growth for clinical
production. We don't know how to create most
cell types from embryonic stem cells.
So we have the potential to create an unlimited number
amount of tissue from the cells, but we need to learn how to
guide differentiation to the desired fate, so we get high
purity functional cells. Then once we can do that, can we
scale up growth and differentiation to get enough
cells for use in clinical therapies, and even clinical
trials? Some of the other problems that
I won't go into today are immunogenicity.
Can we generate solid tissues that won't be rejected.
And one way to do this may be with patient specific stem
cells. And teratomal formation.
An unfortunate consequence of implanting these cells,
embryonic stem cells, directly into the body is that they can
form teratomas, or tumors. Obviously, this is undesired, so
we have to make sure that we're not putting undifferentiated
cells in with our differentiated desired cells.
So the first topic I'd like to discuss, how we can grow these
cells and what factors control what happens to these stem
cells. So from an engineering
perspective, what we're interested in is providing
signals to the cells that will either keep them in an
undifferentiated state, or allow them to form the cell type we
want to make. There are four factors that
we're going to discuss. One is soluble factors.
You can add chemicals to the medium.
These will bind to receptors and tell the cell to either stay a
stem cell or begin to differentiate.
Extra-cellular matrix, so the immobilized protein the cells
bind to can guide the differentiation phase.
Mechanical cues. I'll talk about this in more
detail, along with intercellular contacts, the cells stick to
each other and this signals for them to grow, self-renew or
differentiate. Here's a schematic showing how
we can combine all these different factors to lead to
desired outcomes. So we may want to control
cell-cell renewal as expand and differentiated cells,
differentiate them to the correct phenotype.
Other cell processes, we may be interested in acquiescence, when
cells stop growing, or even apoptosis, where we can select
against certain undesired cells. This raises an issue of the
complexity of the environment. So what we're drawing the arrows
in here suggest that the cells have mechanisms to sense all of
these signals in the environment.
What we don't know is how the cells process this information.
So the context of all these signals, with respect to each
other, is critical. Also critical is the temporal
nature of how you present these cells.
This is not like you have an embryonic stem cell one day and
you sort some factors on and the next day you have a beating
cardiac cell. This process can take weeks to
months. And you're going through many
intermediates. The environment that gives rise
to say, the first intermediate, may not be the same one that
gives rise to the final differentiated cardiac myocyte.
We want to be able to dynamically present these
signals to the cells to get the desired response.
I'd say the vast majority of the field is working in --
factors. And I'll talk about that a bit
today. I also want to address some of
the under-appreciated factors, such as mechanical stress and
cell-cell contacts. So biology is really
identifying a lot of the pathways and sibling molecules
that regulate whether a stem cell decides to stay a stem
cell, or whether it wants to become something else.
There are several pathways that are indicated here.
There's a lot of receptors on the cell surface that bind to
growth factors secreted by other cells.
And then this will turn on self-signaling pathways to gene
transcription that change the cell state.
This is not a complete list. And we're just beginning to
learn how these pathways interact with each other.
Question? ( inaudible )
You mention that human embryonic stem cells have the
potential to go to differentiate into any tissue of the body.
Yet, I haven't heard of, you know, scientists actually making
a three-dimensional tissue structure, like a heart or
something like that. What are the limitations behind
actually seeing published work, as in the first synthesized
human heart from these embryonic stem cells?
Is it the fact that we don't necessarily know every soluble
factor along its differentiation?
Or is it just like the three-dimensional molds,
something along those lines? >> So there's a couple problems
with that, actually taking the cells and forming a functional
organ. The first that you mentioned is
getting enough of the desired cell type.
And I think that's really what the field is concentrating on
now. We can generate cardiac
myocytes, which are the little cell type that causes the
contraction. But I think an even more
pressing problem is how we reassemble those.
So the cells that we can obtain, whether they're cardiac cells or
nerve cells, are randomly oriented and are not connected
to each other in the correct spatial organization.
That's going to be something that we really need to address.
And I'll talk about that in a very basic sense, when we start
to talk about the skin. But in a sense, the field has
to be able to generate, well, there are two general approaches
here. One is to try to generate all
the cell types outside the body and then reassemble them onto
scaffolds and then implant that. That's going to be very hard.
The second approach is to take progenitor cells and put them
directly into the body. And hope that the environment in
the body will help guide their further differentiation and
organization. If that works, it'll be easier.
But it's not clear that's going to work in all cases.
So these pathways primarily respond to chemical growth
factors that are in the medium and bind to receptors on the
surface or inside the cell. What I want to talk about in a
little more detail, are how other signals can affect growth
or differentiation. So we're interested in how
mechanical forces can stimulate cell differentiation.
There are a large number of examples in development, how
mechanical forces are required for tissue to properly form.
One example is cardiac myocytes, the actual beating activity
during heart development is required for your heart to grow
properly. Then there are epithelial and
endothelial cells that form linings of your body.
Your lung epithelia respond to the shear of your breathing, and
then the expansion of your alveoli to completely develop.
That stimulates their growth and differentiation.
Keratinocytes, which form your skin, respond to stretching.
So your skin is thicker in certain parts of your body, like
your knees and your elbows, where they're constantly
stretched and mechanically stimulated.
Bones and cartilage are stimulated by pressure to
strengthen and develop. Also, mesenchymal stem cells
can respond to different types of pressure and strain to
differentiate to bone and cartilage.
So what we decided to do was look at how mechanical forces
affect embryonic stem cells. Our initial hypothesis was let's
apply forces to stem cells and see if they then form those
other cell types that I showed in the last slide.
And we're completely wrong. So that's one point one mentions
about science. Sometimes what you look for
isn't what you see, but it's still going to be interesting.
In our study, we grew embryonic stem cells on a flexible
silicone rubber membrane substrate here.
So we have cell colonies on a flexible silicone membrane.
And we can apply a vacuum onto that membrane.
What that does is stretch the membrane and applies a strain to
the membrane. The strain is transmitted to the
cells. And we do this in a cyclic
manner. So we apply a vacuum, release,
apply a vacuum, release. And we can control the magnitude
of the strain, as well as the frequency of the strain.
What we found is that when you strain the stem cells, it
actually inhibits the differentiation.
They're more likely to stay a stem cell.
Now, one thing I didn't mention, the culture is that if
you keep these embryonic stem cells in colonies, and let them
grow really large, they begin to spontaneously differentiate.
In a typical culture, what you do is every five days or so, you
take all of your cells and harvest them and enzymatically
or mechanically chop them up and re-plate a certain fraction of
them. In this case, we allowed the
cells to grow for a week, and just let them keep growing.
We got these very large colonies and we'd get these areas of
differentiation. The cells that don't express
this Oct4 and aren't in green, are some cell type.
We don't know what they are, but they're no longer embryonic stem
cells, whereas these cells here are still stem cells.
If we apply strain to those cells, so we're stretching them
ten percent, so just elongating them by ten percent longer than
their normal length, at ten cycles per minute.
So every six seconds, we go through a cycle, we keep them
completely undifferentiated. After two weeks, the effect is
more dramatic. So cells that aren't exposed to
strain will undergo spontaneous differentiation.
Almost every cell in this culture is no longer a stem
cell. We don't quite know what they
are, but we know they've differentiated to some
particular lineage. Whereas the cells that we apply
strain, we have some differentiation, but not very
much. We can quantify this.
How we do that is label the cells with an antibody that only
binds to a protein expressed on the surface of embryonic stem
cells. Here, we applied ten percent
strain, ten cycles per minute for ten days.
And here, two populations. So any cell in this region out
here is undifferentiated. The cells that were exposed to
strain, primarily undifferentiated.
The cells that weren't strained are differentiated.
What happens if we drop the strain to eight percent?
Surprisingly, it has absolutely no effect.
So the two strain, non-strained populations are identical.
They both have a large number of differentiated cells and a few
undifferentiated cells. This is a threshold for them.
And we have to be above a certain level of force to keep
the cells undifferentiated. Below that level, it's like not
even having any force at all. We did quite a bit of
characterization here, and I don't want to go too much into
the details. But what we're really interested
in is how are the cells sensing force and what's it doing to the
cells. We looked at a large number of
pathways. And it looks like the force is
somehow stimulating the TGF-beta pathway.
So in embryonic stem cells, TGF-beta is a small protein that
binds to receptors on the cell surface.
Then this turns on a pathway that leads to gene transcription
and keeps the cells in an undifferentiated state.
Their related protein is called bone morphogenic proteins that
stimulate differentiation. A number of other labs have
shown that this TGF-beta signaling keeps cells in
undifferentiated and pluripotent state.
And that they activate this complex called SMAD2/3, which
then goes to the nucleus and turns on gene transcription,
whereas the BMP activates the SMAD1/5 that promotes
differentiation. I won't go into the real
experimental details here either, but what we found was
that in the cells that we apply the stretch to, they turn on
TGF-beta expression by about a factor of ten-fold.
So stretching the cells causes them to make TGF-beta.
They secrete this into the medium, and it binds to
themselves and their neighbors. And then this is an autocrine or
paracrine move that keeps the cells in an undifferentiated
state. If we add this chemical
inhibitor of the signaling, we keep the turnoff signaling
through the TGF-beta receptors, and the cells will
differentiate. We also just looked at
signaling activity. If we look at the level of the
signal downstream of TGF-beta receptor, the strain turns on
this SMAD. If we don't have strain, we have
very little phosphorylated SMAD, and we can inhibit it with this
inhibitor. So the signaling through
TGF-beta directly correlates with the cell state.
We think this is an example of how cells sense mechanical
signals. They sense chemical factors, and
then somehow they process this information to keep them in an
undifferentiated or differentiated state.
The other aspect I'd like to talk to you about in terms of
cell culture engineering is how do we control cell-cell
interaction. The colony I showed earlier, the
small picture here, these colonies grow as
three-dimensional clumps. There's a layer, and it grows
hemispherical clumps of all different sizes and shapes.
There's a lot of anecdotes in the field about how small
colonies may be better for a particular lineage, and big
colonies may give you a different lineage.
But there have been very few studies that really indicate why
or methods that give us methods to actually control this.
One of the first studies that was published in 2005, what that
lab did was quantify the size of clumps that give rise to two
types of blood cells. The myeloid lineage and the
erythroid lineage. The way they do this they take a
plate of cells and enzymatically dissociate these into single
cells. And they count cells, put them
in a small tube and centrifuge them, just spin them together
into a clump. Put these on a plate, expose
them to growth factors that can either stimulate myeloid or
erythroid growth. They found that for the
erythroid lineage, there's an optimum cell number, whereas for
the myeloid lineage, as long as you're above a certain
threshold, you get a lot of differentiation.
This method is very labor intensive.
And it's somewhat artificial. Because as you force these cells
together into a clump, they tend to like to disaggregate.
So we decided to try to develop a method that really lets us
control the size and shape of these colonies as they grow.
This gets us into some of the materials engineering world.
So what we want to do is form these surfaces, they're called
microwells. These little three-dimensional
wells that we can put cells in and they will fill up the wells
to form an aggregate of a certain size and shape.
We create a rubber stamp here using a silicone wafer mold
that's manufactured somewhat like you would manufacture a
computer chip, although it's much simpler than that.
Then this rubber stamp forms a template for polymer molds that
we're going to use to make the microwell.
In this area between the spacer and the stamp, we inject a
liquid polyurethane. This gives us this microwell
shape. What we want is for cells to be
able to attach and grow inside the well, but not the area
between the well. We're going to treat this area
outside the well with a compound that keeps cells from sticking.
We deposit gold at an angle. Gold is vaporized and deposited
at an oblique or steep angle, like this.
The gold will hit the area between the wells, but will be
shaded from the bottom and side, because we're coming at this
angle, it can't hit the bottom. Then we're going to react a
molecule with the gold. This molecule is going to keep
the cells and proteins from sticking.
So this molecule, one end will bond to the gold, and the other
end is a trophyline glycol. The trophyline glycol inhibits
protein cell attachment. When we add protein, it can only
attach to the bare polyurethane in the bottom.
We add cells, they'll attach to the protein and grow to fill
these. What we're left with is
somewhat like an ice cube tray, but all of the trays are filled
with cells. Here's a phase contrast image of
what these wells look like. These are 200 microns on each
side, and about 100 microns deep.
So these wells will hold, on the order of 1 to 5,000 cells.
If we incubate with protein, the protein only goes inside the
well. Then we add cells and the cells
will grow to fill up the well. These areas in here, what you
see are bubbles that are under the surface of the polyurethane.
So if we do a stain for dye, you'll only see cells inside
these wells. What's going to happen is we
put in a relatively small number of cells.
They'll bind and start growing. The cells will grow in three
dimensions and fill up the well. So here you can see, the
differences in color are because you have different levels of
growth. Here we have just a couple
layers. And this dark color indicates
that we're growing above and outside of the well.
You can see the number of cells here are larger.
The Calcein AM just indicates that these cells are actually
alive. The cells have been cultured
for 19 days without passaging. At this point, they mostly
filled up these wells. The cells remain proliferative.
Even as they fill up the wells, they keep dividing.
What they're going to do is grow up outside the well and form
these little mushroom type caps that will eventually break off
into the medium. So a side view, cutting across
the well and showing the colony is provided here.
The green shows the cells are undifferentiated.
The nuclei are shown in red. This sample has been somewhat
mangled, because we took a razor blade and chopped it up.
Eventually what will happen is the colony will fill up the
well. It will grow up above.
And as we add medium or change the medium every day, it's just
going to shear that off. So what we're left with is a
full well of cells. Eventually, we want to be able
to pull the cells out of these wells and then use them, either
in growth or differentiation type experiments.
We can do that enzymatically, if we add an enzyme that's going to
break the contacts between the cells and the substrate.
What we're left with are these little cube in suspension.
These are all of the same size and shape.
Once you get them to differentiate, you culture these
in suspension to keep them from clumping together.
Overnight, they're going to switch from this cube shape to a
spherical shape. One of the things that we
didn't expect is that when we culture cells in small wells,
they remain almost completely undifferentiated.
So this is somewhat analogous to what we saw with mechanical
strain. When we stretched cells, they
don't undergo spontaneous differentiation.
Also, if we keep the colonies fairly small, it keeps them
differentiating. We don't really know why yet.
One theory is that just as the colonies get very large, they
may secrete factors that once you get to a critical
concentration, cause differentiation.
I think where this technology is most useful is that it lets
us get uniform populations of stem cells.
The size distribution of an unconstrained, or just a typical
stem cell culture is shown in these light blue-green bars
here. What you get is aggregates
ranging from 160 to greater than 500 microns in diameter.
Using microwells, we can create these five different completely
dispersed populations using different size features.
This is a way to make the culture much more uniform, which
then lets us control the environment.
What we're interested in doing now is looking at how this
initial starting size of the colony affects what cell type we
get. So by culturing the cells in
suspension, we get this much more homogeneous distribution in
sizes and when you compare it to the standard embryoid body
method. We're working with a number of
people on campus here and around the country to look at specific
lineages. So what size of embryoid body
gives is the most myocytes, for example, insulin secreting cells
or neural cells. So just to talk about the
self-renewal aspect, to summarize that a bit before I
move on to tissue engineering. A large number of labs around
the world have identified soluble factors that keep cells
in the undifferentiated state. The message that we're trying to
get across is that it's not only soluble factors that are
important. But that we can keep cells
undifferentiated by applying mechanical forces to them.
And this appears to work by stimulating TGF-beta cell
signaling. Also, cell size is important.
So regulating cell-cell contact can affect differentiation.
Keeping cells small and minimizing cell-cell contact
will keep them self-renewing. But if you keep them large, you
begin to see spontaneous differentiation.
So I think I'll stop and take any questions or comments here
before we move on to the tissue engineering aspect.
Yep? >> So the mechanical strain, you
said, can keep the cells from differentiating, but isn't that
just a direct effect of the TGF-beta?
Couldn't you just add the TGF-beta to keep the cells from
differentiating? Is the strain just a more
legitimate way to do that, or is there a reason you'd prefer to
do it with the strain rather than just adding the growth
factor? >> Good question.
So I didn't show all the detailed experimentation here,
but adding TGF-beta doesn't completely replicate the effects
of strain. It replicates about 75% of it.
So we think that's the dominant effect.
But not the entire story. Yes, if your goal is to keep
cells in self-renewing state, I think the best way to do it, if
you can add growth factors is to add growth factors.
There's a reason so many people are still doing those types of
signals, because they're much easier to manipulate.
Trying to scale up how you apply strain is difficult.
But one of the messages here is that when you are designing a
culture system, it's important to keep these things in mind.
So other types of forces that are important include shear.
So if you're trying to culture these cells in suspension, you
can't get around the fact that you're going to apply shear to
them. We really need to know what
effect that will have on the cell.
Other questions? So then let's talk about how we
can use these cells in therapeutics.
And there are a large number of labs on campus, in the country,
around the world, that are trying to generate specific
cells for therapeutic applications.
One of the ones I want to talk to you about today, not a lot of
people are studying. This is epidermal keratinocytes.
So, can we make skin cells? And I'd like to introduce why we
want to use stem cells for tissue engineering, and
specifically for skin tissue engineering.
The goal of stem cells in regenerative medicine and tissue
engineering is to take these very early, primitive human
cells and generate the perfect replacement for cells, tissues,
and organs that may have been damaged or diseased.
There are two main ways to this. I think we talked about it in
response to some of the questions before.
But either an in vitro method or an in vivo method.
In the in vitro method, what we do is take these embryonic stem
cells, differentiate them into specific cell types, and
assemble these cell types into an organ, and put that
functional organ into the body and assume it stays relatively
static. This is nice to think about,
because it's a very controlled environment.
You can control what happens at each differentiation, and you
get a fully functional organ that you can put in the body.
The problem is it's too complex at this point to really be
effective. Then there's the in vivo
method, in which we directly place the stem cells, or their
precursors, into the body. And hope the environment at
which you place them will guide their development and
differentiation. This is much easier.
You can take stem cells or their early progenitors, toss them
into a body and see what happens.
So even though it's easy, the problem is that it doesn't work
very often. If you directly put those stem
cells in the body, you'll get teratomas.
If you put early progenitors, the environment isn't quite
right to stimulate their total development.
That makes sense because in the early steps of human
development, the environment is different than it is in the
adult. So there will probably be some
blend of the in vitro or in vivo types of approaches that will
work. And that blend will be different
for different tissues. A lot of different cells have
been shown to be generated from embryonic stem cells.
This is a bit old, and it's very incomplete.
But you get neural cells, skin cells, cornea, cardiac, bone,
blood cells, liver cells. So we're beginning to be able to
make functional adult cells and embryonic stem cells.
I think one of the big questions based in the field is how do we
put these together and use them in a therapeutic sense.
Just because you can make a neuron doesn't mean you can put
it at the right spot of the brain or spinal cord and have it
interact with the local environment and be wired
properly. Some of the challenges that are
facing the field that I think we need to address, as scientists
and engineers, include the low yield.
The cell types I put up on the last slide, we can make them,
but the actual number of each cell type is relatively low and
the purity is low. A lot of this relates to the
complexity of the environment. So the types of signals you give
to cells are going to be important.
And it's not just add this one growth factor and you get your
desired cell type. Defined conditions are going to
be important. A lot of the culture methods now
use animal-derived products, cultures with other cell types
to get these cells and tissues through FDA approval.
These add complications because it's hard to say one batch will
maybe be the same as the next batch.
Also, generating the cell that have the complete, correct
phenotype is important. So often, you get some
properties of a cell type, but not others.
One example, and there's been a lot of excitement in the past
week, that insulin producing cells have been generated from
embryonic stem cells, and that these can "cure" diabetes in
mice. The problem is that these
insulin producing cells don't entirely respond to glucose the
way they should. So there are certain functions
that are missing from these cells that are going to be
critical to their use. And finally, how do we assemble
different cells into functional tissues?
There's a three-dimensional orientation of cells that give
tissues and organs their true function.
This brings us to skin. So why are we studying skin?
Well, there's a market for tissue engineered skin products.
The two types of patients that would benefit most are burn
victims and people with wounds that don't heal.
There are about 10,000 deaths per year due to burns.
And burn deaths are caused by loss of skin function.
People lose the ability to regulate their temperature and
the water content in their body. Another million people
experience permanent disabilities every year because
of burns. So if we can generate
replacement skin that would improve the quality of life, or
perhaps save lives. Non-healing wounds is another
use for skin tissue engineering. And in fact, these types of
wounds are currently being treated by artificial skin
products. The two main types of
non-healing wounds are foot ulcers in diabetics and venous
leg ulcers. Both of these are caused by a
diminished blood flow into the extremities, which then results
in low rates of wound healing and diminished immune response.
And amputations in diabetics are common, typically in the feet.
And venous leg ulcers can also cause amputation.
The costs to the healthcare system runs in the billions of
dollars because of the number of people that have these types of
problems. In trying to engineer skin,
what we want to do is look at the natural tissue, figure out
what it does and see if we can replicate its structure and
function. Or, provide its function through
a slightly different structure. The critical aspects of skin to
keeping people alive are the mechanical barrier and
dessication barrier. You keep micro-organisms out,
infections out. You keep water in.
And some of the current products can do that.
Some of the other functions that would be nice to maintain
include sensory. Your skin contains nerves that
give you the sense of touch. Immune response to fight
infections. Thermal regulation.
This is often controlled by blood supply.
The skin is vascularized, and keeping its vascularization not
only keeps the cells alive, but helps regulate flow in the body.
Then there are also things like pigmentation, which are little
bit more cosmetic. The structure of skin is shown
here. You have the epidermis, which is
the outer-most layer that provides the mechanical
dessication barrier. And most of these other
functions are present in different cell types in the
dermis. We also have sweat glands that
involve thermal regulation, hair follicles.
It's actually quite a complex organ.
The first generation of tissue engineering products really
consist of only one or two subtypes that give us an
artificial epidermis. The maybe not so current skin
substitute, some of these are not currently available.
But historical skin substitutes are shown here.
The simplest type of artificial skin is really just an
artificial membrane. So you have, perhaps, a silicone
rubber membrane that's fused to a nylon mesh.
This has peptides on it that help the cells move and
repopulate the wound. You place this over a wound with
the silicone rubber membrane facing the exterior.
And as the wound heals, the cells will crawl in and
repopulate this mesh. At a specific time point, you
cut off the silicone rubber membrane.
Then there are more biologically based materials.
One is collagen based dermal analogs.
This is cadaver skin that's been treated to kill living cells.
It's just the matrix that you would place down.
Then this would allow cells to crawl in and repopulate the
wound. These are passive materials that
stimulate growth and proliferation of cells from
outside the wound into the wound.
The later generation skin substitutes have actual living
cells in them. One of them contains human
keratinocytes and fibroblasts in an artificial matrix of
extra-cellular proteins. This replicates the dermis and
epidermis structure. The problem with this material
is that when we implant it into a wound, it's not vascularized.
So these cells don't have a blood supply and they don't live
very long. What they do is the time that
they are there, secrete factors that allow cells to repopulate.
So these cells do not become a permanent part of the wound
site. Our vision is to be able to
completely replicate the structure and function of skin,
where you have vascularization, nerves, sweat glands.
How we're starting out though, is just to try to get some of
the cell types that are found in skin from embryonic stem cells.
So what we've been working on is keratinocytes.
These cells form epidermis. And they stratify into multiple
layers. The basal layer of keratinocytes
will grow over the dermis. Their role is to form a
mono layer of cells that are then going to divide and further
differentiate. So as they divide, they move
upward. They begin to secrete keratins,
which are water insoluble proteins, water resistant
proteins. The cells will eventually die
and form this barrier at the outside of your skin.
Typically, you have ten to 20 cell layers thick of
keratinocytes at any point on your skin.
About five years ago, a lab at Harvard was the first to
demonstrate that embryonic stem cells do form these skin
keratinocytes. What they did was take embryoid
bodies, or undifferentiated cells and injected these into
mice. They harvested the tumor that
arose. They found cells in the
keratinocyte lineage. So cells expressed the markers,
P63 and Keratin-14. This indicated that they're
roughly equivalent to these cells at the basal layer of
these proliferative cells. So this was a very important
study. It showed that it's possible to
make the keratinocytes from the embryonic stem cell.
The problem is this isn't a very effective way of doing it.
You have very low yield of these cells.
You end up killing a lot of mice.
And it doesn't give you much insight as to how these skin
cells develop. If you want cells for a
therapeutic application, we need to find a better way to do it.
And our approach is to see if we can induce this factor P63.
P63 is expressed in a lot of epithelial cells, so the cells
that form linings of virtually every organ in your body.
We figured if we can turn on expression of this, it will give
us progenitors that are more likely to form skin cells.
It won't necessarily give us skin, but it will get us closer
along that path. Our first strategy is to use
this embryoid body that we discussed earlier.
You take your undifferentiated cells that are growing in a
dish. And then you pick them off the
substrate and culture them in suspension.
They begin to differentiate to form a large variety of cell
types. The green cells here represent
the cells we want, keratinocytes.
You plate these on a dish coated with a extra-cellular matrix.
We happen to use collagen, because that's the main
component of skin. What happens, then, is your
keratinocytes will migrate out away from the other cells.
This makes sense, because their function is in wound healing.
They're able to crawl and move, close wounds very fast.
So they walk away from these other cell types.
We can then somehow isolate these keratinocytes, purify
them, and induce differentiation and stratification.
Here's an image from that migration phenomenon.
We dropped an embryoid body at this point here and then waited,
I believe a week or a week and a half in this experiment.
So the cells began to crawl out. This panel corresponds to this
image. So these cells still express
Oct-4. They're undifferentiated
embryonic stem cells. Whereas the skin progenitors
walked away from the undifferentiated cells, and
formed these relatively pure regions.
If we magnify the region here, we see that they have this
epithelial like shape. They tend to form tightly packed
sheets of cells. And they also express p63.
Question? Scale?
These individual cells here are roughly 50 microns wide.
So this colony here is on the order of a couple millimeters.
There are thousands of cells in that particular slide.
We did a fairly exhaustive analysis of the different
factors that will guide keratinocyte differentiation.
I don't want to go into all the detail here, but the soluble
media you grow the cells in is important.
So we took these embryoid bodies, put them in collagen for
11 days. Then we put them in different
commercial media formulations that are used to culture
keratinocytes. The idea here is that these
commercial media will contain a number of different growth
factors that keratinocytes like and this may stimulate their
growth. We found that some give us a lot
of keratinocytes whereas others are completely ineffective.
We're currently trying to figure out what factors are the ones
that give us keratinocytes that aren't in the others that
don't. The matrix you plate the cells
on is important. Here, we have the embryoid
bodies cultured for two weeks. We put them on different
matrices and cultured them for an additional month in the
defined certain mediums which we found gives us keratinocytes.
If you put the cells in matrigel, which is commonly used
to keep cells undifferentiated, we get a very low yield.
What happens here is that the cells actually don't
differentiate. You still get a lot of
undifferentiated stem cells. In serum, we get higher level
differentiation. Collagen, about 15%.
Interestingly, gelatin gave us better than collagen.
So we were able to obtain about 25% of the cells in the culture
are keratinocytes. We're not exactly sure what the
others are, probably a mix of a variety of cell types.
Question? >> When you're saying we have
keratinocytes, are you just referring keratinocytes as cells
that express the K14, or whatever or are you going
further than that and looking into more detailed expression?
Good question. Here, we're seeing keratin-14,
and that isn't sufficient to claim that they're actually
keratinocytes because certain other tissues will express
keratin-14. But later we looked at the
ability to terminally differentiate to express other
keratinocyte specific proteins. So it's probably not fair of me
to say that these are keratinocytes at this point.
These are cells that have the potential to form keratinocytes.
This is our best result using embryoid bodies, about 25%.
The problem with embryoid bodies is that by culturing the cells
in suspension in these undefined conditions, you begin to see
differentiation toward a lot of different choices.
So if we want a pure population, we either have to get rid of all
the other cell types or purify the keratinocytes.
It's easier, and perhaps more effective, to do a direct
differentiation protocol. So instead of forming embryoid
bodies and letting them differentiate randomly, you take
the adherent cells and you treat them with different compounds
that direct their differentiation from the start.
One of the factors that's commonly used to direct
differentiation is retinoic acid.
Retinoic acid is a form of vitamin A.
It's structure is shown here. It's been shown to be important
in skin development, as well as development of other tissues,
including neurons. Mice that lack the retinoic acid
receptor don't form basal keratinocytes with the same
structure as mice that have the retinoic acid receptor.
So our hypothesis is that if we apply retinoic acid at the
proper dose and time, we can specify for keratinocyte
formation in embryonic stem cells.
What we do is take these embryoid bodies, these colonies
of cells in suspension and apply retinoic acid as we form them,
keep them in suspension, and analyze for gene and protein
expression after suspension phase, then after plating for an
extended period of time. What we noticed is that if you
apply retinoic acid, you completely change the nature of
what these cells or EB clumps look like.
So without retinoic acid, they're typically very densely
packed. With retinoic acid, they're more
clear. Other labs have sectioned these
and shown that there's hollow space inside of these cells.
What you tend to see is epithelia-like sheets form, and
these make up hollow spheres. The gene expression analysis
that we perform after either two days in suspension and exposure
to retinoic acid over eight days shows that in fact, retinoic
acid applied at this time point gives us more expression of
markers that are epithelial specific and less neural
specific. Oct4 is a marker of
undifferentiated cells. And retinoic acid almost
completely wipes those out, whereas K18 and p63 here are
turned on by retinoic acid. The neural markers Nestin and
Pax6 and Sox1, there's no effect at two days, but after eight
days, we see a down regulation. So we're getting more epithelia
but less neural progenitors. If we plate the cells after ten
days, that have been exposed to retinoic acid, we plate them on
collagen or gelatin, and grow them keratinocyte growth medium,
we can quantify the number of K14 positive cells.
The cells that have been exposed to retinoic acid give us more of
the keratinocyte progenitors than the cells that haven't.
Unfortunately, we're still only roughly at this 30% to 40%
purity here. What we tend to see are these
regions in red that are keratinocyte progenitors
surrounded by these other cells types, in this case, neural
progenitors. We still have the significant
neural progenitors population that we want to get rid of.
The important point I want to make here is that it matters
when you apply the retinoic acid.
If you apply the retinoic acid just earlier in the embryoid
body stage, we get a lot of keratinocytes and we knock down
the number of neural progenitors.
If we apply the retinoic acid, not during the embryoid body,
but during the culture, it doesn't give us keratinocytes,
but it actually enhances neural formation.
If we apply it in both, EB and adherent, we get some
upregulation of keratinocytes, but we knock down the neural
population. So the optimum time for getting
keratinocytes is to give the retinoic acid early, but not
late. So this moves us to the direct
differentiation aspect. If we don't take the cells and
put them into suspension, if we directly add retinoic acid to
the embryonic stem cells, we see the same qualitative effects.
The retinoic acid turns on keratin-specific markers and it
keeps the cells, it turns off the self-renewal.
But what it does is gives us a much higher purity.
So here, we have six days of retinoic acid, and then extended
culture. We obtain an almost pure
population of keratinocytes, these form a very confluent
epithelial sheet. This gets to the last question
that was asked. Do these have the potential to
truly form skin. The answer now is not yet, but
they're getting closer. One of the things that these
keratinocyte precursors are supposed to do is stratify into
multiple layers. They do that, but only in small
islands so that it doesn't occur over the entire surface.
One of the ways to stimulate this is by adding calcium.
If we add calcium, we get stratification and expression in
Flaggrin and Involucrin, which are specific to the terminally
differentiated cells. They also can form these
epithelial sheets. If we detach them from the
surface, we can pick this up with a tweezers and it remains
intact. What we're currently doing is
trying to look at the signals that will stimulate further
stratification and differentiation, things like
mechanical signals as well as chemical signals.
So this is really a work in progress.
We're getting closer in getting regenerative cell types.
Eventually, we'd like to combine keratinocytes with other skin
cell types, endothelial cells, nerve cells, and so forth, to
truly regenerate skin. But we're a ways away.
So we need substantial improvements in both cell
culture design for keratinocytes, as well as
hESC-derived cells to be able to use these in regenerative
medicine. It's going to be a complex
problem, so soluble factors and matrix factors affect the yield.
Things like retinoic acid and collagen and gelatin here.
Mechanical factors may also play a role.
And finally, we've been able to show some function here in terms
of terminal differentiation and stratification.
So before close here, I just want to acknowledge the people
that did the actual work here, the experiments were performed
by Lin, Christian, Yin, Somen, Jeff and Samira.
And this work is really a collaborative effort.
The things I've talked about weren't only from my lab.
Juan de Pablo is our materials expert.
A lot of help from Jamie Thomson on embryonic stem cell culture
and advice on how to keep these cells undifferentiated.
Lynn Allen-Hoffman in the Pathology Department is a skin
expert there. Also Derek Hei, Jeff Jones and
Igor Slukvin have been working with us on other aspects growth
and differentiating. Thanks for your attention and
I'd be happy to take any additional questions you may
have. Yeah?
Hi, Professor. My name is Joe.
So, I can see the rationale of your research, just for
generating skin layers, especially for patients, also
those with diabetes ulcers. But when I thought of those two
different, I would say, injuries, I immediately thought
about infections. Because in the burn unit, it's
very common to have resistance, even MRSA.
And the thing is if you actually just cover the wound site with
new skin layers, it is possible that you're sealing the
infection. Then you can also have bacteria
and even worse symptoms in the person.
That may even cause the death in burn patients more.
So I guess my question is that is it possible to just use your
model and combine it with another treatment to prevent
those bacterial infections? >> Sure, that's a great point.
You would often want to combine, you'd need to combine this type
of model, even if in our wildest dreams are realized and you get
fully functional skin. Once you put that in, it will
take a while to integrate it into the wound site.
By that time, you'd still have a lot of problems with infection.
So you'd need to treat it with antibiotics and other methods.
But I do think that a fully vascularized functional skin
that integrates would be vastly superior to these other types of
dressings, because you'll get blood flow to the area, and
you'd get immune response there. Then you'd also have local
dendritic cells that reside and offer some infection fighting
potential. But that's many generations
away. >> Thank you.
Do you think it would be most advantageous for
patient-specific tissues, if you started out with the induced
pluripotent stem cells rather than the basic human embryonic
stem cells? >> Definitely.
If these induced pluripotent stem cells end up to have all
the functions that we need, it'd be much easier to generate a
bank of cells. There are a couple ways to
envision this working. One is take, if someone needs
treatment, you take their cells, generate an IPS line from that,
then grow the tissues. This may work for someone who
has diabetes who can be treated for an extended period of time,
because it takes a long time to generate the cell lines and
tissues. It would be less likely for a
burn patient who needs something off the shelf.
You may have, for tissues like that, for a heart, for example,
it would be banks of different cell lines that are compatible
with different antigens. So in that case, you have banks.
I'm not an immunologist. But you'd need hundreds and
thousands of lines, and have tissues that have already been
made for those. So if you could have an off the
shelf product, that would be great.
Or if you could just start with a line and a bank and make the
cells as needed, that would be another way to go.
I think that depends on the needs of the specific patient
and disease, which one would be optimal.
Hello, I had a question. You mentioned how the diverse or
different kinds of cells that you can create.
You mentioned how they may not give you full function, such as
the insulin cells that were created.
But is there any knowledge on the life span and longevity of
those cells compared to cells that would normally?
One problem that I didn't talk about in our skin work, is
that these cells have a much shorter life span than primary
cells, and we don't know why. You can get about ten population
doublings. It still gives you a pretty
substantial expansion. But primary cells should have 30
to 40. One of the theories is that
there's something in this selection process that's
prematurely aging the cells. We don't know what it is.
So there's an extensive amount of literature in the aging
field, in terms of -- activity, and other types of pathways that
regulate how much cell divisions a cell has.
If anything, we expected embryonic-derived cells to have
a lot of population doubling, since they're coming from a very
premature cell. In our case, that doesn't
happen. I don't know how general that is
to other lineages though. >> Do we currently have any cell
lines that are anywhere close to being like, having full
expression? Like as far as you were talking
about the K14. I'm sorry, I'm struggling how to
word this. But basically, do we have any
types of cells out there derived from stem cells that, like, that
are anywhere further along than skin.
It depends. I'm definitely further along
than that, but nothing I would want implanted in me.
So, there's room for improvement in a lot of different fields.
I think what's limiting a number of fields, and insulin secreting
cells are pretty far along, neural cells and cardiac cells,
because there's a lot of people studying them and there's a big
market for treatment. What's limiting us is the
ability to integrate them into functional organs.
Again, insulin secreting cells may not be as much of an issue.
The tissue engineering field from the '80s and '90s has
figured out ways of encapsulating these cells and
implanting them. They don't necessarily need to
be in the pancreas. Once they make insulin, as long
as it gets in the blood stream, it's fine.
But neural and cardiac lineages, even if we get cells
that are effective, how are you going to incorporate them?
It kind of brings it back to how far along are we.
One of the advantages of skin is that it is one of the two or
three commercial products that are out there in tissue
engineering. So we know a lot about how, once
we have the cell type, how we can incorporate that into the
current constructs. Cartilage is the only one that
there are products. Our rationale for getting into
skin isn't necessarily because there's a shortage of artificial
skin. You can culture skin from adults
and grow as many cells as you want.
It's more that this is a good model system for looking at how
we can form organs and tissue. While the skin field is a little
bit behind, in terms of generating cell types, I think
once we have them, we'll be able to move forward faster than the
neural or cardiac cells, which are going to have these big
problems in how you design the 3-D constructs.
I hope that kind of gets at what you're asking.
Do we, are they doing any like microwave stuff, to see
what the expression difference is between say, one cell base
versus another, and then seeing how far they are there?
That's a good point. The issue of how do you compare
the embryonic stem cell derived cell with the primary cell you
want to replicate. One way that's commonly used is
microwaves to look at gene expression patterns.
You'd want them to be as similar as possible.
The nice thing about these assays, is that you can, if you
see differences, you can get ideas on what's different
between the cells. The other type of analysis
people will look at is functional tests.
So do the cells make insulin? Do they beat properly?
Do they transmit electrical signals?
Ideally, the gene expression relates to the function.
As far as I know, there is not a current cell type that is far
enough along that it's close to being identical to the primary
cell. And the in vitro differentiation
process, the environment that surrounds it is very different
from what the cells see in people, so it's not surprising
that they're not identical. >> It sounds like two of the
problems that are going on right now is that you're trying to get
these cell to mimic the primary cells and then also scaling that
up. You talked about the challenges.
What's going on in terms of getting these scaled to the
necessary quantities? >> Some numbers here, if you
want to treat a patient, you would need billions of cells.
If you grow the cells in a dish, you can get millions of cells.
That's thousands of dishes of cells at a lab scale, which is
completely unfeasible. That's just to treat one
patient. So if you have tens or hundreds
of patients in a clinical trial, there's no way you can even
culture the cells. Then you multiply on that,
there's an efficiency problem. You don't get 100% efficiency,
so you're dealing with tens, hundreds of thousands of dishes.
I think what the field needs to do is look at what other
adherent cell culture researchers have done.
So this is primarily work that has been done in protein
production. There's very few therapeutic
cells out there that have impacted the cell culture
community as much as protein production.
So here, can we culture the cells, really, in stir tanks and
suspension to get us enough cells?
That involves removing the need for the matrix or being able to
culture cells on microcarriers, which are small spherical
particles that you can stir and keep up in suspension.
And there are people who claim they've been able to culture
cells in these types of configurations for short periods
of time, but not to the scales that we're talking.
So to be able to move on, even to clinical trials, there has to
be some engineering of scale here.
That's something that's hard to get.
There's a chicken and egg argument here.
People aren't interested in the problem until it's time to do
the clinical trial. But then they'll be saying, why
didn't we work on this problem in the mean time?
My idea here is that we should assume that something is going
to come out of stem cells. We may not necessarily want to
work on scaling a particular lineage yet, but if we can at
least learn how we can keep the cells growing in an
undifferentiated state and expand those, it will pay
dividends down the road. I thank everybody for your
attention. ( applause )