cc
talks will be Dr. Sean Palacek, who works with the Chemical and
Biological Engineering
He'll be speaking on tissue engineering, specifically skin
for regenerative medicine.
As much of a discussion as ( no audio )
talking and you from sleeping. possible to keep me from just
with questions, comments, your So please feel free to jump in
Before I get started, though, I thowould like to ask how manyme.
people here are engineers.
So one of the goals of what I want to do this evening is give
you a little bit of an insight
stem cells and regenerative into how emedicine.think about
I'm going to talk a bit about some of the work in our lab
that's trying to generate skin
These approaches really can be from embryonic stem cells.
cell types that are of generalized to a large number of
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
really, why have these cells regenerative medicine; and
engineering. rejuvenated the field of tissue
a big downturn in the 1990s. Tissue engineering went through
into the field didn't end up in A lotclinical products. went
And investment declined dramatically.
This money has started to flow
potential use of stem cells. back in the field because of the
embryonic stem cell self-renewal Then we're going to talk about
we can design culture systems that let us grow up the larget
numbers of cells that we would
applications. And finally, we'll discuss how
we can use stem cells in
specifically, skin tissue therapeengineering.tions,
The first human embryonic stem
here. I think most of us know this, in
Jamie Thomson's lab in 1998.
process so we can better I'd like to go through this
capable of. uMost of the current embryonice
stem cell lines are generated by
fertilization. the process of in vitro
a dish. You grow it for about five days.
It grows to the blastocyst
Here we have on the order of a stage.
There's an inner cell mass hundred cells.
This is before implantation of surrounded by a trifectaderm.
wall would occur in vitro. blastocysts into the uterine
and the cells are then cultured in a dish on fibroblast feeders.
Most of the cells die, but
that survives and proliferates. These form the embryonic steml
cell line.
not quite identical to the inner These embryonic stem cells are
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
Here's a picture of the first tyembryonic stem cell line.dy.
These cells grow in tightly packed clusters.
They really like to have
So if you isolate a single cell, cell-cell contact.
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
A way we can get cells to differentiate is just by pulling
them off a plate and culturing
of the self-renewal factors. them in suspension, if we remove
cells called embryoid bodies. SoYou can think of this asf
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.
want to address this idea of the Before we talk about that, I
adult stem cells. diSo embryonic stem cells havend
certain advantages over adult stem cells.
Adult stem cells are found in a lot of tissue.
They can be harvested and used
And adult stem cells are in therapeutic applications.
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
infinite number of times. aThey do change upon passaging,
however, so there is some effective limit.
Whereas adult stem cells, like
limited number of divisions. the cells in our body, have a
differentiation, as well. Embryonic stem cells can form
any cell type found in the body,
cell types related to the tissue whereas adult stem cells form
So from an engineering point of view, the embryonic stem cells
give us a much more scalable
material. We can, in theory, generate an
unlimited supply of any tissue
single cell source, whereas tadult stem cells, there's aa
limited availability, not very
brains to harvest the stem cell. many people want to donate their
potential here, you can only And because of the expansion
product. generate a limited amount of
properties that give embryonic We've touched on these
perhaps some of the hype that's stbeen associated with them.d
The three main applications for these cells, and one is
biological, gives us a window into some of the early
developmental processes in
We can study this in a dish, humans.
feasible to study it in the whereas it'body. ethical and
These cells can be a source of
effects of drugs and other cells and tissues for screening
Again, it's hard to come up with compounds on human tissue.
but there's a tremendous human heart and brain tissue,
biotech industry in learning how interest in pharmaceutical and
these tissues. different compounds will affect
dish, they give us a source of So if we can grow them in a
And then there's the cells for the screening.
Embryonic stem cells give us a regenerative medicine aspect.
able to be implanted in humans source of tissues that may be
tissue. toThere's also a downside tod
these cells.
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
us a way to address this November, has potentially given
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
But the results are the same. difThese cells return to ans.
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
can generate embryonic stem cell this may mean, is that people
tissue. liThese can then be used fornd
therapeutic applications. So this is really a tremendous
breakthrough that the field has been waiting for.
There's still a lot of
embryonic stem cells in chatherapeutics, though.sing
And today, we're going to hit on
discuss some of the other ones. So one of the problems is that
we really don't have very good
either at the research scale, or tools to culture these cells,
production. We don't know how to create most
cell types from embryonic stem
So we have the potential to create an unlimited number
amount of tissue from the cells,
guide differentiation to the but we need to learn how to
purity functional cells. desired fate, so we get high
scale up growth and Tdifferentiation to get enoughwe
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.
An unfortunate consequence of implanting these cells,.
embryonic stem cells, directly
form teratomas, or tumors. into the body is that they can
we have to make sure that we're Obnot putting undifferentiatedso
cells in with our differentiated
So the first topic I'd like to desired cells.
cells and what factors control discuss, how we can grow these
cells. what happens to these stem
perspective, what we're interested in is providing
signals to the cells that will
undifferentiated state, or allow either keep them in an
want to make. tThere are four factors thate
we're going to discuss. One is soluble factors.
You can add chemicals to the
These will bind to receptors and medium.
stem cell or begin to tell thedifferentiate.r stay a
Extra-cellular matrix, so the immobilized protein the cells
bind to can guide the differentiation phase.
Mechanical cues.
detail, along with intercellular I'll talk about this in more
each other and this signals for them to grow, self-renew oro
differentiate.
we can combine all these Here's a schematic showing how
desired outcomes. dSo we may want to controlto
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.
complexity of the environment. This raises an issue of the
in here suggest that the cells So what we're drawing the arrows
these signals in the have mechanisms to sense all of
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
you sort some factors on and the embryonic stem cell one day and
cardiac cell. This process can take weeks to
months. And you're going through many
intermediates.
to say, the first intermediate, Thmay not be the same one thate
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
factors. the field is working in --
today. And I'll talk about that a bit
the under-appreciated factors, I also want to address some of
cell-cell contacts. such So biology is really and
identifying a lot of the
that regulate whether a stem pathways and sibling molecules
cell, or whether it wants to cell decides to stay a stem
There are several pathways that bare indicated here.e.
There's a lot of receptors on the cell surface that bind to
growth factors secreted by other
And then this will turn on cells.
transcription that change the self-signacell state.ys to gene
This is not a complete list.
learn how these pathways And we're just beginning to
Question? int( inaudible )ch other.
You mention that human
potential to go to differentiate embryonic stem cells have the
Yet, I haven't heard of, you into any tissue of the body.
a three-dimensional tissue know, scientists actually making
something like that. structure, like a heart or
actually seeing published work, Whas in the first synthesizednd
human heart from these embryonic
Is it the fact that we don't stem cells?
factor along its necessarily know every soluble
Or is it just like the differentiation?
something along those lines? three-dimensional molds,
with that, actually taking the >> So there's a couple problems
organ. cells and forming a functional
getting enough of the desired The first tcell type.ntioned is
And I think that's really what
now. the field is concentrating on
myocytes, which are the little We can generate cardiac
contraction. cell type that causes the
pressing problem is how we But I think an even more
So the cells that we can obtain, reassemble those.
nerve cells, are randomly woriented and are not connectedr
to each other in the correct
That's going to be something spatial organization.
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.
The second approach is to take That's going to be very hard.
directly into the body. And hope that the environment in
the body will help guide their
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
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
One example is cardiac myocytes, for tissue to properly form.
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
then the expansion of your the shear of your breathing, and
That stimulates their growth and alveoli to completely develop.
Keratinocytes, which form your differentiation.
So your skin is thicker in skin, respond to stretching.
your knees and your elbows, certwhere they're constantlylike
stretched and mechanically
Bones and cartilage are stimulated by pressure to
strengthen and develop. Also, mesenchymal stem cells
can respond to different types
differentiate to bone and of presscartilage.rain to
So what we decided to do was
affect embryonic stem cells. look at how mechanical forces
apply forces to stem cells and Our initial hypothesis was let's
other cell types that I showed see if they then form those
And we're completely wrong. So that's one point one mentions
about science.
isn't what you see, but it's Sometimes what you look for
In our study, we grew embryonic stistem cells on a flexibleng.
silicone rubber membrane
So we have cell colonies on a flexible silicone membrane.
And we can apply a vacuum onto
What that does is stretch the membrane and applies a strain to
the membrane. The strain is transmitted to the
cells.
manner. So we apply a vacuum, release,
apply a vacuum, release.
of the strain, as well as the And frequency of the strain.tude
What we found is that when you
actually inhibits the straidifferentiation., it
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
In a typical culture, what you spontaneously differentiate.
take all of your cells and do is every five days or so, you
or mechanically chop them up and harvest them and enzymatically
them. rIn this case, we allowed thef
cells to grow for a week, and just let them keep growing.
We got these very large colonies
differentiation. and we'd get these areas of
this Oct4 and aren't in green, The cells that don't express
We don't know what they are, but are some cell type.
cells, whereas these cells here they'are still stem cells.c stem
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.
more dramatic. After two weeks, the effect is
strain will undergo spontaneous So cellsdifferentiation.osed to
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
particular lineage. Whereas the cells that we apply
strain, we have some
much. diffWe can quantify this.very
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.
strain, ten cycles per minute Here, wfor ten days. percent
And here, two populations.
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
undifferentiated cells. This is a threshold for them.w
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
the details. don't want to go too much into
in is how are the cells sensing force and what's it doing to the
cells.
pathways. And it looks like the force is
somehow stimulating the TGF-beta
So in embryonic stem cells, TGF-beta is a small protein that
binds to receptors on the cell
Then this turns on a pathway surface.
and keeps the cells in an thatundifferentiated state.ption
Their related protein is called bone morphogenic proteins that
stimulate differentiation.
shown that this TGF-beta A number of other labs have
undifferentiated and pluripotent signaling keeps cells in
And that they activate this complex called SMAD2/3, which
then goes to the nucleus and
whereas the BMP activates the turSMAD1/5 that promotesion,
differentiation.
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
So stretching the cells causes factor of ten-fold.
They secrete this into the them to make TGF-beta.
themselves and their neighbors. And then this is an autocrine or
paracrine move that keeps the cells in an undifferentiated
state.
inhibitor of the signaling, we If we add this chemical
through the TGF-beta receptors, keepand the cells willling
differentiate.
signaling activity. If we look at the level of the
signal downstream of TGF-beta receptor, the strain turns on
this SMAD.
very little phosphorylated SMAD, If we don't have strain, we have
inhibitor. and we can inhibit it with this
TGF-beta directly correlates So the signaling through
We think this is an example of with the cell state.
signals. how cells sense mechanical
then somehow they process this They sense chemical factors, and
undifferentiated or infodifferentiated state.in an
The other aspect I'd like to
cell culture engineering is how taldo we control cell-cell of
interaction.
small picture here, these The colocolonies grow asier, the
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
colonies may give you a partidifferent lineage. big
But there have been very few
or methods that give us methods stuto actually control this. why
One of the first studies that was published in 2005, what that
lab did was quantify the size of
types of blood cells. The myeloid lineage and theo
erythroid lineage.
plate of cells and enzymatically The way they do this they take a
cells. dissociate these into single
in a small tube and centrifuge And they count cells, put them
into a clump. tPut 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
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
to form an aggregate of a and certain size and shape.ells
We create a rubber stamp here using a silicone wafer mold
that's manufactured somewhat
computer chip, although it's like you would manufacture a
Then this rubber stamp forms a much simpler than that.
we're going to use to make the template for polymer molds that
In this area between the spacer and the stamp, we inject a
liquid polyurethane.
shape. This gives us this microwell
able to attach and grow inside What we want is for cells to be
between the well. We're going to treat this area
outside the well with a compound
We deposit gold at an angle. that keeps cells from sticking.
at an oblique or steep angle, Gold is vaplike this. deposited
The gold will hit the area
shaded from the bottom and side, between the wells, but will be
angle, it can't hit the bottom. Then we're going to react as
molecule with the gold. This molecule is going to keep
the cells and proteins from sticking.
So this molecule, one end will
end is a trophyline glycol. bThe trophyline glycol inhibits
protein cell attachment.
attach to the bare polyurethane When we ain the bottom. can only
We add cells, they'll attach to the protein and grow to fill
these.
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
If we incubate with protein, order of 1 to 5,000 cells.
well. Then we add cells and the cellse
will grow to fill up the well.
see are bubbles that are under These areas in here, what you
So if we do a stain for dye, the surface of the polyurethane.
these wells. you'll only see cells inside
put in a relatively small number What's going to happen is we
They'll bind and start growing. The cells will grow in three
dimensions and fill up the well.
differences in color are because So here you can see, the
growth. yHere we have just a couplef
layers. And this dark color indicates
that we're growing above and
You can see the number of cells ouhere are larger.l.
The Calcein AM just indicates that these cells are actually
alive.
for 19 days without passaging. The cells have been cultured
filled up these wells. At this point, they mostly
Even as they fill up the The cells remain proliferative.
What they're going to do is grow wells, they keep dividing.
these little mushroom type caps that will eventually break off
into the medium.
the well and showing the colony So a sis provided here. across
The green shows the cells are undifferentiated.
The nuclei are shown in red.
mangled, because we took a razor This sample has been somewhat
Eventually what will happen is blade and chopped it up.
well. theIt will grow up above.he
And as we add medium or change
going to shear that off. the medium every day, it's just
full well of cells. Eventually, we want to be able
to pull the cells out of these
in growth or differentiation wells and then use them, either
We can do that enzymatically, if type experiments.
break the contacts between the we add an enzyme that's going to
What we're left with are these cells and the substrate.
These are all of the same size little cube in suspension.
Once you get them to and shape.
in suspension to keep them from differeclumping together.e these
Overnight, they're going to switch from this cube shape to a
spherical shape.
didn't expect is that when we culture cells in small wells,
they remain almost completely undifferentiated.
So this is somewhat analogous to
strain. When we stretched cells, they
don't undergo spontaneous differentiation.
Also, if we keep the colonies fairly small, it keeps them
differentiating.
One theory is that just as the We don't really know why yet.
may secrete factors that once coloyou get to a criticalthey
concentration, cause
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
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
What we're interested in doing environment.
initial starting size of the now is looking at how this
get. colony affects what cell type we
suspension, we get this much So by culturing the cells in
sizes and when you compare it to more homogeneous distribution in
method. We're working with a number of
people on campus here and around
lineages. tSo what size of embryoid bodyc
gives is the most myocytes, for example, insulin secreting cells
or neural cells.
self-renewal aspect, to summarize that a bit before I
move on to tissue engineering.
the world have identified soluble factors that keep cells
in the undifferentiated state.
get across is that it's not only The message that we're trying to
important. soluble factors that are
undifferentiated by applying But that we can keep cells
And this appears to work by stimulating TGF-beta cell.
signaling.
So regulating cell-cell contact Acan 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
So I think I'll stop and take any questions or comments here
before we move on to the tissue engineering aspect.
Yep?
said, can keep the cells from differentiating, but isn't thatu
just a direct effect of the TGF-beta?
Couldn't you just add the TGF-beta to keep the cells from
differentiating?
legitimate way to do that, or is Is the strain just a more
do it with the strain rather there a reason you'd prefer to
factor? than >> Good question.rowth
So I didn't show all the
but adding TGF-beta doesn't completely replicate the effects
of strain.
So we think that's the dominant It replicateffect.t 75% of it.
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
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
But one of the messages here is that when you are designing a
culture system, it's important
So other types of forces that tare important include shear.
So if you're trying to culture
can't get around the fact that these cells in suspension, you
them. yWe really need to know whatto
effect that will have on the
Other questions? cell.
can use these cells in So then let's talk about how we
And there are a large number of labs on campus, in the country,
around the world, that are
cells for therapeutic trying applications.pecific
One of the ones I want to talk
people are studying. to you about today, not a lot of
So, can we make skin cells? And I'd like to introduce why we
want to use stem cells for
specifically for skin tissue tissue engineering, and
The goal of stem cells in regenerative medicine and tissue
engineering is to take these
cells and generate the perfect very early, primitive human
and organs that may have been replacdamaged or diseased.sues,
There are two main ways to this.
response to some of the I thinquestions before. it in
But either an in vitro method or
In the in vitro method, what we do is take these embryonic stem
cells, differentiate them into
assemble these cell types into specific cell types, and
functional organ into the body an organ, and put that
static. aThis is nice to think about,y
because it's a very controlled
You can control what happens at each differentiation, and you
get a fully functional organ
The problem is it's too complex that this point to really be.
effective.
method, in which we directly Then there's the in vivo
precursors, into the body. place the stem cells, or their
which you place them will guide Andtheir development and at
differentiation.
You can take stem cells or their This is much easier.
into a body and see what early progenitors, toss them
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
That makes sense because in the early steps of human
development, the environment is different than it is in the
adult.
blend of the in vitro or in vivo So there will probably be some
work. And that blend will be different
for different tissues.
been shown to be generated from A loembryonic stem cells.have
This is a bit old, and it's very
But you get neural cells, skin incomplete.
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
put these together and use them basein a therapeutic sense.o we
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
and engineers, include the low need to address, as scientists
The cell types I put up on the yield.
but the actual number of each cell type is relatively low and
the purity is low.
complexity of the environment. A lot of this relates to the
to cells are going to be So the types of signals you give
And it's not just add this one important.
desired cell type. growth factor and you get your
be important. A lot of the culture methods now
use animal-derived products,
to get these cells and tissues cultures with other cell types
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
phenotype is important. haSo often, you get somect
properties of a cell type, but
One example, and there's been a lot of excitement in the past
week, that insulin producing
embryonic stem cells, and that cells have been generated from
mice. tThe problem is that thesein
insulin producing cells don't entirely respond to glucose the
way they should.
that are missing from these Socells that are going to bens
critical to their use.
different cells into functional And finally,tissues?we assemble
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
The two types of patients that tissue engineered skin products.
victims and people with wounds would benefit most are burn
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
the water content in their body. regulate their temperature and
experience permanent Another million people
of burns. disabSo if we can generatecause
replacement skin that would improve the quality of life, or
perhaps save lives. Non-healing wounds is another
use for skin tissue engineering.
wounds are currently being And in fact, these types of
products. trThe two main types ofkin
non-healing wounds are foot ulcers in diabetics and venous
leg ulcers. Both of these are caused by a
diminished blood flow into the
in low rates of wound healing and diminished immune response.
And amputations in diabetics are
And venous leg ulcers can also common, typically in the feet.
The costs to the healthcare system runs in the billions of
dollars because of the number of
problems. peIn trying to engineer skin,of
what we want to do is look at the natural tissue, figure out
what it does and see if we can
function. Or, provide its function through
a slightly different structure. The critical aspects of skin to
keeping people alive are the
dessication barrier. mechanical barrier and
infections out. You keYou keep water in. out,
And some of the current products
Some of the other functions that can do that.
include sensory. would be nice to maintain
give you the sense of touch. Your skin contains nerves that
infections. ImThermal regulation.ght
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.
here. You have the epidermis, which is
the outer-most layer that provides the mechanical
dessication barrier.
functions are present in And most of these other
dermis. We also have sweat glands that
involve thermal regulation, hair
It's actually quite a complex foorgan.s.
The first generation of tissue
consist of only one or two engineering products really
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.
rubber membrane that's fused to So you haa nylon mesh.a silicone
This has peptides on it that
repopulate the wound. You place this over a wound with
the silicone rubber membrane
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
This is cadaver skin that's been analogs.
It's just the matrix that you treatewould place down.cells.
Then this would allow cells to
wound. crawl in and repopulate the
stimulate growth and These are passive materials that
outside the wound into the proliferation of cells from
The later generation skin substitutes have actual living
cells in them.
keratinocytes and fibroblasts in One of them contains human
extra-cellular proteins. an artificial matrix of
epidermis structure. This replicates the dermis and
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.
they are there, secrete factors that allow cells to repopulate.
So these cells do not become a
site. Our vision is to be able to
completely replicate the
where you have vascularization, strucnerves, sweat glands.skin,
How we're starting out though,
the cell types that are found in is just to try to get some of
So what we've been working on is skin from embryonic stem cells.
These cells form epidermis. And they stratify into multiple
layers. The basal layer of keratinocytes
will grow over the dermis.
mono layer of cells that are Their role is to form a
differentiate. then going to divide and further
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
Typically, you have ten to 20 ocell layers thick of
keratinocytes at any point on your skin.
About five years ago, a lab at
demonstrate that embryonic stem cells do form these skin
keratinocytes.
bodies, or undifferentiated Wcells and injected these intod
mice. They harvested the tumor that
arose.
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.
study. So this was a very important
make the keratinocytes from the It shoembryonic stem cell.le to
The problem is this isn't a very
You have very low yield of these effective way of doing it.
You end up killing a lot of mice..
And it doesn't give you much
cells develop. insight as to how these skin
therapeutic application, we need If you want cells for a
And our approach is to see if we to find a better way to do it.
P63 is expressed in a lot of can induce this factor P63.
that form linings of virtually epithelial cells, so the cells
We figured if we can turn on every organ in your body.
us progenitors that are more expression of this, it will give
It won't necessarily give us likely to form skin cells.
along that path. skin, but it will get us closer
this embryoid body that we Our first strategy is to use
You take your undifferentiated discussed earlier.
dish. And then you pick them off the
substrate and culture them in suspension.
They begin to differentiate to
types. form a large variety of cell
the cells we want, The green cells here represent
You plate these on a dish coated keratinocytes.
We happen to use collagen, with a extra-cellular matrix.
component of skin. because that's the main
keratinocytes will migrate out What happens, then, is your
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
We can then somehow isolate these keratinocytes, purify
them, and induce differentiation
Here's an image from that migration phenomenon.
We dropped an embryoid body at
I believe a week or a week and a this point here and then waited,
So the cells began to crawl out. half in this experiment.
image. This panel corresponds to this
Oct-4. So these cells still express
embryonic stem cells. Whereas the skin progenitors
walked away from the
formed these relatively pure undifferentiated cells, and
If we magnify the region here, regions.
epithelial like shape. we see that they have this
sheets of cells. They tend to form tightly packed
Question? And they aScale?press p63.
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
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
So we took these embryoid bodies, put them in collagen for
11 days.
commercial media formulations Thethat are used to culturent
keratinocytes.
commercial media will contain a Tnumber 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
that give us keratinocytes that ouaren't in the others thates
don't.
on is important. The matrix you plate the cells
bodies cultured for two weeks. Here, we have the embryoid
matrices and cultured them for We put them on different
defined certain mediums which we an additional month in the
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.
undifferentiated stem cells. You still get a lot of
differentiation. In serum, we get higher level
Interestingly, gelatin gave us Collagen, about 15%.
So we were able to obtain about 25% of the cells in the culture
are keratinocytes.
others are, probably a mix of a We'revariety of cell types. the
Question? >> When you're saying we have
keratinocytes, are you just referring keratinocytes as cells
that express the K14, or
further than that and looking into more detailed expression?
Good question.
and that isn't sufficient to Here, we're seeing keratin-14,
keratinocytes because certain cother 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
in suspension in these undefined is that by culturing the cells
differentiation toward a lot of condidifferent choices.o see
So if we want a pure population,
the other cell types or purify we either have to get rid of all
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
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
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.
receptor don't form basal Mikeratinocytes with the samecid
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
formation in embryonic stem specify fcells.atinocyte
What we do is take these
of cells in suspension and apply embryoid bodies, these colonies
keep them in suspension, and retinoic acid as we form them,
expression after suspension phase, then after plating for an
extended period of time.
apply retinoic acid, you What we noticed is that if you
what these cells or EB clumps completely look like. nature of
So without retinoic acid, they're typically very densely
packed. With retinoic acid, they're more
clear.
and shown that there's hollow Otspace inside of these cells.e
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
to retinoic acid over eight days days in suspension and exposure
acid applied at this time point gives us more expression ofc
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
days, we see a down regulation. So we're getting more epithelia
but less neural progenitors.
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
the keratinocyte progenitors to retinoic acid give us more of
Unfortunately, we're still only troughly at this 30% to 40%.
purity here.
regions in red that are Whakeratinocyte progenitorsse
surrounded by these other cells types, in this case, neural
progenitors.
neural progenitors population Wthat we want to get rid of.t
The important point I want to make here is that it matters
when you apply the retinoic acid.
If you apply the retinoic acid
body stage, we get a lot of keratinocytes and we knock down
the number of neural
If we apply the retinoic acid, progenitors.
but during the culture, it not during the embryoid body,
but it actually enhances neural doesn't giformation.tinocytes,
If we apply it in both, EB and
upregulation of keratinocytes, adherent, we get some
population. So the optimum time for getting
keratinocytes is to give the
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 retinoic acid turns on the same qualitative effects.
keeps the cells, it turns off keratinthe self-renewal. and it
But what it does is gives us a
So here, we have six days of much higher purity.
culture. retinoic acid, and then extended
population of keratinocytes, We obtain an almost pure
epithelial sheet. This gets to the last question
that was asked. Do these have the potential to
truly form skin.
they're getting closer. TOne of the things that theset
keratinocyte precursors are supposed to do is stratify into
multiple layers.
islands so that it doesn't occur They do that, but only in small
One of the ways to stimulate over the entire surface.
If we add calcium, we get stratification and expression in
Flaggrin and Involucrin, which
differentiated cells. areThey also can form theselly
epithelial sheets.
surface, we can pick this up If we detach them from the
intact. What we're currently doing iss
trying to look at the signals that will stimulate further
stratification and
mechanical signals as well as diffechemical signals.s like
So this is really a work in progress.
We're getting closer in getting regenerative cell types.
Eventually, we'd like to combine
cell types, endothelial cells, keratinocytes with other skin
truly regenerate skin. nerve cells, and so forth, to
So we need substantial But we're a ways away.
culture design for improvements in both cell
hESC-derived cells to be able to keratinocytes, as well as
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
And finally, we've been able to show some function here in terms
of terminal differentiation and stratification.
So before close here, I just
that did the actual work here, want to acknowledge the people
by Lin, Christian, Yin, Somen, the experiments were performed
And this work is really a collaborative effort.
The things I've talked about
Juan de Pablo is our materials weren't only from my lab.
A lot of help from Jamie Thomson expert.
and advice on how to keep these on cells undifferentiated.ture
Lynn Allen-Hoffman in the Pathology Department is a skin
expert there. Also Derek Hei, Jeff Jones and
Igor Slukvin have been working
and differentiating. with us on other aspects growth
I'd be happy to take any Thanks for your attention and
have. additional Yeah?ions you may
Hi, Professor.
So, I can see the rationale of My name is Joe.
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
new skin layers, it is possible justhat you're sealing thewith
infection.
and even worse symptoms in the Then you can also have bacteria
That may even cause the death in person.
So I guess my question is that burn patients more.
model and combine it with isanother treatment to preventr
those bacterial infections? >> Sure, that's a great point.
You would often want to combine,
of model, even if in our wildest you'd need to combine this type
fully functional skin. dreams are realized and you get
take a while to integrate it Once you put that in, it will
By that time, you'd still have a into the wound site.
So you'd need to treat it with lot of problems with infection.
But I do think that a fully avascularized 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.
dendritic cells that reside and offer some infection fighting
potential.
away. But tha>> Thank you.rations
Do you think it would be most advantageous for
patient-specific tissues, if you
pluripotent stem cells rather than the basic human embryonic
stem cells? >> Definitely.
If these induced pluripotent
the functions that we need, it'd stem cells end up to have all
bank of cells. be much easier to generate a
envision this working. There are a couple ways to
treatment, you take their cells, One is take, if someone needs
then grow the tissues. generate an IPS line from that,
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.
burn patient who needs something It would be less likely for a
You may have, for tissues like off the shelf.
it would be banks of different that, for a heart, for example,
with different antigens. cell lines that are compatible
I'm not an immunologist. So in that case, you have banks.
thousands of lines, and have But you'd need hundreds and
made for those. So if you could have an off the
shelf product, that would be
Or if you could just start with a line and a bank and make the
cells as needed, that would be
I think that depends on the needs of the specific patient
and disease, which one would be optimal.
Hello, I had a question.
different kinds of cells that You mentyou can create.iverse or
You mentioned how they may not
the insulin cells that were give you fulcreated.on, such as
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
cells, and we don't know why. You can get about ten population
doublings.
substantial expansion. It still gives you a pretty
to 40. But primary cells should have 30
there's something in this Onselection process that'st
prematurely aging the cells.
So there's an extensive amount We don't know what it is.
field, in terms of -- activity, and other types of pathways that
regulate how much cell divisions
If anything, we expected a cell has.
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
expression? Like as far as you were talking
about the K14.
word this. I'm sorry, I'm struggling how to
types of cells out there derived But basically, do we have any
are anywhere further along from stem cells that, like, that
It depends. I'm definitely further along
than that, but nothing I
So, there's room for improvement would want implanted in me.
I think what's limiting a number in a lot of different fields.
cells are pretty far along, neural cells and cardiac cells,g
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
Again, insulin secreting cells functional organs.
The tissue engineering field may not be as much of an issue.
figured out ways of encapsulating these cells and
implanting them.
be in the pancreas. Once they make insulin, as long
as it gets in the blood stream, it's fine.
But neural and cardiac
that are effective, how are you ligoing to incorporate them?ls
It kind of brings it back to how
One of the advantages of skin is far along are we.
three commercial products that that it is one of the two or
engineering. are out there in tissue
we have the cell type, how we Scan incorporate that into thece
current constructs. Cartilage is the only one that
there are products. Our rationale for getting into
skin isn't necessarily because
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.
bit behind, in terms of Wgenerating cell types, I thinke
once we have them, we'll be able
neural or cardiac cells, which to move forward faster than the
problems in how you design the are going to have these big
I hope that kind of gets at what 3-D constructs.
Do we, are they doing any like microwave stuff, to see
what the expression difference
versus another, and then seeing is how far they are there?ase
That's a good point.
the embryonic stem cell derived Tcell with the primary cell you
want to replicate.
microwaves to look at gene One way that's commonly used is
You'd want them to be as similar expression patterns.
The nice thing about these as possible.
see differences, you can get assays, is that you can, if you
between the cells. ideas on what's different
people will look at is The other type of analysis
So do the cells make insulin? Do they beat properly?
Do they transmit electrical signals?
Ideally, the gene expression
As far as I know, there is not a relates to the function.
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
that they're not identical. p>> It sounds like two of theg
problems that are going on right
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
If you grow the cells in a dish, would need billions of cells.
That's thousands of dishes of cells at a lab scale, which is
completely unfeasible.
patient. That's just to treat one
of patients in a clinical trial, So if you have tens or hundreds
culture the cells. there's no way you can even
there's an efficiency problem. Then you multiply on that,
so you're dealing with tens, You don't get 100% efficiency,
I think what the field needs to hunddo is look at what otherhes.
adherent cell culture researchers have done.
So this is primarily work that
production. There's very few therapeutic
cells out there that have impacted the cell culture
community as much as protein
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
cells in these types of configurations for short periods
of time, but not to the scales
So to be able to move on, even that we're talking.
be some engineering of scale to clinical trials, there has to
That's something that's hard to hget.
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
My idea here is that we should in the mean time?
to come out of stem cells. assume that something is going
work on scaling a particular We may not necessarily want to
least learn how we can keep the lineacells growing in anan at
undifferentiated state and
dividends down the road. expand those, it will pay
attention. ( applause ) your