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HAZEL SIVE: I want to
discuss with you today
a very topical and
interesting question, which
is the notion of stem cells.
In fact, I'm going to discuss
two things, the first of which
is another concept
that you need,
following on from the
concepts we had right
at the beginning of lecture.
I feel like this microphone--
The first is a concept that you
need in addition to the ones
we started lecture
with, and then
we'll talk about stem cells.
So today, we'll
talk about potency,
and then we'll talk
about stem cells.
Potency, along with
fate, determination,
and differentiation,
is one of those terms
that you need to know and you
need to understand in order
to understand stem cells.
Potency refers to the
number of possible fates
that a cell can acquire,
number of possible fates
open to a cell.
And this is a very important
concept of development
because, in general,
potency decreases with age,
and decreases as different
parts of the organism
become specialized.
So in general, potency
decreases with age.
But I will put in
here, and we'll
explore this more in a moment,
except for some stem cells.
And we haven't defined a
stem cell yet, but we will.
What kinds of
potencies are there?
There's the big one,
totipotent, where
a cell can become all fates.
And there's really
only one cell that
can do this in the normal
animal, and is the zygote.
And in most animals,
even as the zygote
becomes just two
cells or a few cells,
that full potency is lost.
And cells instead,
in the embryo,
are multi or pluripotent, which
means that they can acquire
many fates, but not all fates.
Embryonic cells, especially
in the early embryo,
and many stem cells
can also become,
are also multipotent
or pluripotent.
And then as time progresses--
is that a hand up?
Yes, sir.
AUDIENCE: How do you reconcile
the fact that human cells,
we can separate them.
Even at the eight cell stage.
HAZEL SIVE: That's
a great question.
The question is
how do I reconcile
what I'm telling
you with the fact
that you can get
identical sextuplets,
or octuplets actually?
It's a good question.
That's true.
In different animals, the
very early embryonic cells
are sometimes totipotent
up to a while.
OK?
And so for example,
in armadillo,
here's a piece of, you know,
fact for your back pockets.
In the armadillo, the
eight-cell embryo almost always
splits into eight single
cells, each of which
becomes a baby armadillo.
OK?
So those cells are totipotent.
In mice, even at
the two cell stage,
the two mouse cells are
probably not equivalently potent
and they're not totipotent.
So very, very seldom--
almost never-- get
identical mouse twins.
OK?
So it's one of
these generalities.
And if you ask me, you get the
specifics are a bit different.
As cell fate
restriction continues,
cells can become
bipotent, or unipotent,
whereby one or just two
fates are open to them.
And so if we look,
taking this concept,
let's now start the
lecture about stem cells.
You're going to
need this concept.
Stem cells, I'll point
out, whatever they are,
got almost 3,000 hits
yesterday on Google News.
This is way below
baseball, which got 45,000.
I checked.
But still, you know,
as science topics go,
stem cells are really up there.
And they're on the covers of
magazines, over and over again.
And we'll talk more
about why that is.
Here's a diagram-- it's
not on your handouts--
that I drew for you.
Let's not dwell on it.
But let's now move
on to Topic Number
2, which will fold in
this concept of potency
and the concepts of
fate, determination,
and differentiation, and
talk about stem cells.
And let's do, as is
our custom, let's
define what a stem cell is.
I think that a stem
cell can be defined
as a cell of
variable potency that
has the capacity to self-renew.
Cells of variable potency
that can self-renew.
They can make more
of themselves.
Despite the hype, despite
covers of Time Magazine
and almost every front
page of every newspaper
across the world, stem cells are
normally found in our bodies.
And normally, as
we'll explore, they're
used for organ maintenance
and repair, organ maintenance
and repair.
But the thing, you know,
that has everyone fired up
is that you can somehow
harness these cells
for therapeutic purposes.
And that you can repair what the
body cannot, by being clever,
and using the power of these
cells as they normally have it,
or as you can give it to them.
And so there's this question
of therapy and therapeutic stem
cells, where the idea, again, is
that you would repair a damaged
organ by introducing
somehow, injecting
or otherwise introducing, extra
or somehow special stem cells--
which I am going to
abbreviate heretofore as SC--
by introducing extra stem
cells into a damaged body.
Does this work?
It does work.
It works for the
hematopoietic system,
as in bone marrow transplants.
And it also works for
skin cell transplants.
Well, let's just put skin cells.
OK.
Skin stem cells can be
grown from your own skin.
And in the case of
burn victims, this
has really saved
countless lives.
The original technology
began to be developed here
at MIT by Professor Howard
Green, who is now at Harvard.
But the idea is
to take your skin
and grow it on
something like gauze
or some kind of
some solid support,
and then to cover a burn patient
with layers of support on which
there are some stem cells.
And these stem cells will
help fill in the holes
in the skin left by the burn.
Normally when a wound heals,
as I'm sure you've noticed,
it heals from the sides.
The only way a wound can
heal is from the side.
And if it's a big wound, it
can take a very, very long time
to heal.
And you can get
infections and so on
while the healing
process is going on.
So seeding the inside of a
wound with stem cells that
can start the skin
regeneration process
and seal up the body
against infection,
that's been incredibly useful.
And we'll talk more about bone
marrow transplants in a moment.
OK.
We previously talked about this
process by which cells decide,
are undecided
initially, they decide
what they're going to become.
And then they differentiate
into their final function.
Stem cells fit into
this litany somewhere
between the commitment stage
and the differentiation stage.
And in this diagram,
these multiple arrows
are there for a reason.
There are multiple steps between
commitment and differentiation.
And somewhere along the way, a
group of cells with capacities
we'll talk about,
leaves this lineage
and sits around and waits,
partially determined,
so that it can go on and make
more differentiated cells when
they're needed.
And I've added on there the
potency timeline, decreasing
with age, of the
developing animal.
But let's diagram the notion
of stem cells on the board.
Stem cells generally
divide slowly.
Here's one.
It's a variable potency.
It may be multipotent.
It may be bipotential.
And it is somewhat
committed, which is
a slightly difficult concept.
Because last time
we talked about
committed versus uncommitted.
But now I'm telling
you something
can be somewhat committed.
And that gets to
this multiple arrows
there were as cells progress
in their fate decisions,
they change their
molecular signature
and they really do
become closer and closer
to a cell that's
made a decision.
But it's kind of like, you
know, if you're weighing up
going to med school or
going to graduate school
in bioengineering,
you know, you have
decided that it will
be one or the other,
but you haven't decided which.
You're somewhat committed.
And then when you
make the decision
to go to graduate school, you
have now become committed.
OK?
So the cell is doing the
same kind of notion there.
There's your stem
cell, variable potency.
Under the correct
stimulus, that stem cell
will divide to give rise
to two different cells.
One is another stem cell.
And the other is something
we'll call a progenitor.
The progenitor is more
committed than the stem cell.
The progenitor cell
is going to go on
and divide, usually a lot.
Progenitors divide rapidly.
And their progeny
will eventually
go on and differentiate into
one or more different kinds
of cells, maybe a stripey
cell, and a spotted cell type,
and a cell type with squiggles.
And so here are the
differentiated cell types.
And the number of different
differentiated cells
that comes out of
this process is
a reflection of the
potency of the stem cell.
OK?
So here you've got
these progenitors.
The idea is that
these progenitors
will have similar potency.
But as I'll show you, there's
a whole variation on this.
But here the number of
differentiated cell types,
the number of cell
types reflects
potency of the stem cell.
OK?
This kind of diagram is
called a lineage diagram.
It tells you what--
not only what the final
fate of the cell is,
it tells you something
about the progress
towards that final fate.
So a lineage we can
define as the set
of cell types arising from
a stem cell or a progenitor.
Let's talk about the discovery
of stem cells, because this
is something that really
was pivotal in helping
understand whether or not there
was some way that the body
normally repaired itself.
It was clear that during
early development,
there was lots of cell
division and lots of changes.
Cell types were formed
and organs were formed.
But it really wasn't
clear in the adult
how much repair there was,
how much turnover of tissues
there were, and really what
the whole dynamic process
of maintaining the adult was.
And the discovery of
stem cells came about
because people looked to
see how long cells lived.
And what they found
was found using
a turnover assay that measures
the half life of cells.
And they found that
in almost all organs,
in fact, probably in all organs,
cells did not live forever.
They turned over.
They died.
And they were
replaced by new cells.
And this turnover assay
implied that there
was some kind of replacement.
And the cells doing
the replacement
were called stem cells.
You find this by a pulse/chase
assay, which we'll go over
on your handout in a moment.
And what was found was really
variable for different organs.
Firstly, all organs,
about, show cell turn over.
Red blood cells have a half
life of about 120 days.
There are a lot of red
blood cells in your body.
And in fact, that implies
that there are about 10
to the seventh new red
blood cells made a day.
In your intestine,
the half life of cells
is three to five days,
in the small intestine.
And the hair on your head has a
half life of about four years.
So it's variable for
different kinds of cells.
If you look at
your first handout,
it diagrams a
pulse/chase assay where
a cell population is labeled
with a nucleotide analog.
It's a normal nucleotide, but
it's got a bromine added to it.
And it acts like deoxythymidine,
gets incorporated into DNA,
and you give just a short pulse
of this nucleotide analog.
So only some of the
cells get labeled.
And you only give
it for a short time.
So you get a labeled
cell population.
And then you stop the
labeling by adding
lots of unlabeled thymidine,
and that's called a chase.
And you follow the cells
over this long chase period.
And then you can watch and see
what happens to those cells
that you initially labeled
over a very short time.
And so in this example, I've got
four cells initially labeled.
Over time, they're
only two cells left.
And if you measure the time
from going from four cells
to two cells, you can get to
the half life of that cell
population.
OK?
You can also-- if you don't
have a handout for this,
just look on the screen--
you can also follow the
labeled cell population
and see what those cells become.
And you can see that they
go on to differentiate
as particular cell types.
So this is a kind
of way of labeling
the lineage of these cells.
And that is useful, too.
This was the theory behind
stem cell definition.
But what is a stem
cell look like,
and how do you isolate one?
It turns out that
that's really difficult.
So isolation and
assay in the adult
stem cells are very, very rare.
And that is one of
the issues with using
stem cells for therapy.
There are very few of them
and they're hard to isolate.
Hematopoietic stem
cells comprise
about 0.01% of the
bone marrow, which
is where the stem
cells reside, and where
the precursors of your whole
blood and immune system reside.
The way that this
was dealt with was
through a really
clever technique
that has the acronym of FACS.
I'll give you a
slide in a moment.
It stands for Fluorescence
Activated Cell Sorting.
We'll go to a slide
in a moment so we
don't have to spell it out.
And the idea behind FACS is
that you label stem cells.
And you might be guessing
what to label them with.
But you label them usually via
their cell surface proteins
with some kind of tag,
often an antibody tag.
And then you can use that tag
to make them different colors.
And you can then sort
them, cell by cell,
through the Special Fluorescence
Activated Cell Sorter.
Sort individual
cells, and then you
can assay individual cells
or small groups of them
for stem cell properties.
Let's look at a slide of how
the FACS, the Fluorescence
Activated Cell Sorter works.
No, let's not.
I've really gotten
ahead of myself here.
And I'm going to go back
because I want to show you this.
Hold on to that
thought and let's
go back to this notion
of a pulse/chase assay.
I forgot that I had this here.
This is really important.
This is a pulse/chase
assay of intestinal cells.
And so your small intestine
lies inside your belly.
And if you look
at its anatomy, it
contains many
tubes whose surface
is thrown into folds
to increase the surface
area for food absorption.
And if you look at these folds,
they are very closely packed.
So you get a huge
surface area increase.
And the cells of
these folds that
are doing the food
absorption turn over
every three to five days.
Those are the ones
I was talking about.
So if you blow up one
of these folds, which
is called a villus,
there's the part
that's sticking up
into the cavity,
into the lumen of
the small intestine.
And then there's this
part that kind of dips
down into the lining
of the intestine, which
is called a crypt.
The stem cells lie somewhere
at the base of the crypt.
It's not exactly clear where.
But the idea is that somewhere
at the bottom of this crypt
there are these stem cells
that under specific conditions
will start to proliferate.
And they will move
up into the villus
and replace the villus
cells that are dying.
You can monitor this by doing
a pulse/chase experiment.
So here's the crypt.
And they've given,
in this experiment,
a pulse of thymidine
has been given.
And you're looking
kind of at time 0,
right after the pulse of
thymidine has been given.
Here the cells, the black
cells are in the crypt.
They're labeled.
And then if you
look over time, you
can see that these black cells
move away from the crypt.
They're moving up
into the villus.
And here they are actually
right on top of the villus,
replacing the cells
that were turning over.
So that's a really
beautiful demonstration
of a pulse/chase assay.
OK.
Now we can go to our
Fluorescence Activated Cell
Sorter.
The idea is that you
take a mix of cells--
you don't have this
on your handout,
just look on the screen--
the cells are labeled
with fluorescent antibodies.
And you put them into
a reservoir and droplet
generator.
And the cells drop out of
this reservoir, one at a time.
And as they drop out,
they go past a laser.
And you can tune the laser to
whatever wavelengths you want.
It excites the cells.
And if the cells emit in
the particular wavelength
you're interested in, the
detector will detect that.
And then it actually
gives a charge to the cell
that it is the
correct fluorescence.
And as the cells
are dropping down,
the cells of the correct
color are deflected
by an electric charge.
And different color
cells can be collected
into different flasks.
OK?
This really works.
It's a fantastic machine.
You can collect cells
about, you know,
you can collect millions
of cells an hour.
It's pretty quick.
But you do it one
cell at a time.
And in that way, you
can isolate cells, which
have got stem cell properties.
OK.
So you've used
your FACS machine.
You've got cells that
look as though they've
got stem cell properties.
And now, let's look at the
assays that may be used.
And there are three assays
that you should know.
One is a repopulation assay
to test stem cellness.
And this is a transplant assay
where you're transplanting test
cells, test stem
cells, into the adult.
And you have removed from the
adult, endogenous cells that
might be competing
with those stem cells.
That would include
the stem cells.
We'll go through a
slide in a moment.
I'm going to list them here.
Another one is an
in-vitro induction assay,
where you are going
to take isolated cells
and you're going to treat
them with various inducers,
various signaling
molecules, and you're
going to test and see what
fates those cells can acquire.
And a third assay is called
an embryo incorporation assay,
where you are going to take
cells that may be stem cells,
and you're going to test
them in a chimeric embryo.
Let's go through
your next slides
to discuss each of these points.
Bone marrow transplants resulted
in a Nobel Prize in 1990
for E. Donnall Thomas
and Joseph Murray.
It's a technique that
saved millions of lives,
and here how it
works in a mouse.
You take the mouse and
you irradiate the mouse
to destroy the bone marrow
and the stem cells associated
with the bone marrow.
If it's a person, to destroy
the diseased burned bone marrow.
And then you replace that bone
marrow with normal bone marrow
to either try to make the
person better, or in this case,
to test something
about stem cells.
The irradiated mouse
or person would die,
but the normal bone marrow
will cause the mouse to live.
And if you've put stem
cells into that mouse,
you can start getting them out
of that mouse whose life you
have saved, and isolate
more stem cells.
Those kinds of assays
led to the definition
of the hematopoietic stem cell.
Here it is.
It's a pluripotent stem
cell that gives rise
to all of these different kinds
of cells, the immune cells,
and all of the blood cells.
It's a very, very
powerful stem cell.
And the ability to actually make
this diagram and say that there
was a single cell that gave rise
to all these different lineages
was because of a titration
assay where you could take these
putative hematopoietic stem
cells that were difficult
to isolate-- and still haven't
been isolated in their purity--
but you can titrate them down.
And you can introduce what you
think is one stem cell, 10 stem
cells, 100 stem cells, and so
on, into an irradiated mouse,
and ask how many
stem cells does it
take to repopulate the entire
blood system and immune system.
And it turns out,
you have to mix
these cells with carrier cells,
otherwise it doesn't work.
But it turns out that
one cell can repopulate
the entire hematopoietic system,
which is really extraordinary,
and led to the diagram that I
showed you in the slide before.
OK.
Here's another assay.
This is an in-vitro
induction assay.
And the idea here is
that you start off
with something, which you
think might be stem cells,
by various criteria.
And then to test what
these cells can do,
you put them into plastic
tissue culture dishes,
and you add some
nutrients and so on
to allow the cells to divide.
And then you add some inducers.
And you remember a
couple of lectures back,
inducers are just ligands for
various signaling systems.
You might add fibroblast
growth factor to this one,
and retinoic acid to
that one, and then you
ask what happens to the cells.
They will go on, in
general, to differentiate
into different cell types.
And depending on what
they differentiate into,
you can say something about the
potency of these putative stem
cells.
You can't test if they're
really stem cells,
but you can say something
about their potency.
You can do a similar experiment,
but in a whole mouse.
The mouse is made
from, the embryo
is made from a part of the
very early embryo called
the inner cell mass.
And you can inject labeled,
putative stem cells
into an early mouse embryo,
into this inner cell mass
part of the embryo, put it into
a mother, a recipient mother,
and then ask what comes
out, what kind of embryo
comes out of that process.
And if you see that
the baby that comes out
of this chimeric embryo has got
a green liver and green ears
and green whiskers, you'll
know that these cells that you
put into the chimeric
embryo, that you made
the chimeric embryo
with, had the capacity
to give liver,
ears, and whiskers.
OK?
So this is a powerful
assay to, again,
look at the potency of
cells, not, in this case,
the stem cellness of cells.
One of the things
about stem cells
is that you only
want them to work
when you want them to work.
If you cut yourself,
in the normal process
of keeping your
liver the right size,
in the normal process of keeping
your heart muscle correct,
you want your stem cells
to be working and keeping
everything homeostatic.
You don't want them to be
dividing out of control,
because then you'll get cancer.
And so something has to
control what stem cells do
and when they do it.
And this is a
question of regulation
and the notion of
a stem cell niche.
Stem cells are kept
quiescent, usually in G0
that we talked about in
the cell cycle lecture.
And they're kept quiescent by
signals from the surrounding
cells.
So their cell-cell
interaction, and by signals
from the surrounding cells.
And those are given a special
name by stem cell biologists.
They're not that special, but
they're given a special name.
They're called niche
cells, or the niche.
OK?
They're just surrounding cells
that are secreting signals.
On some kind of activation--
you cut yourself, your organ
normally needs repair--
things change.
So on activation, by some
kind of environmental input--
local or less local--
niche cells induce the
stem cells to divide.
And they do this--
and they induce the
stem cells to divide
into a stem cell plus a
progenitor, which then goes on
to do all the things that I
diagrammed that progenitors do,
on the first board.
And the niche cells
do this because they
have changed their signaling.
This is yet another use,
or a very related use,
of the notion of cell-cell
signaling in controlling life.
Here's a diagram.
Here are niche cells.
You don't have this.
Just look on the screen.
Surrounding cells,
maintaining stem cells quiet.
When there's an environmental
input, the niche cells change.
They activate the stem cells
to divide and form more stem
cells and progenitor cells.
One really fantastic
example of the niche,
and the interaction between
stem cells and the niche,
is in the hair.
This is from my colleague
Elaine Fuchs at Rockefeller,
who over many years has figured
out that in the hair follicle--
this is the hair sticking
out of the skin--
in the hair, there's
a small group
of cells on one side of the hair
shaft called the bulge cells,
and these bulge cells
are the stem cells.
Her investigators
isolated these bulge cells
and did the following
experiment to show
that they were hair stem cells.
There's a kind of a mouse
called a nude mouse that
has a very bad immune system.
So it's useful for immune
system experiments.
But it also has no hair.
And you can do a
transplant into this mouse
of these bulge cells.
And you get little tufts
of hair growing where
the rest of the mouse is nude.
And you've done a
careful experiment
where you've labeled the cells
you have transplanted in so
that you can show
that they actually
came from the transplanted
tissue and not
from the mouse, the
nude mouse tissue.
And you can do that because
you labeled them with GFP
and they're green.
So this the green hair.
And it shows you that these
bulge cells are stem cells.
During the life of a hair--
your hair has a life,
we discussed four years
on your head--
during the life of a hair, the
bulge cells and the niche cells
are in different places.
And depending on whether they're
touching or far apart from one
another, there's induction
of hair growth or not.
So at a particular stage
of the hair cycle--
this is called the hair
cycle, look on the bottom
of the screen here--
the bulge cells and
a group of cells
called the dermal
papilla that lies right
at the bottom of the hair
shaft are touching one another.
And at that point, a
particular signaling pathway
called the Wnt
pathway is activated.
And these dermal papilla
cells tell the bulge cells
to start dividing and start
making a new hair shaft.
After that's happened,
the bulge cells
move away from the
dermal papilla cells.
Here they are during
growth, the growth period.
You see the bulge and
the dermal papilla cells
are no longer touching.
At this point, the stem
cells become quiescent.
And there are enough progenitor
cells in the hair shaft
to give you formation
of the hair.
And then the stem cells remain
quiescent until the next hair
cycle starts and they get in
contact with the dermal papilla
again and start making new hair.
This is a really
beautiful story that's
shown us quite clearly
how the niche cells can
control these stem cells.
All right.
So let's spend the last minutes
talking about therapeutics.
Here's the dream.
You know, the dream is
that you have a stem cell
population for every
organ in the body,
including things
like limbs, such
that if your limb gets
severed or your heart
becomes really diseased or
your spinal cord is injured
and you can't walk anymore,
that you can just inject
into a patient the correct
stem cells and everything
gets repaired.
That's the dream.
And that's really the
holy grail of what
thousands and thousands of
investigators are going after.
And it's given precedence by
bone marrow transplants, which
really are very successful.
Turns out that
it's kind of tough.
It's tough because these adult
stem cells are really rare.
The hematopoietic stem cells
are special because they're
kind of liquid.
They're single cells.
They're not attached
to anything.
And they are relatively
easy to identify.
But other stem cells
are very difficult.
So the idea is that you inject
stem cells to repair a damaged
organ.
You need stem cells of
the correct potency,
otherwise you're not going
to repair the specific organ.
But adult stem
cells are very rare.
And so the quest has been to
find some kind of substitute
for adult stem cells.
And those substitutes
come in two flavors.
One are embryonic stem
cells, abbreviated, ESCs.
Embryonic stem
cells are cells that
grow from the inner cell mass
of an early mammalian embryo.
And you can grow
from them groups
of cells that will keep
growing in the laboratory
for a long time and
have variable potency.
So the embryonic stem
cells are derived
from the inner cell mass of
an early mammalian embryo--
human in the case of human.
And you grow out
so-called ESC lines,
which means that these cells
grow continuously in culture.
And each embryonic stem cell
line has a unique potency
and can be used to do
different things, in terms
of theoretical repair.
If you look on
your next handout,
the idea is that you take
this inner cell mass,
you plate the cells
as single cells,
they grow and grow and grow.
And if you treat them
with various inducers,
you can get them to become
heart or neuron progenitors,
inject those into animals
and make them better.
There are some issues
with embryonic stem cells.
And the problems are twofold.
One is ethical in that you
have to harvest human embryos.
You have to obtain and
harvest human embryos
to get these human
stem cell lines.
And currently, you're
really not allowed
to do much human embryo work
to obtain human stem cells.
But the second,
even if you were,
is that these cells
are non-autologous.
They do not match the person
into which you're putting them.
They do not match the immune
system of the recipient.
And so they'll be rejected.
And it's the same as
an organ transplant.
You have lots of
problems of rejection.
So the latest thing that is
very exciting and wonderful
and potentially might be very
useful, is the use of things
called IPS cells, in which
you convert adult cells
into stem cells.
And you do so, as I'll tell you,
by adding some transcription
factors to them.
The advantage of these is
that they are self cells.
You could do them from yourself.
The disadvantage is that
they're really not proven.
And there is still, I will
just say, lots of problems.
But there are a lot
of people, including
some of the top
investigators in the world,
working on these IPS cells.
So look at your last handout.
We will remove our interesting
calendar reminders there.
Adult differentiated
cells, which are unipotent,
can be treated with three or
four transcription factors.
And finding these transcription
factors is the key.
And once you express these
transcription factors
in these adult cells, like
magic, they become stem cells.
It really was like magic.
You can test the potency
of these stem cells
in the same way we discussed.
And Professor Yamanaka in
Japan and Professor Jaenisch
here have shown that these are
really very powerful cells.
And those are the
promise of the future.
And we'll stop there
and meet on Friday.
