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OK, so now that I've told you about embryonic stem cells
let me tell you a little bit about adult stem cells.
And I'll tell you about the work that my laboratory does.
So we're going to dig a little bit deeper into the biology of stem cells.
So here we're going to tap into the potential of adult stem cells.
We'll talk about their biology and we'll talk about
these cells as potential for regenerative medicine.
So what are adult stem cells? Let's just refresh our knowledge again.
These are cells, remember, of the body that can repair tissues when it's injured
or when a tissue undergoes normal wear and tear.
So, some tissues naturally have more stem cells than others and replace it's cells continuously.
Examples of that would be skin stem cells that regenerate the epidermis and the hair follicles,
blood stem cells that are able to regenerate red blood cells and immune cells.
And in fact, every two weeks time you've got a brand new surface of your epidermis
and of course, if you're lucky, your hair follicles not only undergo periods of rest
but they also undergo periods of growth.
And blood cells have an enormous capacity to continually regenerate.
So other tissues, though, have very few stem cells.
Those are the tissues that we were talking about earlier in the presentation;
cells of the central and peripheral nervous system.
In fact, it's only the last couple of years that we've even known that there are, in fact,
stem cells in the adult nervous system.
Pancreatic tissue is even more problematic.
Doug Melton's laboratory has been studying pancreatic islet formation and
found that the only way to get islets is by damaging existing islets
and getting islets to produce more islets.
That there appear not to be any of these multipotent adult stem cells, for instance,
that could regenerate an islet if necessary.
And so that's one of these reasons why
I spent the first part of my talk discussing embryonic stem cells.
So, but for those tissues that do have an abundance of adult stem cells,
we can think about those types of stem cells for regenerative medicine.
So, the characteristics of adult stem cells then are the ability to divide to produce more stem cells.
So, whether they're adult or whether they're embryonic stem cells,
all stem cells have self-renewal capacity.
And the ability to produce differentiated tissue.
In this case, for my laboratory, epidermis or hair or sebaceous glands.
One of the characteristics of adult stem cells is that they tend to be immature relative to their progeny.
So when we study the hair stem cells,
those cells are less mature than the rest of the cells within the hair.
Adult stem cells are typically multipotent
and they can make a subset of cell lineages as I've just described to you
in the case of hematopoietic stem cells or skin stem cells.
So the cytoplasm determines the gene expression pattern that the nucleus will have.
We talked about the fact that the adult skin stem cell
has a nucleus and properties of gene expression that are quite different
from when that adult skin stem cell nucleus
is placed into an environment of the oocyte.
In the case of taking the adult skin stem cell, putting it into the oocyte
to make a hybrid cell that then goes on to generate
embryonic stem cells, the nucleus gets reprogrammed.
In the case of the adult skin stem cell, the cytoplasm of this cell is different
and presents a different program of gene expression
for this nucleus versus this nucleus.
So, even though this nucleus and this nucleus have the same genetic content,
the pattern of gene expression is different.
At present, the only cell that we know about and the only cytoplasm that we know about
that is capable of producing an unlimited different variety of gene expression patterns
afforded to all of the cells of the body is that of the egg's cytoplasm.
As far as we know, adult skin cytoplasm does not have that capacity.
So we'd really like to know more about what's the difference between this cell and this cell.
And why these cells have different patterns of gene expression
and just what those different patterns of gene expression are.
So, my laboratory does study skin and we use that as a model system.
And I think of all of the various different tissues and organs of the body, there's no question that
nature clearly has had a lot more fun and fancy in creating body surfaces
than she has in creating any of those ugly organs
that are tucked underneath it and that most scientists work on.
So my laboratory has been fascinated, for my lifetime, in skin
as is all of nature and I think many of you.
It's the one tissue of our body that we really feel intimate about.
We see it every day. We notice when there's even the slightest change in our skin.
And then of course, when you consider that virtually every society
in the world has adorn upon what nature has given us and I think it's kind of interesting
that often, when you consider different societies
and different cultures and different adornments
that societies have given to their skin and their hair,
that one often uses appendages from other animals
the feather, in many examples, as a further case of enhancement of our skin surface.
So that's just one of the reasons why I always like to remind my audience
that you too have an interested in skin and we only go just a little bit deeper into the skin.
So where do adult skin stem cells come from?
During embryonic development cells become
increasingly more restricted in what tissues they can make.
So, back in early development,
the skin's surface begins to form after gastrulation of the embryo.
And at that stage there's a single layer of multipotent embryonic skin stem cells.
And these cells can give rise to the epidermis, the hair follicles, the sebaceous gland.
In the case of the epidermis, for the unipotent stem cell,
those cells exist within the epidermis.
And when we go from the single layered epithelium to the stratified epithelium,
this is the structure of the epidermis that is going to be maintained in your adult skin.
And this single layer located in the innermost, called the basal layer of the epidermis,
is going to be the only layer of cells that remains unipotent and remains proliferative.
These cells are going to differentiate and as they differentiate, they start to move upward
and these cells way at the top are going to be the cells then that ultimately are going to be
sloughed from the surface of your skin,
continually being replaced by inner cells differentiating and moving upward.
So this is the reservoir down here of where the
unipotent cells of the adult skin epidermis are going to be.
So these are unipotent, adult, epidermal progenitor cells.
These are multipotent embryonic skin stem cells.
If adult stem cells reside within a tissue, they're usually tucked as safely and deeply
as possible within the tissue and that's a property of many different types of adult tissues.
So stem cells in the skin making epidermis and hair and more.
The skin stem cells provide the nearly endless supply of cells
to replenish the epidermis and hairs of our body.
So can we isolate them?
Can we coax them to become hair as well as epidermis?
Can we coax them to become other types of cells
without deriving embryonic stem cells by nuclear transfer into oocytes?
So, back when I was a post-doctoral student, I began to study skin stem cells.
And at that time I was studying almost exclusively human epidermal stem cells.
And one of the reasons why I became a skin biologists is because skin epidermal stem cells
are amongst the few stem cells of our body that have the capacity to
become cultured in the laboratory and
to be maintained and propagated as adult stem cells of the skin.
Now, I just talked about, in the previous part of the presentation,
embryonic stem cells that we could culture and which maintain their properties endlessly.
Adult human epidermal stem cells aren't like embryonic stem cells
in that we can't expose them to neural factors and get neurons
but these stem cells can grow and be maintained in the laboratory and this is how we do it.
We take these cells and we place them into a petri dish and then we provide them with medium
and it's very similar to that of the nutrients that are moving through your bloodstream all the time.
We then add those cells to our cultures of skin stem cells.
And then we put them into this incubator and this incubator then has the right environment,
the right temperature, 98.6 degrees Fahrenheit, the temperature that our body temperature is in,
and then in addition, the right mixture of carbon dioxide and oxygen that allow us then,
in this incubated environment, to be able to culture and maintain and propagate these cells.
So this is what we do, then, and if we now take a look at what happens after a week or so,
after we've cultured these cells, if we grow these cells on plastic,
and now we've stained these with hematoxilyn and eosin
which allow us a histologic stain so we can visualize these stem cells,
what we see is that these cells have the capacity to undergo division.
And here you can see an anaphase cell and here you can see a metaphase cell.
And here we can see other epidermal cells within the petri dish
and these cells divide, then, and can be propagated.
We can passage them from one petri dish to another petri dish to another petri dish
and keep getting more and more skin cells.
In addition, we've figured out ways in which we can recreate the environment so well
that in fact, the skin epidermis pretty much
behaves and looks like a normal human skin epidermis.
And in this case, down here, what we've done is
instead of plating these cells on plastic, instead we plate these cells
now in mixture of collagen and fibroblasts, which creates the dermis,
and then we plate these epidermal cells down on that.
We let them grow to confluence and then at confluence,
we raise the collagen and plop it onto a raft, a floating raft and now feed the cells from media below.
Because your epidermis is at your skin surface,
the blood vessels that are supplying the nutrients for your epidermis
are actually located deep in the dermis and those blood vessels then provide the nutrients
and that allows the epidermis to grow.
And it turns out that one of the features of epidermal differentiation
is basically predicated on the fact that the cells move away from their nutrient source.
So many years ago one of my graduate students in the laboratory
was studying some of the factors that are existing within the blood vessels
and learned that, in fact, if you float these cells at the air-liquid interface
that now these cells can undergo a normal process of differentiation.
And these cells up here would be the cells that in our body would be
sloughed from the skin surface, continually being replaced
by inner layer cells moving outward.
But in a petri dish, these cells have no where to go.
So these cells keep on pilling up and
that's really about the only difference between this tissue and the skin epidermis of our bodies.
So we can do pretty good about recreating the epidermis
from scratch in a culture dish.
So, what can we use this for?
Well, back when I was a post-doctoral fellow at MIT, many years ago,
my mentor, Howard Green, developed the technology
for growing human epidermal cells in culture.
And, in fact, way back then, in the late 1970s, he and another investigator,
Eugene Bell, also at MIT at the time, devised methods in which they could adapt
the use of cultured, human epidermal stem cells
for burn therapy to treat badly burned patients.
In many cases, patients' body surfaces would be so badly burned
that there wasn't enough good skin to be able to graft.
But if we took small amount of the good skin and put those cells into culture
within a few weeks time we could generate many culture dishes
of epidermal cells and since they're the patient's own cells, the cells would not be
rejected on a graft and therefore we could take a small biopsy from the patient's skin.
Place those cells into culture. Passage those cells.
Produce more large petri dishes full of skin stem cells.
And then basically graft those cells onto the back, for instance,
or the burned area of the victim.
And then since it's the patient's own cells just passaged in culture,
the patient doesn't reject them.
They recognize the cells as self and basically the burn is repaired.
The cells that are coming from these petri dishes are epidermal stem cells.
They are unipotent stem cells that generate epidermis.
So the graft of these cells basically produces epidermis but the patient's grafted skin
does not contain hair follicles nor does it contain sweat glands.
But that's the least of this patient's worry in a burn therapy.
So the fact of the matter is that this type of technology, then,
has been used across the world now in many various different burn clinics
in conjunction with other classical means of skin grafting
to be able to help patients and to be able to treat the most badly burned of patients.
And I just point out to you that there are a number of biomedical engineering groups
that are generating artificial skin.
You might have heard of various different methods for artificial skin.
You might have heard of various different doctors using pig skin
or various different other types of non-human skin.
Those skins are basically used as protective means until the patient's
skin cells can be repaired or for instance, until these cultured cells
can be able to take on the patient and regenerate epidermis.
Pig skin cannot be used forever on a patient
that's recognized as foreign and would be rejected.
Similarly, artificial skin is useful for a transient period of time.
But basically one needs to have normal, human cells in order to recreate skin epithelium.
So this is one of the few early on successful examples
of the use of adult stem cells in a clinical setting.
OK, so epidermal cells can be grown for months in a dish
and forever when grafted onto a burn patient.
I'll just show you a few examples of this.
So, here is an example, then, that was taken,
again way back in the early days when people were
just beginning to develop this technology for the use in regenerative medicine.
And here is an examples where the cells, then, were enzymatically
removed from the petri dish once they were cultured
and then they were grafted on to this patient's badly burned leg.
All of the cells that you see encompassing this entire area here
were entirely generated from cultured epidermal cells
from an area of good skin taken from this patient, placed into culture
and then grafted into the bad, wounded area.
Again, here's an example of one of the cultures.
The cells that you see in pink here are the epidermal cells.
And when those cells were grafted onto the patient's skin
here we see a cross-section of the patient's skin
from the badly burned...what was a badly burned area
and now has perfectly healthy, regenerated epidermis.
And then if we follow this technology over time
and say, "Well, a year later how good is this epidermis?"
A year later, if you take a look at the epidermis of a normal patient
and the epidermis of repaired...culture repaired epidermis grafted onto a person
a year later you cannot tell the difference between
the epidermis and the repaired epidermis from culture.
The problem still is that there's problems with scarring.
Problems with scarring are dermal problems. They're not epidermal problems.
So scientists have long taken care of the epidermal problems
using cultured epidermal stem cells for burn therapy.
There's still a lot of work to go in the area of improving the quality of skin for burn patients
and in particular in the area of dermal repair
and in the areas of generating hair follicles and generating sweat glands.
So what does it take to make a hair follicle, then?
What I'm going to show you here is very furry mouse.
This is a mouse that my laboratory generated a number of years back
and here is it's brother. Its brother looks a lot skinnier.
This looks like a very fat mouse. In fact, this mouse has less fat than its brother.
All of that puffiness was, basically, excess hair.
So we can do this in a mouse.
Ideally, we'd like to be able to do this with regards to help for human hair therapy.
OK, so now let's talk a little about the hair follicle.
So, here, again, is our schematic. This is the hair follicle and here is the epidermis
and then over here is a sweat gland.
So, what are these structures doing?
Well, as I already talked to you about, the epidermis acts as a barrier.
And the epidermis acts to keep harmful microbes out and essential bodily fluids in.
And the hair follicles provide warmth, protection.
The sweat glands and sebaceous glands provide lubricants.
So in fact, if you didn't have the epidermis and its appendages,
you'd be in pretty bad shape. You'd either die from dehydration or you'd die from infection.
So, we absolutely have to have the epidermis, which is about cellophane thickness of your skin
and we have to have hair follicles; at least with regards to most animals.
So most animals, in fact, spend most of their time making hair.
It's only humans that spend most of their time making epidermis.
And we think, we just think, that we need our hair.
So, all of these cells are derived from a common embryonic progenitor cell,
an embryonic skin progenitor cell which I've already alluded to in a previous slide.
And, in the adult, there's constant turnover and regeneration
not only of your epidermis, as I've described to you, but also of your hair follicles.
And that means that there have to be adult epithelial skin stem cells.
I've already talked about the unipotent stem cells of the epidermis.
What about the stem cells of the hair follicle?
And, again, just to remind you, the stem cells of the skin, like any good stem cell,
are cells with the capacity to self-renew and to differentiate into one or more tissues.
So one of the big questions of the field was
where are the skin stem cells and how can a skin stem cell form different tissues of the body.
I mean really, when you look at these structures, the epidermis and the hair follicle,
these are such incredibly different structures
it's fascinating to me as a scientist how these structures can derive from one common stem cell.
So let's find out where these stem cells are.
Well, what we knew when we began this research,
and this is now roughly at the time of about the turn of the century,
the year 2000, what we knew about stem cells, multipotent stem cells of the skin
is that these cells are used sparingly, like most stem cells, and that they divide infrequently.
These stem cells are basically kept as reservoirs of fountain of youth cells.
They are able to create tissues so the body wants
to be able to preserve the stem cells--use these stem cells infrequently.
Another thing that we knew about the skin stem cells, and this is work that
I had done back when I was a starting assistant professor at the University of Chicago
quite a few years ago now ago, what I had learned at that time was that
the keratinocytes that have proliferative potential express keratins 5 and 14
which we had cloned and characterized a number of years ago.
And so, based on just these two biological principles, Tudorita Tumbar in my laboratory,
then in my laboratory, she's now at Cornell University in Ithaca, NY with her own laboratory,
she and Geraldine Guasch, another post-doctoral fellow in my laboratory
worked out a method
by which they could identify the multipotent or putative multipotent stem cells of the adult skin.
So what they did was to make two genes, first.
One of these genes encoded an inducible repressor protein
called the tetracycline-inducible repressor protein. This means we can add tetracycline
and induce the activity of the protein, in this case, repression of gene expression.
And we took this gene and we placed it under the control of the keratin 5 promoter.
meaning that this tetracycline-inducible repressor protein is now going to be expressed
in all of those cells that have proliferative potential within the skin.
And then we made another gene and this gene, down here,
is a gene that encodes a fluorescently tagged histone protein under the control of
a tetracycline regulatory element. So now, this transcriptional repressor protein
in the presence of tetracycline, is going to be able to bind
this tetracycline regulatory element and drive the expression of histone H2B-GFP.
So now what we need to do it be able to, in step two,
make a transgenic mouse that is expressing both of these two genes that we engineered.
So, in this case, what we do is we inject the genes into what is called
the fertilized oocyte at the one cell stage and this fertilized oocyte
now has a male pronucleus. You can tell it's male because it's bigger.
Unfortunately I'm always in favor of the female and the strength.
But basically, this male pronucleus is larger than that of the female pronucleus.
It's before these nuclei have fused and what we do is we inject those two genes, now,
into the male pronucleus and obviously all of this technology has to be done under a microscope
because this, at the single cell stage, is smaller than what we can see.
And so then what we do is we now that this injected, fertilized oocyte
and we now implant that into what we call a pseudo-pregnant mother.
So this mom thinks she's pregnant but she's not.
She's been mated the night before with a vasectomized male, so not much was happening.
But basically she's got all the right kinds of hormones circulating through her body
so she's ready for embryos but she doesn't have any herself.
And so we provide her with these embryos
and basically now what we're left with is what we call transgenic mice.
Transgenic because now they contain two genes that have integrated into their chromosomal DNA
and now every single cell of this animals body now basically has these two genes
but only those cells of the skin that are keratin 5 promoter active, that are expressing keratin 5
are also now going to express that tetracycline-regulated repressor protein.
And then only when tetracycline is going to be there
is that repressor going to be able to function as a repressor to be able to control
the expression of the other gene which is basically the histone H2B-GFP gene.
So how does this work then?
So what we're now going to do is called a pulse-chase experiment.
This was type of technology that was first engineered to be able to label DNA
rather than chromosomal proteins. Many people used pulse-chase experiments by basically
pulsing with tritiated labeled thymadine or with bromo-deoxyuridine
to incorporate into DNA and then chase it over time to look at
which cells are dividing rapidly and which cells are dividing infrequently.
So what we're going to do know is take our mouse, our bi-transgeneic mouse, and now
what we get is in the absence of tetracycline, this repressor protein is
going to be produced because it's under the control of the keratin 5 promoter.
So it'll be produced in all the skin cells.
But, basically, the repressor protein is not going to be functional because it needs
tetracycline to act as a repressor and so what happens is that this gene, then, is on,
producing lots of histone H2B-GFP
and those histone proteins incorporate into the chromosomal DNA
and so this mouse has skin that's glowing green with histone H2B-GFP.
And now if we take a cross-section of the skin what we see is that
all of the nuclei of the skin epithelia but only the nuclei of the skin epithelia are green.
There are about twenty different cell types of the skin and we only have one cell type,
that of what we call the keratinocyte, which consists of the cells of the epidermis,
the hair follicle, the sebaceous glands, the sweat glands,
which are basically now expressing histone H2B-GFP
because the cells with proliferative potential that exist within
the epithelium of the skin are basically the cells that are
expressing keratin-5 which basically allows this protein to basically be expressed.
OK, so now when we add the tetracycline, now this protein acts as a repressor protein
and basically shuts off the expression of Histone-H2B-GFP.
And so now the gene turns off.
So we start out with all of these nuclei green but now all of those cells that are
dividing very rapidly are no longer producing Histone-H2B-GFP
and so they're going to start diluting out the label.
All of those cells that are differentiating, those cells that are
moving outward, being sloughed from the skin's surface,
the hairs that are being produced are basically all going to be lost
from the skin surface because the tissues are undergoing turnover.
Those cells are no longer going to have H2B-GFP expression.
But those cells that exist within this entire population
of green glowing cells that are dividing infrequently, rarely dividing,
the characteristics of what we know about stem cells,
those cells are going to be the only cells of the skin that are
still going to retain the label, the fluorescence label after an extended period of time.
So if we add tetracycline to the animal's diet and now we wait four weeks,
now what we're left with is just those label retaining cells now
that are keratin-5 promoter active that are expressing histone-H2B-GFP
and these are cells that exist within the hair follicle.
These are cells that are known...in a region known as the bulge.
These are bulge follicle cells and these cells reside just above...
just below the sebaceous glands. So these would be the sebaceous glands of the hair follicle.
And then, this is the growing hair follicle that produces the hair.
And so these bulge cells are part of the hair follicle
but as you can see these are the only cells within the skin epithelium
that are able to retain their label, that have the properties of being infrequently cycling.
So this is just a 3D image of a section of skin and you can really see that the skin
is really divided into proliferative units
where every unit has surrounding epidermis and a single hair follicle
and every hair follicle, then,
has one of these label retaining compartments known as the bulge.
So, what can we do now as scientists with these labeled bulge cells?
Well, let's consider what the adult stem cells can do.
We know that adult stem cells should be able to repair wounds
as well as to, in this case, produce a new hair follicle during the normal hair cycle.
And so let's consider, first of all, whether or not these cells can repair wounds.
And, in fact, here we've just scratched the surface of the animal's skin
and now you can see that these fluorescently labeled bulge cells have basically
been mobilized to move upward where they re-epithelialize the epidermis.
They repair the wound that was created.
And so this is one characteristic of adult stem cells--what you might
expect for adult stem cells to have; the ability to repair tissue upon injury.
So what's the the other characteristic, then?
The other characteristic is the normal wear and tear of the tissue.
And in this case, your hairs, as we mentioned before,
undergo periods of rest and then periods of growth again.
So after a while you probably notice some of your hairs fall out.
Those hairs are able to regrow.
And that property is known as the hair cycle.
So what happens to these label retaining cells, then, during the hair cycle?
Well, what happens is that now we can see that these bulge cells
still remain bright green even during the activation step of the new hair cycle.
And what happens is, at the beginning of the new hair cycle,
these cells down here, called dermal papilla cells,
which are specialized mesenchymal cells of the body,
find themselves right adjacent to this bulge compartment of fluorescently labeled cells.
And there's something about these cells that stimulate the regrowth of the new hair follicle.
So what happens then is that the cells exit the niche and then they rapidly divide
to regenerate the new hair follicle and we know that these cells are rapidly dividing
because we basically take a look at these cells. These are characteristics of quiescent cells.
These cells are no longer green. They label with a variety of different proliferating markers
such as Ki67 or Basonuclin but these cells have lost their fluorescence and
that must mean that they've divided rapidly and diluted out the label.
And so what we're learning, then, is that very few cells
from the bulge are activated with each new hair cycle.
And when they exit they undergo rapid division.
Now what do we know about these cells?
Well, we know that they have to be...that these cells have to be
derived...the cells of the new hair germ have to be derived from these bulge cells
because now if we simply go underneath the microscope and expose them a little longer
we can see that in fact these cells down here are, in fact, green.
They just have much less green than the bulge cells.
So when they exited they rapidly divided and
they lost their histone-H2B-GFP as a consequence of, basically, diluting out the label.
So very few, then, label retaining cells are activated with each hair cycle
and they exit at the base, they rapidly divide and they differentiate.
So right away, this now tells us that these label retaining cells
of the bulge have the other property that we would expect of stem cells
and that is the ability to regenerate a tissue in normal homeostasis or normal wear and tear.
So we view, then, the bulge as a transition zone.
These cells reside in a particular niche of the hair follicle.
And, these cells find themselves in a growth and differentiation inhibited environment.
And so, there must be some kind of environmental changes in the niche
that are activating the stem cells which are otherwise quiescent.
In the course of a wound stimulus,
these cells are prompted to move upward to repair the epidermis.
In response to a dermal papilla or a mesenchymal stimulus these cells
are activated to exit at the base of the niche where they give rise to the hair follicle
and that hair follicle then produces the new hair.
So, what we did next was to take advantage of the power of the ability
to culture various different cells from the skin.
And here, we could take our label retaining cells
and place them into culture and ask what happens to these cells.
Well, if we do that, what happens is that these cells give rise to colonies.
These colonies of cells are relatively large colonies
composed of small, relatively undifferentiated cells.
And now, if we pick a colony whose contents are derived from one single bulge cell,
and we passage that colony into another petri dish,
what we get are more large colonies and what that means
is that, basically, those are properties of self-renewal.
We start with one bulge cell. We get a colony of cells.
We passage that colony of cells and we get more large colonies and,
basically, properties that you would expect of stem cells.
By contract, if we now take those cells expressing about 100-fold less fluorescence,
the progeny of the bulge cells, and we now place those cells
into culture and ask, "Well, what happens now?"
These cells, by contrast, give rise to relatively small colonies composed of
large, more differentiated cells and, on passage, these cells undergo scenesence.
Basically, they cannot produce large colonies and they basically cannot grow endlessly.
Again, properties that are more typical of what we call a transit amplifying cell;
a cell with a limited number of cell divisions before these cells then go on to differentiate.
So, these data all tell us that the bulge really does appear to be a reservoir of stem cells.
But are all of these cells within the bulge multipotent stem cells?
Or is it that we simply have a bag of stem cells,
some of which migrate upward to give rise to the epidermis on injury
and others of which produce the hair follicle?
Are we dealing with a bag of a heterogenous mixture of different stem cells?
Or are we dealing with a homogenous mixture of stem cells that are multipotent?
And so if you want to address that question, you have to use what is know as
a clonal analysis to answer that question.
You have to ask, "Is one bulge cell capable of doing it all?"
So here we have our ability by virtue of the ability to culture these cells.
Now what we do is we start with a single labeled bulge cell
derived from our bulge, fluorescently labeled bulge,
and we pick one of these colonies whose progeny are all derived from that single labeled bulge cell.
And the next thing we do is we combine that GFP labeled colony, now, with
unlabeled dermal cells and now we remove...do a full thickness graft of a plug of skin.
We take off a plug of skin from a nude mouse which cannot make hair follicles
and we introduce, now, our mixture of cells.
So let's first do the experiment called the control of the experiment.
And that is to ask what happens if we just take unlabeled dermal cells
and we create a wound, effectively, in the nude mouse and see what happens.
Well, here's our answer. Basically, the nude mouse can repair
it's own skin and if we just use unlabeled dermal cells we do get repair of the epidermis
and what that means is that the nude mouse's epidermis can grow in and fill in a wound.
And what that means to us is that, basically, there have to be adult stem cells
that are unipotent stem cells that exist within the epidermis and that's a good thing
because you wouldn't want injury repair to be predicated on the basis of having hair.
So, if we now take our contents of a single bulge colony
and we combine that with unlabeled dermal cells,
and we now graft that onto the back of a nude mouse,
now what you see if a tuft of hair, lots of different hairs.
All of those hairs are derived from one single bulge stem cell.
And, of course, the hairs happen to be green.
This wasn't really intended to be used in a late night bar in the bar scene.
But basically, we could color code the hair.
We could make it all sorts of different colors of fluorescence.
But, that wasn't our intention in this particular experiment.
Our intention was to demonstrate that all of these cells, basically,
came from that single bulge cell. Now, "Why are these cells green?" you might ask.
In fact, aren't those cells supposed to be losing their label when they start to divide?
Well, remember, that we're using a tetracycline regulatory system and in this case,
what we're doing is using cells in the absence of tetracycline so that all of those cells
derived from the bulge cell are fluorescently green.
So it takes tetracycline to be able to turn off the histone-H2B in these cells.
So, if we now take a cross-section of the skin and take a look at what we get
what we see are green epidermis. We see green sebaceous glands and we see green hair follicles.
And since all of these cells came from a single bulge cell,
what that tells us is that these bulge cells are multipotent.
They have the capacity to repair the epidermis.
They have the capacity to repair and produce sebaceous glands and the capacity to make hair.
They're multipotent cells able to make at least three different tissues.
So, how do we then isolate and characterize these cells?
How do we get these cells to be able to do our culture system?
Well, basically, what we use is a technology known as Fluorescence Activated Cell Sorting.
We can take advantage of the fact that these cells are fluorescent to be able to
isolate and purify them with high efficiency.
And the way in which we do this is, in step one, we enzymatically separate
the epidermis and discard it from the dermis.
And so, basically, what we have here is the epidermis. We throw that out.
Now we have the hair follicles sitting in the dermis
and now what we do in step two is, basically, to enzymatically digest that tissue
and we make a single cell suspension of follicle cells, now, with some dermal cells.
And of that, only a very few of these cells are green.
Those are the cells that are derived from the bulge.
And now we purify those green cells from all of the other cells by
passaging this cell mixture through what we call a Fluorescently Activated Cell Sorter.
And this is called a FACS machine. FACS for Fluorescently Activated Cell Sorting.
What this machine does is it basically takes the population of the cells,
and it identifies which ones are the brightest fluorescently labeled
and puts them into one test tube
and takes the other cells which are weaker labeled and put them into another test tube
and cells that don't have any label at all and put those into yet another test tube.
And so basically, by this type of technology, we can purify
get very cleanly purified populations of our labeled bulge cells.
So now what can we do with that?
Well, we can take these cells and now we can isolate messenger RNAs.
If we isolate the messenger RNAs we can
produce two different...we can isolate messenger RNA populations from
the very brightly fluorescently labeled cells, the bulge cells, and their progeny.
And now we take those two groups of messenger RNAs and
we can use those two groups of messenger RNAs as templates to make fluorescent cDNA.
And now we can take that fluorescent cDNA and hybridize it to microchips that
contain oligo nucleotides to coding sequences of genes.
This type of technology, now, is referred to as microarray technology.
And what this allows us to do is to ask the question of what are the genes
that these very brightly fluorescently expressing cells make relative to
these weaker fluorescently labeled cells. What's the difference in gene expression patterns?
And so this advanced technology, now, allows us to say,
"What's the difference between bulge cells and the bulge cell progeny?"
And from this type of technology we've come up with, now, about 150 new genes
that are expressed specifically by the bulge cells and not by their progeny.
So these gene expression patterns, then, allow us to
learn a lot more about these quiescently labeled bulge cells
and also look at these quiescently labeled bulge cells in their activated state.
One of the things that we learned about these fluorescently labeled bulge cells was that they
express a large number of different family members from two signaling pathways.
One is know as the TGF-beta signaling pathway and
the other is known as the Bone Morphogenic Protein signaling pathway.
Sounds like it should be in bone but in fact many different cell types utilize this pathway.
Signaling is active based on antibodies that are antibodies for transcription factors
that are activated by TGF-betas called phospho-Smad2 and by factors that are
activated by BMPs which are phospho-Smad1.
And we can tell that, by using both of those antibodies,
that these signaling pathways are active in these quiescent cells.
We can also take our cells in culture
and we can expose them to these various different growth factors.
And when we do that, what we can show is that either one of these growth factors
transiently withdraws the cells from a proliferative state to a quiescent state.
And if we wash away the growth factors, the cells resume their proliferative activity.
And so what these data tell us is that these up-regulated genes,
patterns of gene expression, and these up-regulated signaling pathways
have..are contributing to maintaining the quiescent state of these bulge stem cells.
What we also found was a number of up-regulated genes that
encode for inhibitory molecules for another signaling pathway.
And this signaling pathway is called the Wnt pathway.
And we found that activated Wnt signaling pathway members tend
to be expressed at a reduced level in these bulge cells.
So what's the significance of this?
Well, my laboratory has been interested in this pathway for quite a few years now.
The pathway was first identified in a genetic screen in Drosophila fly
where Wnt, the Wnt pathway, in flies is called Wingless.
And you might imagine from the phenotype that flies that
basically are deficient for one of these various different Wnt signalling members
turn out have defects in their wings and in fact in something called the dentical of the fly
which we think is quite similar to that of the mammalian hair follicle.
So, what is this pathway?
So what this pathway utilizes is a protein called beta-catenin.
And beta-catenin has two functions. It's an interesting protein.
Although now we're learning that there are many different types of proteins of the body
that are used for multiple different functions.
This was one of the first ones that was discovered
and normally, in the absence of any Wnt signal, or wingless signal,
then beta-catenin sits out here in the membrane where is interacts with a protein called E-cadherin
and another protein called alpha-catenin
and a bunch of other proteins that we're now learning about.
And it basically acts in cell-cell adhesion.
But, in the presence of a Wnt signal, now there's a complex of various different events,
it's like a domino of events, a series of events that happen,
whereby a series of different phosphorylations results, in the end, in an accumulation
of beta-catenin protein in the cytoplasm and this excess beta-catenin can now be
utilized as a nuclear transcription factor
for proteins of the Lef1/Tcf family of DNA-binding proteins.
So, when beta-catenin interacts with a Lef1/Tcf DNA-binding protein, together that
protein constellation acts as a transcription factor and together
those complexes can then activate downstream target genes.
So, by receiving a Wnt signal, the cell can change its program of transcription
and start to express a new set of genes.
So, how do we ever detect, in a mouse, whether or not
a particular cell is receiving a Wnt signal or not?
When do we know that there happens to be a Lef1/Tcf DNA-binding protein available
and the cells receiving a Wnt signal and stabilizing beta-catenin
and activating these target genes?
Well, several years ago Ramanuj Dasgupta, who was a graduate student in my laboratory
and is now actually a faculty member at New York University School of Medicine,
made a mouse, engineered a transgenic mouse.
And this mouse encoded a beta-galactosidase or LacZ and this is a bacterial enzyme.
And he put this bacterial enzyme encoding beta-galactosidase under the control
of an enhancer element containing multimerized Lef1 DNA binding sites.
So, now, in the animals that are harboring this gene, whenever a particular cell
happens to be expressing a Lef1/Tcf family member of binding proteins
and the cell has stabilized its beta-catenin by receiving a Wnt signal,
now, basically, these transcription factors start to be activated on this enhancer
expressing LacZ and now producing lots of beta-galactosidase.
And beta-galactosidase is an enzyme that cleaves a colorless dye and it turns blue.
And so by using this colorless dye, exposing our tissues to the colorless dye,
we can tell whether or not the cell is blue or whether the cell is not
as to whether or not the cell is receiving a Wnt signal.
And so let's take a look at an example of this.
And here is an example where we're taking a look at a cross-section of the skin
and if we first take a look at whether TCF-3 is expressed or not,
here we have an antibody to TCF-3 and we can see that
TCF-3 is preferentially expressed in this bulge.
We can also now take a look at the Wnt reporter activity and we see just a few cells.
Here are a few blue cells within these bulges that are turning blue.
And curiously, they're only at the stage
just before the activation stage of the new hair cycle
where the mesenchymal dermal papilla cells come into contact with the bulge
to stimulate a new round of hair growth.
And we wonder whether or not the activation of these blue cells
might have something to do with that activation of hair growth.
Could Wnt signalling be stimulating hair growth?
And in fact, if we take a look at what happens, then,
at the very earliest stages of stem cell activation to make the new hair,
we see an up-regulation in stabilized beta-catenin
and this new hair germ ends up producing excess beta-catenin as it comes into contact
with the dermal papilla cells and the bulge to
make this new hair structure that then grows downward and will make the new hair shaft.
So now, let's make another transgenic mouse that's expressing elevated levels
of stabilized beta-catenin and ask what happen now, again, in the hair cycle
now when the animal is, basically, expressing a little too much beta-catenin
in its bulge stem cells.
And when that happens, then, now at the same stage
at which dermal papilla cells come into contact
with the bulge to reactivate the new hair growth cycle,
now we see many more cells turning blue, indicative of Wnt signalling.
So these data are starting to tell us that there's something about
genes that are regulated by Lef1-beta-catenin complexes that are driving the cell
from its quiescent state to starting a new round of hair growth.
And when one thinks about how we would make a hair, this type of knowledge is important.
So, Wnt reporter activity, then, in the follicle stem cells
is hair cycle dependent, Wnt dosage dependent, and that then raises the questions:
What are the consequences to that? And what happens is that we can now
basically test whether or not Wnt is an important message that stem cells need to become hair cells.
And so here just to describe the technology, what we did then was to use a skin regulator,
like our keratin-5 promoter, this time driving stabilized beta-catenin rather than LacZ
and now asking the question of whether or not
we...the hair follicles have a consequence
when they're, basically, expressing slightly elevated levels of stabilized beta-catenin.
So this is called a gain of function study.
We're expressing an excess of stabilized beta-catenin in the mouse.
So what happens in the gain of function study?
Well, the hairs still undergo their normal periods of growth.
This is the wild type hair in its growing stage. This is the transgenic mouse hair
in its growing stage. The hair follicles can still grow.
They still produce hair. And the hair follicles still undergo their resting stage as well.
At the end of a period of a spurt of hair growth the hairs become dormant.
The lower two-thirds of the hair follicle degenerates.
And what we're left with is the quiescent bulge
just sitting there as basically the bottom of the hair follicle at the end of its growth phase.
And you can see that the transgenic mice follicles also undergo a normal round of hair cycle.
They enter quiescence normally.
But now what happens is that in the presence of slightly elevated levels of stabilized beta-catenin
now the hair follicles cannot hold their quiescent state
as long as their wild type counterparts do.
And so all of the hair follicles precociously say,
"I'm going to go ahead and start making hair follicles now."
They enter the hair cycle precociously.
And so that data tells us that, basically, stabilized beta-catenin and Wnt signalling
is starting to precociously coax these cells to
become hair cells sooner than their wild type counterparts do.
And again, this just reminds us that by beta-galactosidase reporter activity
there are more cells that are stimulated at the start of the new hair cycle.
So, beta-catenin and Tcf then function in stem cell activation
and we conversely then did a conditional loss of function experiment
where we removed the beta-catenin gene in the adult skin.
And what happens when you remove the beta-catenin gene is that
in the absence of beta-catenin the stem cells can no longer be maintained
and one loses all...the animal loses all of its hair.
So if you don't have beta-catenin, you can't stabilize beta-catenin.
Basically, all of the hair become lost and the stem cells cannot maintain themselves.
Conversely, if you express a little too much beta-catenin,
the stem cells are activated to make hair precociously.
These data are really starting to tell us something about
what controls the dormancy of these stem cells;
making these stem cells, coaxing them, to making hair.
So, in this case, instead of making a mouse expressing excess protein,
we remove the gene from the mouse altogether and this is referred to as a knock out mouse.
So, since beta-catenin is so important to the mouse we must engineer a mouse
so that we can remove and control it in the tissues that we want to
and this is called a conditional inducible knock out mouse
where, again, we use a keratin-5 promoter now to control the enzymes that are basically
going to end up removing or excising the beta-catenin gene and then we use an inducible system,
like a tetracycline inducible system,
in order to be able to activate the enzyme whenever we want to.
So, scientists can really manipulate the mouse. That's really one of the reasons why we use mice
rather than humans which obviously you can't do any of these studies for.
So we're learning a lot about the hair follicle and ultimately we hope that
this work is going to be useful
in terms of understanding the properties of human hairs.
So, when we remove the beta-catenin gene, then, from the mouse's skin
the stem cells do not survive and the mouse becomes bald.
So, what happens to the mice when we express way too much beta-catenin?
Well, this really gets us into the issue of hair growth.
And as I mentioned to you, in the presence of excess beta-catenin,
now these mice develop very super furry coats. You might imagine that this mouse over here is
a lot thinner than it's brother and it turns out that this mouse is now able to basically, just
produce a lot of excess hair and
this is the mouse that is basically expressing elevated levels of stabilized beta-catenin.
Well, in this first experiment that we did,
we didn't try to control the level of Wnt signaling at all.
And in many different types of cancers, human cancers as well,
if you allow growth factor signaling pathways to go amok, basically just be uncontrolled,
then it often gives rise to cancers.
And Wnt signaling is not any exception to that.
There are many types, different types of tumors
where excess Wnt signaling gives rise to the cancer.
In fact, colon cancer, familial colon cancer is actually a case where defects,
not so much in beta-catenin,
but in the proteins that are associated and involved in stabilizing beta-catenin
are defective in colon cancer and that leads to excess stabilized beta-catenin
and excess activation of some of the genes I've been telling you about.
And it turns out that in these mice they develop something called pilomatricoma
and it's basically a hair tumor. It's a hair ball growing underneath the skin.
These tend to be benign tumors so they're not cancerous.
But basically, they are lumps or bumps on the scalp and we went on to demonstrate that
the human condition, human pilomatricomas,
are indeed due to stabilizing mutations in beta-catenin.
So the mice are telling us something about various, different human disorders.
as well as about hair growth.
So clearly if one ever wanted to adapt the activation of Wnt signaling
in a clinical setting,
you definitely have to be able to control Wnt signaling to be able to do that.
But again, like many growth factors there are many ways of controlling a growth response
and the important thing is that we're understanding that beta-catenin stabilization
and Wnt signaling along with several other growth factor signaling pathways
are playing important roles in hair growth.
So, to be useful clinical, Wnt signaling would need to be very carefully regulated.
So what I've told you is basically that it is the levels of stabilized Lef1 and beta-catenin
that are determining the outcome of these stem cells.
We start stem cells at the low end of the scale where
Wnt signaling or Tcf/Lef1/beta-catenin activity is very low
and then as we start to elevate the levels the stem cells can become activated.
But, then if we really start to elevate the levels we can push these cells
into a hair follicle fate to make new hair follicles and in fact,
we can make additional hair follicles at very high levels;
many more hair follicles than normal in that furry mouse that I described to you.
We've learned that Wnt signaling plays an important role in hair differentiation,
in actually making the dead hair cells that produce the hair shaft.
And then finally, that if you use way too much Wnt signaling,
that this then leads to tumorigenesis in the form of pilomatricomas.
So, if one wanted to control the levels of Wnt signaling in activating the hair cycle,
you'd need to be somewhere around the low end of the scale.
So, we're also learning that there are different sets of genes depending upon
how much Lef1/Tcf/beta-catenin is active in the cell;
that there are different sets of genes that are switched on.
In fact, in some cases, they're proliferation associated genes whereas in other cases
they happen to be genes like hair keratins, which are, basically, involved in producing the hair shaft.
So, I've now told you, then, in summary, that the bulge is a transition zone.
It contains infrequently cycling cells that are keratin-5, keratin-14 positive.
These are stem cells and what we've learned about these stem cells
is that TGF-betas and BMP signalling pathways maintain these cells in an undifferentiated state.
And what we're learning is that Wnt signaling, along with Wnt's various different partners,
are involved in stimulating these cells to make a hair follicle.
These cells then produce rapidly proliferating cells that go on to use additional signaling pathways
that we're learning about in producing the hair shaft.
We've learned about the hair growth stimulus
by learning methods where we can purify these dermal papilla cells.
We've also engineered various, different mice where we can purify these cells
and we now have a good idea about what genes these cells are making.
And, basically, what types of factors those cells are producing and secreting
that might give us clues as to how these stem cells become activated
in these mesenchymal-epithelial interactions.
And so that's going to be another important aspect about understanding more about hair biology.
So additional questions, then, that we'd like to address in the future is:
Can we make hair cells culture and use these for hair replacement
like we can for generating burns as I described to you earlier?
We can take skin stem cells and produce epidermis and in fact we can
take hair follicle stem cells and produce epidermis.
We can take hair follicle cells and produce hair.
But we can also hope that in the future we might be able to, for instance,
expose stem cells in culture
to various different hair producing growth factor signaling pathways
and generate hair cells that then could go on to be useful in a clinical setting.
And then, of course, one wonders, "How far can you take these adult stem cells?"
Could we get other types of stem cells or other types of cells such as a nerve cell.
So, coaxing skin stem cells to become hair cells we think might be something that
we could consider for the future. We certainly are working on this type of a possibility
as are other researchers who work in this area.
In addition, we're learning various different growth factors
and various constellations of growth factors that are important in switching on
or controlling nuclear Lef1 and nuclear beta-catenin which we can already
do when we place these cells in a culture dish.
So now we need to know: Can we learn to maintain and propagate
and differentiate follicle stem cells and their associated mesenchymal cells?
And then, how many options;
epidermis, hair, sebaceous glands, sweat glands possibly corneal epithelial cells.
In the early embryo, it's the mesenchym that dictates
what types of an epithelium is going to be forming and in the early embryo one could take
specific corneal mesenchym, for instance,
and differentiate non-corneal cells to be able to produce corneal cells.
We don't know yet whether that's a property of adult stem cells but if it is,
these adult skin stem cells, hair stem cells,
could be useful for other potentials in regenerative medicine
perhaps treating blindness one day.
That's something that scientists don't know about yet.
We continue to work on the mouse to ask whether that might be possible
and again, underscoring the importance of this type of research.
We can already take our stem cells, as I've shown you examples of,
and basically generate hair follicles in mice
and obviously we don't yet know whether that potential can be realized or
whether it can be valuable in the future in a clinical setting in humans.
So, what's the big deal about this role? Well, all of the cells of the body have the same genes
and cells, as I mentioned, differ by the patterns of genes they express.
And so, what will happen if cultured follicle stem cells are treated with nerve growth factors?
Well, at present, let me emphasize that, in fact,
scientists don't think that that's going to be possible.
We think it might be possible to be able to push the plasticity
of these skin stem cells to be able to perhaps produce other epithelial types of cells.
We don't think it's going to be possible to take these epithelial stem cells
and make neurons. There might be other stem cells within the skin that have that potential
be we don't think that that's a potential for epithelial stem cells. Why not?
Well, this gets back to the issue of: what do I mean by reprogramming?
We talked early on in the presentation about nuclear transfer studies
the ability to take an adult skin stem cell nucleus, place that into an oocyte,
and basically, reprogram the nucleus to act
as if it was an embryonic stem cell when we then culture the cells.
What is it that happens in that reprogramming?
And, what happens are, basically, a number of marks that are placed upon
the chromosomes, placed upon the proteins, placed upon the DNA
that basically give those marks history, of saying during embryogenesis,
with all these different marks that are given to the chromosomes, to the DNA,
what genes are supposed to be expressed when.
That's important for our body, for all the cells in our body to function.
This is called epigenetics, it's an emerging field.
And what we know is that to reprogram means to basically strip the cell
of all that memory of information of different marks, different post-transcriptional marks,
but marks such as methylation, acetylation, ubiquitination, phosphorylation,
various different types of marks that basically tell the cell what genes it's supposed to express.
And what you're asking in a reprogramming experiment is, basically, to ask the cells to forget
what they were and by the time you get to be an adult stem cell of the skin,
you've gone through a lot of marks and you've got a lot of epigenetics
carried in that nucleus, carried in the DNA that is basically saying
"I'm a stem cell of the skin and I ain't gonna make a neuron!"
And it's really that property of the oocyte that can really do this reprogramming
in a way that we just don't think that we know enough yet nor are we even optimistic that
we'll ever know enough to be able to reprogram, for instance, an adult skin epidermal cell
to be able to make a neuron.
So we don't yet know whether we're going to be able to reprogram
skin stem cells in the future to be able to make neurons.
So, are there ways to get around this problem?
Well, in the future, scientists may be able to engineer human embryonic stem cells from
skin stem cell nuclei as we've already done
and as I've already told you about with mouse skin stem cells.
The oocyte cytoplasm does the best job at reprogramming the chromatin
even if it isn't yet perfect.
There are multiple types of stem cells of the skin
and there may be some stem cells of the skin that have this potential.
So, just in wrapping things up, then, I always tell my own students
that the Golden Rule of scientists is that:
Make sure you don't get too engrossed in the details to miss the big picture.
It's often, when we're looking for something in science,
that we can't find it no matter how hard we look
and yet when we're not looking for something,
that's when we most often discover something really exciting.
and so that just emphasizes the importance of
a broad base of a lot of different science
going on in the field of stem cell research. Not only people studying adult stem cells but
people studying embryonic stem cells,
people studying the biology as well as people studying the clinical applications.
We need to know much more and the future looks very bright for this type of technology
but it's going to be a long time before scientists really know:
will it be possible, as we think it might be,
as we've got it written in the books, as what it should be, and really research
is the only way we're going to sift our way through the answers to those questions.
But I do tell my students, as well to keep an open mind, to be receptive to new ideas.
And as well, we need to tell the public and teach the public of the importance
of keeping an open mind and being receptive to these new ideas
that are emerging and that scientists are so excited about in the whole field
or embryonic and adult stem cell research.
So, at this point I'm going to conclude my talk and say
I've enjoyed talking to you today about the kind of science that we're interested in
and that I'm passionate about and the science
that my graduate students and my post-docs do in the laboratory
at the Rockefeller University, Howard Hughes Medical Institute,
in New York City, in Manhattan.
Thank you.