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Differentiation.
For someone who didn't see the

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board.  Yeah?

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Yeah.  So what a cell will become
eventually.  And differentiation is

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the process by which
it becomes that.

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OK.  Good.  So these terms are
essential for you to know for

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today's lecture.
And if they are a little murky for

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you then you should really make sure
they become clear.

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OK.  I'm going to talk to you today
about stem cells.

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This is the how-to module,
our second how-to module.  We have

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two lectures, stem cells
and cloning.

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And what I want to do today is to go
beyond the media,

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beyond the hype and tell you about
stem cells, what we really know and

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why there is such a fuss about them.
And to put it in perspective of

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where you are in the course,
in our game board of life we're

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moving right along past foundations
up through formation and now into

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our second how-to module.
So let's start with a question.

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And you have a handout.  If you do
not have a handout, Shamsah,

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could you be mail person?
And on the other side it looks like

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we need another mail person.
You will realize that I typically

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do give you a handout of the most
important slides so that you don't

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have to grapple with things and that
you don't, in fact,

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have to print out the PowerPoint
handout before you come.

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So you should always just check.
OK.  So what is a stem cell?

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Well, a stem cell is something that
has two important capacities.

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Firstly, it can self-renew.  That's
a very popular term.

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It can make more of itself.
And, secondly, it can give rise to

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a number of different,
one or more different cell fates.

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So it can give rise --

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-- to one or more differentiated
cell types.  Schematically,

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I've represented it for you in this
way.  So here is something that's

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called an uncommitted stem cell.
And you should be familiar with the

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term uncommitted, naive
and so on.

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And this uncommitted stem cell can
undergo cell division to make more

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of itself.  And it can also go on to
become something that's termed in

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the field a "progenitor cell".
It's just a term.  It means a cell

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that is going to give rise to
something else.

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And I've depicted it here as a
determine progenitor.

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It knows what it's going to become.
And this term uncommitted stem cell,

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the uncommitted part is a little
misleading.  I'll talk about that

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more in a moment.
This determined progenitor then goes

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on to give rise to a whole bunch of
one or more, actually,

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differentiated cell types.
And the number of cell types that a

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stem cell can go on to give rise to
is a measurement of its potency.

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That is the definition of the term
potency.  So this is the notion of

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the stem cell concept.
You have this.  It's the first

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figure on your handout.
And you'll find this in many

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newspaper articles and books.
So what's so special about this and

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why are there newspaper articles and
newspaper articles and newspaper

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articles and television programs and
covers of Time Magazine about stem

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cells?  What's the hype?
Well, the deal is that it is

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believed by some that stem cells are
going to be some kind of

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universal repair kit.
That somehow because stem cells can

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give rise often to differentiated
cell types and make more of

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themselves that they are in a
position to repair parts of the body

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when the body needs repair.
You have a heart attack, you throw

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in some stem cells,
repair the heart, you have a brain

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lesion, you throw in some stem cells
and you repair the damage.

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And if you go through the
newspapers, as I did very,

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very briefly, it's very easy to find
many, many different headlines.

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One of the big ones last week,
I'll talk about this at the end,

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is that the Massachusetts House
passed a law expanding the use of

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stem cells in research.
Hopkins is beginning human trials

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with donor adult stem cells to
repair muscle damage from heart

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attack.  This is an enormous amount
of interest in these cells because

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of this notion of universal repair
kit-ness.  There are also

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advertisements.
The Stem Cell Bank and the Cord

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Blood Registry would love you to pay
them to save your child's umbilical

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cord stem cells,
or the blood cells from a newborn's

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umbilical cord in the possibility
that you'll use these later on to

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save the life of this child or
somebody else.

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And the other reason that stem
cells have got so much hype is that,

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as we'll discuss towards the end of
the lecture, they involve the use,

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or are likely to involve the use of
human embryos.

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And that's a real fire point.
But let's step back and let's talk

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about stem cells and biologically
what are these things and what do

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you need them for?
And the thing we have to consider

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is the question of organ maintenance.
Now, I've told you several times,

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oh, you see, it popped up.  There
you go.  We have to give these frogs

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a moment to pop up.
OK.  That must have been somebody's

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frog that popped up.
I've told you several times that

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our bodies consist of about ten to
the twelfth cells.

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That's a lot of cells.
But it's actually much worse than

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that because it is not the same ten
to the twelfth cells that you

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started with, once you got there,
that you keep throughout your life.

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The cells in your body are
constantly dying and are constantly

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being replenished.
And, in fact, if you look at your

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ten to the twelfth cells,
over the course of your lifetime you

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will make many times ten to the
twelfth cells.

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You're not going to replace every
cell in your body because some of

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them life forever,
but for many populations of cells in

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your body you will replace those
organs completely.

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OK?  So we're talking about making
lots and lots of cells.

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And the issue really is one of
organ and tissue maintenance.

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And if one considers how organs and

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tissues are maintained,
they fall into three categories

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loosely.  Those that have a high
turnover rate where cells do not

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live for very long and need to be
replenished rather frequently to

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maintain the size and function
of the organ.

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Those organs that have a low
turnover rate and those that are

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said to be static.
Organs with a high turnover rate

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include, as I'll tell you a moment,
the blood, some of your hair.

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Organs with a low turnover rate
include things like your liver and

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pancreases.  And organs that were
thought to be static,

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but in fact that may not be true,
was the nervous system.  And now we

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know that that's not true.
So let's talk about how we get to

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these terms.  High turnover.
Low turnover.  Let me just write it

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out.  Turnover.
And static natures of organ

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maintenance.  And the way we do this,
or the way this has been done is by

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an assay that's called
a pulse-chase assay.

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And a pulse-chase assay,
and this is the second slide on your

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handout, goes like this.
In a cell population one can add a

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nucleotide analog bromodeoxyuridine.
It's uridine but it's incorporated

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into DNA because it's got that deoxy
there.  And bromodeoxyuridine is a

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nucleotide that is incorporated and
can then be detected using

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various stains.
So you can tell which cells have

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incorporated BrdU by staining them
appropriately.

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So you can tell which cells are
dividing, which cells have gone

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through S phase by whether they are
labeled or not.

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And if you give a cell population a
short pulse, if you give BrdU for

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just a short time,
this is called a pulse,

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it gets incorporated into the DNA,
and then the BrdU is gone.  And so

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you label your cell population.
And then you follow the cells over

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a long time without
any added label.

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And so the only cells you look at
are the ones that were labeled

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during this pulse period.
And if you do that, you see that

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your initially labeled cell
population lives for a while,

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and then the cells start to
disappear because they die.

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And you can monitor the half-life a
population by counting how long

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cells live for.
OK?  So this is a pulse-chase

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analysis.  And this will give you
the cell turnover time of

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a particular organ.
And this is very useful in trying to

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figure out whether cells have a high
turnover rate or a low turnover rate.

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Now, you can do something else with
this.  You can look at your labeled

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cell population,
and you can actually look not just

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at the numbers of cells but you can
look at what those cells become.

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And sometimes your initially
labeled cell population will be the

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final form, the differentiated state
of the cells, but sometimes it won't.

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And then it's very informative to
follow that labeled cell population

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and see what they do.
This is your third handout,

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your third slide on your handout.
And if you follow them you might see

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that this labeled cell population,
during the chase, differentiates

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into something.
And that's very interesting.

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And then later on those cells will
die and you'll be able to measure a

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half-life as previously,
but this is very interesting because

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it tells you that this labeled cell
population was not the same as the

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final fate of the cells.
And it implies that there is some

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kind of precursor cell,
some kind of progenitor in your

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labeled cell population that's
giving rise to the differentiated

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cells.  And it implies that these
initially labeled cells either are

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stem cells or are progenitors
derived from stem cells that are

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responsible for repopulating a
tissue as the tissue or organ needs

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to be maintained.
So these are two assays that are

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very useful and are used to,
have been used to define high

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turnover organs.
So what are some of these organs

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that have, or tissues that
have high turnover?

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In fact, they've been used,

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these assays have been used to
define many tissues,

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those with high and those with low
turnover.  But it's the high

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turnover ones I want to talk about.
The red blood cells in your body

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have a turnover time,
half-time of about 120 days.

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And if you do the calculation,
because you have a lot of red blood

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cells, you're making more than a
billion cells a day.

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OK?  As you are sitting here,
you are probably, during the course

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of this lecture,
making several million new red blood

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cells.  Your intestine,
some of the cells in your intestine

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that are responsible for food
absorption turnover every

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three to five days.
Your skin cells,

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the epidermis of your skin turns
over with a half-life of about 14

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days.  And your hair,
depending on where it comes from,

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turns over with a half-life of about
14 days to about four years.

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And the sense in many of these
organs, and I'll go through this a

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bit more in a moment,
is that this high turnover and the

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replenishment of these cells as they
disappear is driven by a stem cell

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population.  And I'm going to
abbreviate stem cells SC.

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And I'm going to put forth the
hypothesis that stem cells replenish

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these organs.
Now, in some cases you can actually

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look at the organ and you can do
your pulse-chase,

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just like I told you,
and you can see that this must be so.

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And an example that we've talked
about previously,

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not in this context but previously
is the testes,

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the spermatagonia are the cells
sitting right at the edge of the

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seminiferous tubule,
which we'd label with BrdU.

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And as you do your chase you would
see those cells as labeled cells

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moving towards the lumen and
differentiating into the

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stem cells.  OK?
And so you can actually see those

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cells giving rise to the
differentiated cells.

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Another tissue where this is very
beautiful is in the epidermis of the

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skin.  So the epidermis of the skin
comprises multiple layers stacked up

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on top of each other.
And as you get towards the outside

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of the epidermis,
the cells in the outer layers are

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dead and they form the layer that we
feel as skin.  But,

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in fact, there are a lot of cells
that are living underneath

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the outer layer.
In the very deepest layers of the

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epidermis reside a dividing
population of cells that are,

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that include the stem cells, and
I'll show you an assay for some of

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this in a little bit.
These dividing cells,

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if you follow them, give rise to the
cells in the layers above.

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So they either are the stem cells
or they are progenitor cells that

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are replenishing the epidermis.
Now, in the blood, I told you that

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you can do a pulse-chase assay.
And you can very accurately measure

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00:15:31,000 --> 00:15:35,000
the half-life of red blood cells.
And it's clear that red blood cells

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must come from some precursor.
In ourselves, in most vertebrates,

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in many vertebrates red blood cells
do not have nuclei so they cannot

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divide themselves.
And so there must be some kind of

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progenitor.  But the progenitors of
red blood cells and,

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in fact, many other cells in the
blood lie deep within the bone

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marrow, the cavity of the bones.
And so you really cannot observe

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them well to come to the conclusions
that I've just given you about the

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testes and the epidermis
of the skin.

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So you have to do some kind of other
assay to figure out where these

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cells are coming from.
And over the years a huge body of

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data has given rise to this notion.
In the bone marrow of the long

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bones, and some of the shorter bones,
too, there is some kind of

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pluripotential or pluripotent
hematopoietic stem cell,

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that I've abbreviated HSC.
And this HSC gives rise,

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through a series of progenitors
which then go on to differentiate,

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into all of the cell types that are
found in the blood.

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The red blood cells.
The so-called white blood cells.

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The immune cells.  A huge spectrum
of differentiated cell types.

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And this hematopoietic stem cell has
been very, very useful in helping

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people who have got blood associated
illnesses in that it can,

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after removal of diseased blood
cells, it can repopulate the tissue

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and help people get better.
And it's done so, and I'll go

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through how this is done both in an
assay sense and point out that this

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is exactly what's done in humans,
it's done so in a bone marrow

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transplant.  So in assays,
let's just write down a couple of

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things here.

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I've told you one assay already
for stem cells.

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OK?  So assays for stem cells ask

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how do you know if you've got a stem
cell?  Well, you can do that.

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You can ask this using the
pulse-chase by direct observation

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with your pulse-chase.
You can also do it by a repopulation

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assay.  And the notion in a
repopulation assay is that you try

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to restore a particular cell type by
putting in what you think is the

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precursor of that cell type.
Now, in order to do these assays

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you usually have to get rid of the
normal cells that were there so that

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you can assay your repopulation.
OK?  Otherwise,

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the normal cells can out-compete the
cells you're putting in.

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OK?  But that's an aside.
So what is a bone marrow transplant?

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The notion here is that one takes
an animal, and this is done in

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humans, and you irradiate the animal
so that you destroy all its bone

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marrow.  This will eventually kill
the animal, OK,

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because you cannot live without a
hematopoietic stem cell and without

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all the cells that
derive from that.

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And so, although this irradiated
animal would die,

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you can rescue it by injecting
normal bone marrow.

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You inject it into a mouse in the
tail vein, into people it goes into

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a central line that goes into your
blood system.  And then those bone

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marrow cells, it's extraordinary,
know where to go.  They home to the

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bone marrow and they repopulate it
and they make again the

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hematopoietic stem cell and all the
lineages that come from that.

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Or they are the hematopoietic stem
cell, they maintain themselves and

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they give rise to all the other
lineages.  One of the criteria that

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I told you about stem cells is that
they need to be self-renewing.

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You can ask whether or not these
stem cells are self-renewing in an

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assay like this,
where you take a mouse that you've

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rescued by bone marrow transplant,
and you can ask whether it can serve

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as a source of marrow of
hematopoietic stem cells that will

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rescue another irradiated mouse.
In other words,

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you could have rescued that first
mouse just by giving it a whole

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spectrum of almost differentiated
red blood cells,

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white blood cells,
immune cells and so on.

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OK?  In order to test whether you
actually rescued it by giving it a

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stem cell, you can ask whether or
not you can take this rescued mouse

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and use it to rescue another mouse.
And, in fact, you can.

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And this is consistent with,
not unequivocal, but it's consistent

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with a sense that you have generated
more stem cells in the rescued mouse

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that can go on to rescue another
mouse.  Another set of experiments

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that has been really pivotal in
understanding stem cells is to try

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to purify the stem cells and to
assay what are these pure stem cells.

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And part of this is asking how many
cells do you need to rescue in a

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repopulation assay?

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00:21:06,000 --> 00:21:10,000
OK.  And so let me
make a note here.

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That when it comes to stem cell

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isolation the overriding conclusion
from many assays done by many labs

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is that stem cells are rare and they
are hard to find.

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00:21:32,000 --> 00:21:36,000
In this experiment,
which is number five on your handout,

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00:21:36,000 --> 00:21:40,000
you can assay for how many stem
cells you need to rescue a mouse by

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removing bone marrow from one mouse
and staining that bone marrow,

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if you can, for a stem cell marker.
Now, what is a stem cell marker?

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OK?  If I have a stem cell marker I
would be able to go and pull out the

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stem cells.  Well,
there are no markers.

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00:21:57,000 --> 00:22:01,000
A marker could be a protein or an
RNA that is expressed in a

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particular group of cells that you
suspect are the stem cells.

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OK?  This is very much an
experimental-driven field.

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00:22:10,000 --> 00:22:14,000
There are markers that always
appear, proteins that are always

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00:22:14,000 --> 00:22:18,000
there, antigens on the surface
stained with antibodies that are

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always there where you have a
population of cells that is able to

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00:22:22,000 --> 00:22:26,000
rescue a mouse.
And so you can use these to

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hypothesize that this stem cell
marker is, in fact, marking

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00:22:30,000 --> 00:22:35,000
the stem cells.
And you can go and purify cells that

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are expressing a particular marker.
And the way you do this is by using

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00:22:39,000 --> 00:22:43,000
a really fantastic machine called
the Fluorescence Activated Cell

294
00:22:43,000 --> 00:22:47,000
Sorter (FACS).
I'll go through it very briefly in

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00:22:47,000 --> 00:22:51,000
a moment.  But what it is able to do
is to purify cells that have got

296
00:22:51,000 --> 00:22:55,000
particular fluorescences.
And they have these particular

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00:22:55,000 --> 00:22:59,000
fluorescence spectra because of the
way you have stained them for

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00:22:59,000 --> 00:23:03,000
particular stem cell markers.
And what you get out of your FACS

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00:23:03,000 --> 00:23:07,000
machine is a population that is much
more enriched for what I've called

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00:23:07,000 --> 00:23:11,000
stem cells, but they're actually
cells that are expressing this

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00:23:11,000 --> 00:23:15,000
punitive stem cell marker.
And then you can take these cells

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00:23:15,000 --> 00:23:19,000
and say, well,
have I really enriched for the stem

303
00:23:19,000 --> 00:23:23,000
cell, by asking how many cells you
need to introduce into an irradiated

304
00:23:23,000 --> 00:23:27,000
mouse to rescue that mouse.
And you can do a dilution assay

305
00:23:27,000 --> 00:23:31,000
where you put in one cell,
ten cells, a hundred cells.

306
00:23:31,000 --> 00:23:35,000
Obviously, when you're down in the
one cell range,

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00:23:35,000 --> 00:23:39,000
you do your dilution assay,
you get into a Poisson distribution,

308
00:23:39,000 --> 00:23:43,000
and you may not have exactly one
cell.  But you can do this

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00:23:43,000 --> 00:23:47,000
statistically and you can figure out
how many cells from your population

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00:23:47,000 --> 00:23:51,000
it takes to rescue a mouse.
OK?  And the data to date tells us

311
00:23:51,000 --> 00:23:55,000
that one cell of the correctly
purified population is sufficient to

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00:23:55,000 --> 00:23:59,000
rescue an irradiated mouse.
You have to do some tricks.

313
00:23:59,000 --> 00:24:02,000
You have to add some extra cells to
this one cell.

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00:24:02,000 --> 00:24:06,000
You cannot just put one cell into
the mouse and rescue.

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00:24:06,000 --> 00:24:09,000
You have to mix this one cell with
some other cells that help it do its

316
00:24:09,000 --> 00:24:13,000
work.  OK?  But this is really an
extraordinary assay that tells us

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00:24:13,000 --> 00:24:17,000
we're very close to purifying a
hematopoietic stem cell.

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00:24:17,000 --> 00:24:20,000
What is a FACS machine?  Very
briefly.  You can look at this later.

319
00:24:20,000 --> 00:24:24,000
Cells that are labeled with
fluorescent antibodies are broken up

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00:24:24,000 --> 00:24:28,000
into a stream of tiny droplets each
of which will contain one cell.

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00:24:28,000 --> 00:24:32,000
And the cells then pass in a stream
through a laser which activates the

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00:24:32,000 --> 00:24:37,000
fluorescence.  There's a
fluorescence detector that you can

323
00:24:37,000 --> 00:24:41,000
program to respond to a particular
wavelength.  And what happens is

324
00:24:41,000 --> 00:24:46,000
that when the detector finds that it
has responded to a cell passing

325
00:24:46,000 --> 00:24:50,000
through it because of the
fluorescence emitted,

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00:24:50,000 --> 00:24:55,000
a charge is given to that cell,
and that cell with a particular

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00:24:55,000 --> 00:25:00,000
fluorescence moves down the stream
through some deflecting plates where

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00:25:00,000 --> 00:25:04,000
it will be deflected,
according to its charge,

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00:25:04,000 --> 00:25:08,000
into a collecting bin.
And that way you can really get some

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00:25:08,000 --> 00:25:12,000
very highly purified cell
populations.  You can purify about

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00:25:12,000 --> 00:25:15,000
300,000 cells an hour using the FACS
machine.  And we have a lot of them

332
00:25:15,000 --> 00:25:19,000
here at MIT and use them.
Many laboratories use them for

333
00:25:19,000 --> 00:25:22,000
various things.
A really cool machine.

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00:25:22,000 --> 00:25:26,000
OK.  But we need to move on.
So we talked about some assays for

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00:25:26,000 --> 00:25:30,000
stem cells.
Talked about the potency of

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00:25:30,000 --> 00:25:34,000
hematopoietic stem cells.
I want to tell you something now

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00:25:34,000 --> 00:25:39,000
about the control of stem cells and
how a stem cell decides what to do.

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00:25:39,000 --> 00:25:43,000
So if you think about organ
maintenance, there is an implicit

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00:25:43,000 --> 00:25:48,000
understanding that the organ knows
how much it needs to be maintained.

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00:25:48,000 --> 00:25:53,000
It needs to know how many cells are
being lost, it needs to know when to

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00:25:53,000 --> 00:25:57,000
make more cells,
and it also needs to know if there

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00:25:57,000 --> 00:26:02,000
has been some catastrophe.
If there's been a liver,

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00:26:02,000 --> 00:26:07,000
some kind of a resection of the
liver, or if there has been skin

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00:26:07,000 --> 00:26:12,000
wounding, OK, the organ needs to be
able to respond.

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00:26:12,000 --> 00:26:16,000
And the stem cells in an organ need
to be able to respond to this

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00:26:16,000 --> 00:26:21,000
catastrophe.  And this is where this
term niche comes in.

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00:26:21,000 --> 00:26:26,000
Niche is one of these fields in the
term, terms in the field.

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00:26:26,000 --> 00:26:31,000
It's like the term progenitor.
And niche is nothing that you

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00:26:31,000 --> 00:26:37,000
haven't heard about before.
What it refers to are the cells,

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00:26:37,000 --> 00:26:42,000
as written up there, that surround
the stem cell.

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00:26:42,000 --> 00:26:48,000
And what I'm going to tell you is
that the niche cells,

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00:26:48,000 --> 00:26:53,000
the surrounding cells control,
by induction, stem cell fate.  And

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00:26:53,000 --> 00:26:59,000
I'm also going to point out that
there can be some input

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00:26:59,000 --> 00:27:04,000
of the environment.
So this is a cartoon that I drew for

355
00:27:04,000 --> 00:27:08,000
you.  It's number six on your
handout.  It's six across on your

356
00:27:08,000 --> 00:27:13,000
handout.  And I have here some stem
cells that I've called quiescent.

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00:27:13,000 --> 00:27:17,000
They are not dividing.  Or they may
be dividing just a little bit just

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00:27:17,000 --> 00:27:21,000
to maintain themselves but not to
give rise to any progenitors that

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00:27:21,000 --> 00:27:26,000
will go on to differentiate.
And these quiescent cells are

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00:27:26,000 --> 00:27:30,000
maintained in this quiescent state,
we believe, because they are

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00:27:30,000 --> 00:27:35,000
surrounded by a group of cells that
is maintaining them quiescent.

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00:27:35,000 --> 00:27:40,000
What happens then is that these
quiescent cells sometimes get a

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00:27:40,000 --> 00:27:45,000
stimulus, some kind of environmental
input, be it wounding,

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00:27:45,000 --> 00:27:50,000
be it some kind of hormonal input
from another part of the body that

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00:27:50,000 --> 00:27:55,000
influences the surrounding cells and
changes the surrounding cells such

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00:27:55,000 --> 00:28:00,000
that they now change their patterns
of gene expression and start

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00:28:00,000 --> 00:28:05,000
secreting stuff,
most likely, that changes the

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00:28:05,000 --> 00:28:11,000
activity of the stem cells that they
are surrounding.

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00:28:11,000 --> 00:28:15,000
I've shown you arrows there
indicating induction from the stem

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00:28:15,000 --> 00:28:19,000
cells, from the surrounding cells to
the stem cells.

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00:28:19,000 --> 00:28:23,000
And what this inductive process
does, just like in the early embryo

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is to activate the stem cells to
divide and to form progenitor cells

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00:28:27,000 --> 00:28:31,000
which will go on to do their thing
and differentiate into

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00:28:31,000 --> 00:28:35,000
various fates.
And also, of course,

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00:28:35,000 --> 00:28:39,000
the stem cells are renewed.
So this notion of the surrounding

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00:28:39,000 --> 00:28:43,000
cells telling the stem cells what to
do is kind of new in the stem cell

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00:28:43,000 --> 00:28:48,000
field.  If one is a developmental
biologist, it's one of these,

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00:28:48,000 --> 00:28:52,000
OK, kind of observations because we
know that cells signal to one

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00:28:52,000 --> 00:28:56,000
another.  And it's not surprising
that stem cells are told what to do

380
00:28:56,000 --> 00:29:01,000
by the cell surrounding them.
But there has been not very much

381
00:29:01,000 --> 00:29:05,000
information on this.
And I want to give you one system

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00:29:05,000 --> 00:29:09,000
where it's been very  beautifully
looked at and where the contribution

383
00:29:09,000 --> 00:29:13,000
of the surrounding cells has been
looked at.  And this system is the

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00:29:13,000 --> 00:29:17,000
hair follicle.
So your hairs come from a sheath of

385
00:29:17,000 --> 00:29:21,000
cells that starts off in the
epidermis and moves deep down,

386
00:29:21,000 --> 00:29:25,000
extends deep down into the deeper
layers of the skin called

387
00:29:25,000 --> 00:29:30,000
the dermis.
And there is a shaft of cells of

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00:29:30,000 --> 00:29:35,000
which these matrix cells down here
are important because these matrix

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00:29:35,000 --> 00:29:40,000
cells divide and they give rise to
cells that will form the center of

390
00:29:40,000 --> 00:29:44,000
the shaft and secrete,
or not secrete but synthesis

391
00:29:44,000 --> 00:29:49,000
proteins called keratins.
The keratins are this stuff.

392
00:29:49,000 --> 00:29:54,000
And eventually the cell gets so
full of keratin that it dies.

393
00:29:54,000 --> 00:29:59,000
And at the same time more cells are
coming in the bottom here and this

394
00:29:59,000 --> 00:30:04,000
whole shaft, this whole hair shaft
is pushed out of the hair follicle.

395
00:30:04,000 --> 00:30:08,000
OK?  And so that is why your hair
grows.  It has been pushed out from

396
00:30:08,000 --> 00:30:13,000
the bottom.  And all the stuff that
we are so fond of is dead cells.

397
00:30:13,000 --> 00:30:18,000
It's actually protein.  It's a very
interesting polymer of various

398
00:30:18,000 --> 00:30:22,000
keratins.  Now,
what's very interesting here is that

399
00:30:22,000 --> 00:30:27,000
over the years researchers have
shown that the hair,

400
00:30:27,000 --> 00:30:31,000
well, let me back track for a moment.
You know your hair falls out,

401
00:30:31,000 --> 00:30:34,000
right?  You brush your hair and your
hair falls out.

402
00:30:34,000 --> 00:30:38,000
OK?  Ergo, it must be replenished
somehow.  And over the years it has

403
00:30:38,000 --> 00:30:41,000
been shown that these bulge cells,
or cells in a little bulge, which

404
00:30:41,000 --> 00:30:44,000
was a rather insignificant little
bulge but it's there,

405
00:30:44,000 --> 00:30:48,000
sitting on the side of the hair
shaft is actually the source of the

406
00:30:48,000 --> 00:30:51,000
stem cells for the hair.
And another region, I'm going to

407
00:30:51,000 --> 00:30:54,000
tell you about in a moment,
is down here.  It's labeled DP.

408
00:30:54,000 --> 00:30:58,000
Bear it in mind as I tell you
something and then we'll

409
00:30:58,000 --> 00:31:02,000
come back to it.
So let me first show you some data

410
00:31:02,000 --> 00:31:06,000
that tells us that the bulge cells
are the cells that contain or that

411
00:31:06,000 --> 00:31:11,000
include the stem cells for making
hair.  One can do an experiment

412
00:31:11,000 --> 00:31:16,000
where one takes these bulge cells
and does essentially a repopulation

413
00:31:16,000 --> 00:31:20,000
assay, just as in the case of the
bone marrow transplant.

414
00:31:20,000 --> 00:31:25,000
And one can do this into a strain
of mice called nude.

415
00:31:25,000 --> 00:31:30,000
So nude mice have got multiple
issues.

416
00:31:30,000 --> 00:31:33,000
But one of their issues is that they
have no fur.  OK?

417
00:31:33,000 --> 00:31:37,000
And one can take bulge cells from a
normal mouse and transplant them

418
00:31:37,000 --> 00:31:40,000
into a nude mouse.
Here's the control transplant,

419
00:31:40,000 --> 00:31:44,000
and you can see there's no fur.  And
here is a transplant of bulge cells

420
00:31:44,000 --> 00:31:47,000
from a normal mouse.
And here you see this tuft of fur

421
00:31:47,000 --> 00:31:51,000
growing out on the back of the nude
mouse.  And, in fact,

422
00:31:51,000 --> 00:31:54,000
some very cool assays have been done
where these transplanted cells are

423
00:31:54,000 --> 00:31:58,000
labeled green.
And you can look at these tufts of

424
00:31:58,000 --> 00:32:02,000
fur and see them growing
fluorescently green.

425
00:32:02,000 --> 00:32:06,000
So you know that the new fur came
from the transplanted cells.

426
00:32:06,000 --> 00:32:10,000
OK.  So what about niche?  Well,
you know that your hair falls out.

427
00:32:10,000 --> 00:32:15,000
But, actually, there's a lot more
to it than that.

428
00:32:15,000 --> 00:32:19,000
And it turns out there is a whole
hair cycle of when the hair grows

429
00:32:19,000 --> 00:32:24,000
and when it doesn't grow.
And it goes like this.  There's a

430
00:32:24,000 --> 00:32:28,000
time called anagen which is a period
of growth when these matrix cells

431
00:32:28,000 --> 00:32:32,000
down here are dividing and they are
pushing out this hair

432
00:32:32,000 --> 00:32:37,000
and it's growing.
After a while the cells down here

433
00:32:37,000 --> 00:32:41,000
stop dividing and the hair follicle
doesn't grow anymore,

434
00:32:41,000 --> 00:32:45,000
or the hair shaft doesn't grow
anymore.  This is called catagen.

435
00:32:45,000 --> 00:32:49,000
And it's a time that's termed
regression.  It is when there is no

436
00:32:49,000 --> 00:32:53,000
more growth.  And if the hair is
going to fall out,

437
00:32:53,000 --> 00:32:57,000
it might fall out then,
or it might stick around and the

438
00:32:57,000 --> 00:33:02,000
next step might take place.
And this is a step called telogen

439
00:33:02,000 --> 00:33:06,000
which is a resting state.
And then after that there is a new

440
00:33:06,000 --> 00:33:10,000
state, a new anagen where the hair
will start growing again.

441
00:33:10,000 --> 00:33:14,000
And it may either be a new hair or
it may be the one that's there that

442
00:33:14,000 --> 00:33:19,000
just grows longer.
It depends on the part of the body.

443
00:33:19,000 --> 00:33:23,000
OK.  Now, what I'm going to tell
you is that a region called the

444
00:33:23,000 --> 00:33:27,000
dermal papilla induces stem cell
activation at certain times during

445
00:33:27,000 --> 00:33:31,000
the hair cycle.
So look at this.

446
00:33:31,000 --> 00:33:35,000
At anagen, during growth,
the bulge and this dermal papilla,

447
00:33:35,000 --> 00:33:39,000
OK, this DP, it doesn't matter what
the name is but just look at the red,

448
00:33:39,000 --> 00:33:42,000
are far apart from each other.
At catagen they're still far apart,

449
00:33:42,000 --> 00:33:46,000
but look at what happens at telogen
in this resting stage.

450
00:33:46,000 --> 00:33:50,000
The dermal papilla and the bulge
are touching one another.

451
00:33:50,000 --> 00:33:53,000
And this is the phase, although
it's called a resting phase,

452
00:33:53,000 --> 00:33:57,000
this is the phase when the hair
follicle is actually getting ready

453
00:33:57,000 --> 00:34:01,000
for the next big spurt of cell
division and differentiation that is

454
00:34:01,000 --> 00:34:05,000
going to make the hair longer or
make a new one grow.

455
00:34:05,000 --> 00:34:08,000
And then, subsequently,
the dermal papilla and the bulge

456
00:34:08,000 --> 00:34:11,000
stay close together,
but eventually they will start

457
00:34:11,000 --> 00:34:15,000
pulling apart again.
Well, it's been shown very

458
00:34:15,000 --> 00:34:18,000
elegantly that this dermal papilla
signals to these bulge cells when

459
00:34:18,000 --> 00:34:22,000
they're touching one another and
tells the bulge stem cells to

460
00:34:22,000 --> 00:34:25,000
activate and start making more hair
or a new hair follicle.

461
00:34:25,000 --> 00:34:29,000
And the signaling is done through a
pathway called the wnt

462
00:34:29,000 --> 00:34:33,000
signaling pathway.
And I point this out because it uses

463
00:34:33,000 --> 00:34:37,000
a protein that should be familiar to
you, which is our old friend

464
00:34:37,000 --> 00:34:41,000
beta-catenin that we talked about
way back when as being important for

465
00:34:41,000 --> 00:34:45,000
dorsal-ventral patenting in the
early embryo.  And I point it out to

466
00:34:45,000 --> 00:34:49,000
you to make the point that proteins
are used over and over again.

467
00:34:49,000 --> 00:34:53,000
Regulatory proteins can be used
over and over again in the body.

468
00:34:53,000 --> 00:34:57,000
OK.  So this is a very lovely
example of what happens

469
00:34:57,000 --> 00:35:02,000
in a stem cell niche.
So let's move on and let's ask how

470
00:35:02,000 --> 00:35:08,000
plastic adult stem cells are.
OK.  So here we have some issues.

471
00:35:08,000 --> 00:35:13,000
Well, the goal of the medical
community is to get a stem cell that

472
00:35:13,000 --> 00:35:19,000
can repair every tissue.
And it may be that every adult

473
00:35:19,000 --> 00:35:24,000
tissue has a stem cell population in
it, but these can be difficult to

474
00:35:24,000 --> 00:35:30,000
isolate.  It's very difficult to
isolate these hair stem cells.

475
00:35:30,000 --> 00:35:35,000
You can get enough to get tufts of
hair on another mouse,

476
00:35:35,000 --> 00:35:41,000
but it's hard to get enough of them
to repair someone's bald head for

477
00:35:41,000 --> 00:35:47,000
example.  OK?  So the Holy Grail of
stem cell-ness is to try to find a

478
00:35:47,000 --> 00:35:53,000
cell that is a multi-potent adult
progenitor.  OK?

479
00:35:53,000 --> 00:35:59,000
And this has been the real goal of
the adult stem cell field.

480
00:35:59,000 --> 00:36:07,000
And the question that has been posed
is, is there a multi-potent,

481
00:36:07,000 --> 00:36:15,000
a highly plastic, multi-potent also
termed plastic,

482
00:36:15,000 --> 00:36:23,000
adult stem cell?
And if there was then in theory one

483
00:36:23,000 --> 00:36:29,000
could use it to repair all organs.
So here again is our stem cell

484
00:36:29,000 --> 00:36:33,000
concept where some kind of stem cell
goes on to give rise to a set of

485
00:36:33,000 --> 00:36:37,000
differentiated cells.
Now, the set of differentiated

486
00:36:37,000 --> 00:36:41,000
cells forms what's called a lineage
where all the cell types that come

487
00:36:41,000 --> 00:36:45,000
out of a single stem cell are
related to one another because they

488
00:36:45,000 --> 00:36:49,000
come from a common heritage,
from a common progenitor, from a

489
00:36:49,000 --> 00:36:53,000
common stem cell.
And you can, for different lineages,

490
00:36:53,000 --> 00:36:57,000
make different trees of stem cells
progenitors and differentiated

491
00:36:57,000 --> 00:37:02,000
progeny.
And it has been hypothesized that

492
00:37:02,000 --> 00:37:06,000
there is some kind of
interconvertibility between

493
00:37:06,000 --> 00:37:11,000
different stem cells in the adult,
and that one stem cell for one

494
00:37:11,000 --> 00:37:15,000
lineage can be under the right
circumstances converted to a stem

495
00:37:15,000 --> 00:37:20,000
cell for a different lineage.
And that maybe there's even some

496
00:37:20,000 --> 00:37:24,000
kind of master stem cell floating
around in the adult that can give

497
00:37:24,000 --> 00:37:29,000
rise to all other stem cells.
Maybe there is some totipotent or

498
00:37:29,000 --> 00:37:33,000
pluripotent very,
very highly potent adult stem cell

499
00:37:33,000 --> 00:37:38,000
that can give rise to all these
other stem cell lineages and can,

500
00:37:38,000 --> 00:37:43,000
therefore, be used as this universal
repair kit.  And this has been

501
00:37:43,000 --> 00:37:47,000
assayed.  And the prediction from
this is that adult stem cells from

502
00:37:47,000 --> 00:37:52,000
one lineage, for example the
hematopoietic lineage,

503
00:37:52,000 --> 00:37:57,000
can contribute to another lineage.
For example, the muscle cell

504
00:37:57,000 --> 00:38:02,000
lineage.  And this prediction has
been tested in the following way.

505
00:38:02,000 --> 00:38:06,000
It's particularly been tested with
hematopoietic stem cells.

506
00:38:06,000 --> 00:38:10,000
And the question has been posed can
these hematopoietic stem cells

507
00:38:10,000 --> 00:38:14,000
contribute to other lineages?
Here's a mouse expressing green

508
00:38:14,000 --> 00:38:19,000
hematopoietic stem cells,
or making green hematopoietic stem

509
00:38:19,000 --> 00:38:23,000
cells because of expression of the
protein GFP under an appropriate

510
00:38:23,000 --> 00:38:27,000
tissue-specific promoter.
You can isolate bone marrow cells

511
00:38:27,000 --> 00:38:32,000
from the GFP expressing mouse.
Transplant them into an irradiated

512
00:38:32,000 --> 00:38:36,000
mouse.  You get rescue,
as we've discussed, but that's not

513
00:38:36,000 --> 00:38:40,000
the point of the experiment.
The point of the experiment,

514
00:38:40,000 --> 00:38:44,000
and this is the second to last slide
on your handout.

515
00:38:44,000 --> 00:38:49,000
The point of the experiment is to
ask not about the bone marrow cells

516
00:38:49,000 --> 00:38:53,000
in the rescued mouse,
but to ask whether any of the other

517
00:38:53,000 --> 00:38:57,000
tissues in the mouse contain these
green cells that would have arisen

518
00:38:57,000 --> 00:39:02,000
from hematopoietic stem cells.
OK.  So we're asking whether these

519
00:39:02,000 --> 00:39:06,000
hematopoietic stem cells can
contribute to lineages other than

520
00:39:06,000 --> 00:39:10,000
the blood and the immune system.
And this has been an enormous focus

521
00:39:10,000 --> 00:39:15,000
of research.  And the answer
initially appeared to be yes.

522
00:39:15,000 --> 00:39:19,000
And this was a cause of tremendous
excitement.  Here's one piece of

523
00:39:19,000 --> 00:39:23,000
data.  This is a picture of some
cells from a mouse that was rescued

524
00:39:23,000 --> 00:39:28,000
with green hematopoietic
stem cells.

525
00:39:28,000 --> 00:39:32,000
And this is a picture of the muscle
from the mice.

526
00:39:32,000 --> 00:39:36,000
The blue are nuclei and the green
is the GFP that would have come from

527
00:39:36,000 --> 00:39:40,000
these transplanted hematopoietic
stem cells.  And you can see green

528
00:39:40,000 --> 00:39:45,000
cells also in the nervous system,
nerve cells that appear to come from

529
00:39:45,000 --> 00:39:49,000
these hematopoietic stem cells.
Well, it turns out alas that this

530
00:39:49,000 --> 00:39:53,000
is an artifact of the experiment.
And, in fact, what happens is that

531
00:39:53,000 --> 00:39:57,000
these green hematopoietic stem cells
rescue the mouse in

532
00:39:57,000 --> 00:40:02,000
the correct way.
But then they also fuse together

533
00:40:02,000 --> 00:40:06,000
with other cells.
They fuse with muscle cells and

534
00:40:06,000 --> 00:40:11,000
with nerve cells.
The two cells actually join

535
00:40:11,000 --> 00:40:16,000
together.  Their cell membranes
mingle and you get a kind of hybrid

536
00:40:16,000 --> 00:40:20,000
cell that is a mixture of the
hematopoietic cell and the regular

537
00:40:20,000 --> 00:40:25,000
muscle cell.  And this whole notion
of the adult hematopoietic stem cell

538
00:40:25,000 --> 00:40:30,000
contributing to muscle,
to nerve, to various other lineages

539
00:40:30,000 --> 00:40:34,000
has completely fallen to disfavor in
most fields, in most

540
00:40:34,000 --> 00:40:38,000
people's opinion.
Although, there is still data,

541
00:40:38,000 --> 00:40:42,000
there is still much experimentation
to do to really address this,

542
00:40:42,000 --> 00:40:46,000
both in the hematopoietic stem cell
and other lineages.

543
00:40:46,000 --> 00:40:49,000
But no data currently supports that
adult stem cells can interconvert

544
00:40:49,000 --> 00:40:53,000
one to another.
And that leads us to the difficult

545
00:40:53,000 --> 00:40:57,000
position of asking,
so where is this universal repair

546
00:40:57,000 --> 00:41:01,000
kit going to come from?
And this is where we move into the

547
00:41:01,000 --> 00:41:06,000
field of, I'm going to just leave
that now, of embryonic stem cells.

548
00:41:06,000 --> 00:41:12,000
So you know now that embryos have
got many cells that are multipotent,

549
00:41:12,000 --> 00:41:17,000
pluripotent.  And indeed the zygote,
as we discussed,

550
00:41:17,000 --> 00:41:22,000
is totipotent.  These cells can give
rise to many, many different tissues

551
00:41:22,000 --> 00:41:28,000
because during embryogenesis that's
the deal.  That's what

552
00:41:28,000 --> 00:41:33,000
has to happen.
And so it came to the attention of

553
00:41:33,000 --> 00:41:37,000
people long ago that embryonic stem
cells might be a great source of

554
00:41:37,000 --> 00:41:41,000
stem cells that can be used as this
universal repair kit.

555
00:41:41,000 --> 00:41:45,000
Now, I want to tell you,
I'm not going to go through this,

556
00:41:45,000 --> 00:41:49,000
you can go and look on the
PowerPoint, you can go and look on

557
00:41:49,000 --> 00:41:53,000
the PDF file on your website.
I'm going to tell you about a type

558
00:41:53,000 --> 00:41:58,000
of cell, in the last few minutes,
called the embryonic stem cell.

559
00:41:58,000 --> 00:42:07,000
And what you really need to know

560
00:42:07,000 --> 00:42:11,000
about this is it's almost totipotent.
And the first slides,

561
00:42:11,000 --> 00:42:16,000
I'm not going to go through,
show you that this embryonic stem

562
00:42:16,000 --> 00:42:20,000
cell type is almost totipotent.
Now, what about these embryonic

563
00:42:20,000 --> 00:42:25,000
stem cells?  They come from,
in taking a very early embryo,

564
00:42:25,000 --> 00:42:30,000
at a stage called the inner cell
mass stage --

565
00:42:30,000 --> 00:42:35,000
-- when there is a little mass of
cells in the developing embryo,

566
00:42:35,000 --> 00:42:40,000
that's going to give rise to the
whole embryo.  And taking this cell,

567
00:42:40,000 --> 00:42:46,000
removing these inner cell mass cells
and putting them into a Petri dish

568
00:42:46,000 --> 00:42:51,000
with various culture media,
what happens is that the cells from

569
00:42:51,000 --> 00:42:57,000
this early embryo divide and they
make various clumps.

570
00:42:57,000 --> 00:42:57,000
And the clumps are descended from
one particular cell.
And each of these clumps of cells
is called an embryonic stem cell
line.  Now, what's a cell line?
A cell line is a population of

571
00:42:58,000 --> 00:42:58,000
cells that can grow continuously in
culture.  In other words,
it self-renews.  What's a stem cell
line?  A cell line,

572
00:42:59,000 --> 00:42:59,000
this is on your PDF file so you
might want to sit and listen to this
and then go and get this afterwards.
OK?  What is a stem cell line?

573
00:43:00,000 --> 00:43:09,000
A stem cell line is a cell line that
has the potential to go on and

574
00:43:09,000 --> 00:43:18,000
differentiate into various different
lineages.  OK?

575
00:43:18,000 --> 00:43:27,000
And so from this embryo that has
been put in a Petri dish and the

576
00:43:27,000 --> 00:43:36,000
cells allowed to grow you get out a
number of stem cell lines.

577
00:43:36,000 --> 00:43:45,000
And the notion is that each of
these stem cell lines,

578
00:43:45,000 --> 00:43:54,000
if treated with the correct factor
that's called here a differentiation

579
00:43:54,000 --> 00:44:03,000
factor, can go on to give rise to
different tissues.

580
00:44:03,000 --> 00:44:07,000
Heart muscle, pancreas,
cartilage and so on.  And the notion

581
00:44:07,000 --> 00:44:11,000
is that different ES cell lines will
have different potencies.

582
00:44:11,000 --> 00:44:15,000
So some will be really good at
making hearts,

583
00:44:15,000 --> 00:44:19,000
some will be really good at making
muscle and so on.

584
00:44:19,000 --> 00:44:23,000
And it's not quite clear how one
gets these different ES cell lines,

585
00:44:23,000 --> 00:44:28,000
but they exist from the mouse.  And
some of them exist from the human.

586
00:44:28,000 --> 00:44:32,000
And this is where we get into the
other major issue of stem cell

587
00:44:32,000 --> 00:44:36,000
biology and the reason that there is
so much legislature.

588
00:44:36,000 --> 00:44:40,000
Because in order to get these human
stem cell lines you have to use a

589
00:44:40,000 --> 00:44:45,000
human embryo.  To get human embryos
you use methods of assisted

590
00:44:45,000 --> 00:44:49,000
reproductive technology.
I sent you some notes on this and

591
00:44:49,000 --> 00:44:53,000
you can read this in the book.
And the notion is that you remove

592
00:44:53,000 --> 00:44:57,000
eggs from a female,
you make embryos in vitro,

593
00:44:57,000 --> 00:45:03,000
in a test-tube.
And then when the embryos are balls

594
00:45:03,000 --> 00:45:09,000
of cells you harvest them,
you kill them and you turn them into

595
00:45:09,000 --> 00:45:15,000
ES line.  And the thing that is
sticking people right now in the

596
00:45:15,000 --> 00:45:21,000
legislature and is something that is
really worth thinking about is

597
00:45:21,000 --> 00:45:27,000
whether ethically it is OK,
guys, it's a 11:54, so stay.

598
00:45:27,000 --> 00:45:32,000
Stay.  OK.
And the thing that is killing people,

599
00:45:32,000 --> 00:45:36,000
is really sticking people ethically
is whether it's OK to take these

600
00:45:36,000 --> 00:45:41,000
human embryos and turn them into
cell lines, because this does

601
00:45:41,000 --> 00:45:45,000
involve killing an embryo and this
is an ethical issue.

602
00:45:45,000 --> 00:45:50,000
And I'm going to leave this here,
and we'll come back to these ethical

603
00:45:50,000 --> 00:45:53,000
issues when we talk about cloning.