Good morning, class. Nice to see you here.
Yesterday morning I was in Australia and today I'm here.
And it's nice to be back. It's a long way home.
We're talking today about the issues of growth and differentiation
about normal cells becoming specialized into different types of
cells throughout the body and how that leads ultimately to the issues
of organismic cloning. And, to give you an overall
background about this, I want to show you the way that a
worm is put together. This is the worm C. elegans.
I don't know how well this overhead will come out.
You can see it reasonably well. So, this is the worm Caenorhabditis
elegans. It's been an object of much study here.
Bob Horowitz in our own department just got the Nobel Prize
for his work on this.
And the reason it's been the object of so much study is,
in fact, that it's a relatively simple organism.
It's the way that you and I looked about 600 million years ago when
Metazoa, remember Metazoa means multicellular organisms,
first arose on the face of the planet. Let's see if I have too
much overlap here. Well, that's about the way it should
look. Now, what's remarkable about this organism is it has something
like 965 cells in the adult. The cells all descend from a
fertilized egg. It happens to be that this worm is
a hermaphrodite. So, you probably know
hermaphrodites are both male and female and it can fertilize itself
or two worms can get together and fertilize each other.
Because, as you can see, it makes both eggs and it makes
sperm. And what's most interesting is the finite number of cells in the
body of the adult. As I said, it's 965,
whose lineage can be traced through a pedigree that's given right up
here. So, one can plot out, with great precision, how all the
cells, how the egg cell and the fertilized egg divides in two,
each of those cells divides again in two and how ultimately one has a
whole series of different descendents from different branches
of this very elaborate family tree. Now, one of the issues that we want
to pursue is the fact that the genomes, in principle,
all the cells in this organism are the same. That is to say whenever
the cell goes through a cycle of growth and division the cell is
genetically identical, yet phenotypically, yet the behavior
reach of these cells becomes increasingly different.
And here you can see the different lineage of cells.
This organism devotes a disproportionately large amount of
its anatomy to reproduction, much more even than we do. Here,
this is the cuticle, the outer coat. Here's the vulva, the female organ.
This is the male organ and a variety of other cells types.
Here's the pharynx. And yet all the cells in all these organisms,
with the exception of the gametes, remember gametes means sperm and egg
are genetically identical. The gametes have only half the
genetic content. Everybody else is identical,
has a diploid genome, that is to say two copies of each gene.
And one can trace this all out. And this represents one of the
great mysteries of developmental biology, which is to say how are
cells that genetically identical to one another genotypically identical
to one another, phenotypically quite different one
from the other? What makes them so different?
In fact, this image that we have here, which in itself represents a
stunning achievement, that is being able to trace the
pedigree of each cell in the adult body is very different from our own
lineage because, as I may have mentioned to you in
the past, each of us goes through ten to the sixth mitoses in a
lifetime, you and I. That's ten to the sixteen cell
divisions. And at any one time we have roughly three times ten to the
thirteenth cells in our body. So, that means, if you think about
that carefully, the ratio between these two suggests
there's roughly a hundred times more here than here,
three hundred times more It means, roughly speaking, that our body
turns over roughly a hundred times in our lifetime.
That is to say the cells turnover. Not all of the cells,
but that there's a continuing replacement of existing cells with
new cells. After all, if, in fact, there were no such
replacement than we might, as an adult, be formed of this many
cells, and we would stay with the exact same number of cells
throughout our lives. But this, in fact, is the number of
cell divisions. And so, there's an enormous turnover
which, right away on its surface, independent of the fact that it's an
enormously large number, precludes one from really being able
to draw out a pedigree like this, prevents anyone from really
understanding how each particular cell can trace its line of decent
back to the fertilized egg. So, what we really want to explore,
in this lecture and the next one, is this major puzzle that one starts
out with a fertilized egg. Let's say this is a fertilized egg.
It divides in two. It divides in four. And through subsequent cycles
of growth and division we ultimately end up with the adult.
Already at the four-cell stage, here in a vertebrate embryo, these
cells have begun to take different phenotypic paths.
That is to say cells have begun to commit themselves into entering into
one or another differentiation lineage. And when I say a
differentiating lineage, I mean a group of cells which has
already made the decision to become blood cells, to become gut,
to become nerve cells and so forth. And these commitments already start
here at the four-cell stage, and they continue to play themselves
out until one reaches a newborn, and then thereafter one just grows
bigger. One other important thing to show here is that this pedigree
that I showed you here is not simply the result of exponential expansion
of all the cells, because many cells,
during the course of development, are actually weeded out from
embryonic tissue. And this happens even in our own
development. For example, here, if we look at our fingers,
I remember now I talked about fingers about two weeks ago and got
myself into some hot water. If we look at our fingers, you'll
see here we have five fingers, God willing, and but early in
embryogenesis our hand looks like a solid flap of tissue.
And what happens, during the course of vertebra development,
is that the tissue in between the beginning fingers is eliminated
through the process of apoptosis. Apoptosis means programmed cell
death. Apoptosis is equivalent to cell suicide. And what I mean by
that is to say that development involves not only the exponential
proliferation of cells but it involves the selective elimination
of cells here in a very obvious anatomical way.
It sometimes can be defective, in which case individuals are born
with large webs between their fingers. And this is part of normal
development. And the same can be said here. If there were no
apoptosis during the development of this worm embryo then there would be
vastly larger numbers of cells. We mentioned implicitly apoptosis
during the development of the immune system, because recall there that
bee cells, which are destine to produce antibodies,
if they produce inappropriate kinds of antibodies,
if they produce antibodies that are self-reactive,
i.e., recognize some of the body's own proteins, those cells are
eliminated by apoptosis. If they produce defective antibody
molecules, they're eliminated by apoptosis. And,
therefore, differentiation, which is what we're talking about
here, involves not only the commitment of cells to a certain
lineage, but the purpose of elimination of cells in certain
parts of the organism in order to carve and sculpt out properly
shaped tissue. Again, our fingers are one dramatic
example of that. It turns out we can learn an awful
lot about this process by studying one specialized adult tissue,
which is to say the organs of hematopoiesis.
And they'll teach us a lot about some of the lessons we need to learn
about organismic development and differentiation.
And when I use the word hematopoiesis,
the term hematopoiesis, or the adjective hematopoietic
refers to the creation, the formation of different kinds of
blood cells. In fact, we know that all the cells
in the blood descend in the organism from a common progenitor.
And this progenitor is called a pluripotent stem cell.
Pluripotent means that this stem cell, and we'll define a stem cell
momentarily, this stem cell is able to create descendents which can
commit themselves in a number of distinct directions.
They can differentiate in a number of distinct directions.
In this case, we see all these various kinds of white and red blood
cells which descend from this pluripotent stem cell.
And, as a consequence, we call it pluripotent because it
has these multiple distinct types of differentiation lineages.
So, here we talk about pluripotent. Later on I'll talk about totipotent
cells. Totipotent are cells that can do everything.
Therefore, in fact, what's a totipotent cell?
Well, a cell in the early embryo, including a fertilized egg is
totipotent in that it can direct its descendents into all of the
differentiation lineages in the body. Here we have a cell that's already
more limited. It's only pluripotent, pluri in the sense of multiple
but not total. Totipotent obviously means it can do
everything. And here we see the different kinds of derivative white
blood cells that exists in the bone marrow and in the circulation,
and there's a whole series of different ones of them.
We'll talk about some of them shortly, but we've already
encountered some of them up here in the form of T cells and B cells.
When we use the word stem cell the essence of the definition
is as follows. A stem cell is a cell that can
self-renew and it can also have a differentiated daughter.
So, here's the way one can diagram a stem cell. Here's a stem cell
that has two daughter cells. One daughter cell is exactly like
Mom and the other daughter cell has undertaken a program of becoming
differentiated. So, here we have an asymmetric cell
division on the part of this stem cell up here.
We'll prove later on that these two
cells are genetically identical, but clearly they're reading out
their genes in quite different ways. This cell is absolutely the same as
the mother cell. This cell has already committed
itself. It's made the commitment to differentiate in one
or another lineage. And another way of noting this
graphically is the following. We can draw a picture like this,
as we did before, and here we'll have a second arrow that goes around
like this. It loops around back on itself, and that implies the whole
program of self-renewal. Now, the whole concept of
self-renewal is a simple one. If a stem cell can self-renew,
that implies that the process of growth and division does not deplete
the pool of stem cells in the body or in a particular tissue.
So, let's imagine what we're looking at here. Here we have a stem cell.
It has one cell that is just like Mom. This is a differentiated cell.
Once again, you can have a growth and division. This is,
once again, a cell like Mom. This is a differentiated cell and
so forth. And what you notice here in this arrangement is that the stem
cells perpetuate themselves. They are self-renewing.
And, as a consequence, the pool of stem cells is never depleted in the
best of all possible worlds. It turns out that in most of the
tissues of our body there is self-renewing stem cells going on,
because most of the differentiated cells in our body have a finite
lifetime. Not all of them but most of them. And when I say finite,
I mean it can be measured in a matter of days or weeks
or months. In the case of the brain,
things turnover very slowly. Even in the case of our bones,
our bones actually turn over roughly once, 10% a year.
10% of the matter in the bone is actually turnover in every year.
So, almost all tissues in the body are in a process of continuing
self-renewal and repair. And that self-renewal and repair is
maintained by this stem cell compartment, as is indicated here.
This has certain kinds of great advantages, and one of the
advantages is indicated by the following. Let's imagine that we
draw a picture, just for the sake of argument,
of one of the most highly proliferative tissues in the body,
which is to say the lining of the colon or of the duodenum.
So, here we have, let me draw it slightly differently,
here's the way the lining of the small intestine looks like.
Out here are the contents of the small intestine.
So, let's say here is the lumen of
the small intestine. And here we have, protruding into
the lumen, when I talk about a lumen I'm talking about the bore or the
channel of a cylindrical or tube like organism.
So, here is the lumen of the small intestine. Here are these
fingerlike projections, they're called villi, that protrude
into the lumen of the small intestine. And down here at the
bottom of this are these cavities that are called crypts,
C-R-Y-P-T-S. These are the crypts. Now, what's important to realize is
that what goes through our intestines is not that pleasant.
It's pretty corrosive stuff. I probably told you this already,
more bacteria living in there than we have in our entire cells in our
entire body. There are all kinds of digestive juices.
And so the cells out here at the tips of these villi are continually
exposed to all kinds of corrosive material, including the junk that we
eat everyday which is flowing by like this. And this indicates how
critically important it is that we have self-renewal,
because the cells out here, being continually exposed to the
most corrosive kinds of influences, are rapidly damages.
And, therefore, the cells out here have a lifetime
of only three or four days and are then induced to jump off the end of
a gangplank and commit apoptosis. So, the cells at the tip of the
villis are continually jumping off and dying. And what's happening is
that down here in the bottom of the crypts we have stem cells.
The stem cells are continually
producing progeny that have committed themselves to
differentiate. And the progeny,
as you might guess from what I've just said, are continually migrating
up the sides of the villis up to the end here. And this whole migration
takes four or five days, and by the time they get to the tip
and have stuck their heads into the contents of the lumen of the
intestine for that period of three or four days.
Finally, they're eliminated and they jump off into the abyss.
So, there's a continuing action going on here.
The stem cells are continually dividing. And what advantages does
this have for us? Well, it means that cells that are
damaged are not allowed to hang around for a very long period of
time, i.e., cells up here in the top that are exposed to,
for example, potential mutagenic influences are rapidly eliminated.
Why is that good? Because the mutagenic influences up here could
well create a mutant cell that, in principle, is able to become
cancerous. And the body says,
well, I don't mind if that happens because these cells up here are
going to be eliminated anyhow. They're going to be pushed off the
end of the diving board or the gangplank into the abyss,
so they are continually undergoing apoptosis, not as a pathological
process. As a normal process. They're continually being pushed
out here. And what that means is that the
cells down here, in the bottom of the crypt,
are actually physically protected from the contents of the lumen of
the small intestine because some of the cells in this crypt are
continually secreting a kind of mucus in this area right here.
It's called a mucin. And this mucin here creates a physical
barrier, so the cells that are in the bottom of the crypt are never
directly exposed to the contents of what's flowing by in the
small intestine. And that is extremely important
because, in fact, it means that these cells down here
are shielded from the mutagenic influences of what might be present
in the lumen of the small intestine. In theory, one might be able to
evolve cells that don't mind being up here in the lumen of the small
intestine. But, in fact, that's never been possible.
That is to say evolution has just said, well, we can't really evolve
cells that are resistant to the corrosive influences of what happens
in the small intestine. And, therefore,
we're just going to use these cells for a very short period of time and
then get rid of them. What that also means is the
following. The stem cells down here stay within that crypt.
They don't migrate out. They stay there in that shielded
site. And, in fact, if you think all this through,
it's very important to protect these cells from becoming mutated because
if they do become mutated they could become the precursors
of cancer cells. If these cells become mutated out
here, it doesn't matter because they're going to die anyhow.
And so, we now have the following kind of dynamic.
Here's the stem cell. I'll draw it again. I'll just
abbreviate it stem cell. And one of the ways by which we
want to preserve the genetic integrity of the stem cell is to
insure that the stem cell divides as infrequently as possible. Why?
Because the whole process of cell division is itself a fallible
process. Every time a cell grows and divides, as we learned from the
cell cycle, there's the possibility of different kinds of genetic
disasters happening in which case we might end up with a mutant stem cell.
And that mutant stem cell could in turn, I argue,
become involved in creating a tumor. So, we have the following kind of
arrangement. Here is the stem cell. It has one daughter that has
committed herself to differentiate and the other that remains
a stem cell. Well, you're saying this enormous
amount of activity must involve a frenetic amount of cell division on
the part of the stem cell. But that's not really the way it
happens because this daughter cell undergoes a series of exponential
divisions, I can't fit them all on the blackboard here,
and might even yield a hundred descendents which then become the
ultimate differentiated cell. This daughter cell becomes one stem
cell. This daughter cell undergoes these exponential expansions in a
process of creating a population of cells that are called transit
amplifying cells.
And at the bottom of this hierarchy, I can't draw all hundred, there may
be a hundred of these cells, ten to the second, and these cells
then differentiate.
At the bottom, the hundred cells at the bottom,
they go into the last stage of differentiation.
They become the specialized cells that line the tips of the villis.
Now, why is there this arrangement? Well, look at what the advantage of
it is. The stem cell has just divided once, but this cell has
generated a hundred progeny. And that means that the stem cell
doesn't have to divide that often. The stem cell can divide once.
Every time the villis needs a hundred new cells it needs to divide
only once. And, therefore, the stem cell actually is
one of the most slowly dividing cells in the entire gut because it
only needs to divide episodically. Each time it divides it generates
this enormous array of progeny. One other aspect of differentiation,
and when I talk about differentiation here,
I mean the acquisition by these cells of all of the traits they need
to line the colon. When I talk about differentiation in
the skin, I talk about the ability, the acquisition of the cells of
becoming fully competent, fully functional skin cells.
The same thing with neurons in the brain. And one additional important
concept I'd like to introduce is that when cells differentiate they
often become post-mitotic. Post-mitotic means that these cells
give up the option of ever dividing again. In other words,
as they acquire more and more specialized traits they say to
themselves now I'm a nerve cell, now I'm a cell in the tip of villis
in the intestine, now I'm a muscle cell,
I'm not going to divide ever again. And this is generally true. The
most differentiated cells in the body in general end up losing the
ability to divide. They become post-mitotic.
They've exited irreversibly from the cell cycle.
They can't go back in. As you recall, we talked about
cells going from G0 back into the active G1 phase of the cell cycle.
That's a reversible exit from the cell cycle. Post-mitotic cells are
irreversibly committed never to divide again. And,
again, that holds true for almost all cells of the body.
One exception to that is, interesting enough, in the liver.
Because what you can do with a mouse, or even with a human,
is you can cut away a couple lobes of the liver, major league surgery.
And when you do that, what happens is that all the remaining cells of
the liver, and these remaining cells in the liver are called hepatocytes,
these hepatocytes, which until that time had been highly specialized
differentiated cells in the liver, many of them divide, they double
again. And in short order one ends up with
a liver which is exactly the same size as one had before.
And that's actually quite remarkable because there are very
few organs in the adult human being where that will happen.
There is, by the way, an interesting puzzle here,
and that is the following. Let's say you cut away half the
liver and many of the hepatocytes, which were already highly
differentiated, began to divide again,
so they were not serious post-mitotic cells,
they could reenter into proliferative phase.
How do these cells know when to stop dividing so that they end up
regenerating a liver of exactly the right size? People have been
looking at that for 30 or 40 years. Nobody has any idea why. Why
doesn't the liver when all these cells divide become one and a half
times the size of its former diameter or half the size?
Nobody really understands that. In any case, I just want to
indicate that there is this dynamic between differentiation and
proliferative capacity, one in opposition to the other.
Well, how much hematopoiesis is taking place here in our bone marrow,
for example, where a lot of this takes place? So,
here are just some interesting numbers. There are roughly five
times ten to the twelfth red blood cells --
-- per liter of blood.
And red blood cells, you may recall, are called erythrocytes.
Remember, it's never good to use a short Anglo-Saxon term if you can
use a long complicated Greek one. And each of these red blood cells
has roughly a lifetime of 120 days. That is to say after it's made it
sits around in the blood for roughly 120 days.
It gets warn out. It gets gobbled up by the cells in
the spleen. Much of the contents are recycled. The pigment in our
stool comes from the recycling of the hemoglobin,
the iron and the hemoglobin, and we're constantly renewing that.
Well, the average human being, let's say, has roughly ten liters of
blood per person.
And if you put all those numbers together, I once calculated that the
number of red blood cells that is being generated in the body is
roughly ten to the tenth per hour. And you can go through the
calculations if you want, it is fine with me, but take my word
for it's roughly what's going on there. Ten to the tenth per hour.
Each time I utter a word there's probably, I don't know,
ten to the seventh new red blood cells being made in my bone marrow.
So, this is not minor league proliferation.
Now we begin to understand why there are ten to the sixteenth cell
divisions in the lifetime of a human being. In fact,
a great majority of them, to be fair, are occurring in the
bone marrow and in the intestine. And if you have an individual who's
being exposed to certain kinds of chemotherapy, chemotherapy,
as you may know, is very toxic for dividing cells,
and the side effect toxicity of anticancer chemotherapy is largely
felt, first of all, in the bone marrow where individuals
tend to become anemic. Anemic means they have lower than
normal numbers of red blood cells and also they lose a lot of the
lining of the intestine which creates all kinds of also
unpleasantness as well. So, we're not talking about
something that happens on rare occasion in the life
an individual. This is a staggering amount of
mitosis that's happening every day of our lives. Let's go back for a
moment to this diagram here and realize that when I'm talking about
erythrocytes, I'm only talking about one of the branches of this
multi-branch pathway. And here we see some other
interesting aspects of what's going on here, and I'll give you some
proofs very shortly that this actually is what it says it is.
It actually is organized this way. The pluripotent stem cell is capable
of self-renewal, and it can spew off daughters which
actually can go in two different directions. Its daughter may decide
that it might become the precursor of the lymphoid cells in the blood
or it might commit itself to becoming a myeloid precursor.
So, that's already the beginning of a bifurcation.
These cells are not yet differentiated.
They've just made the commitment that each of them can,
in principle, become the ancestor of highly differentiated cells.
And these cells, we can imagine, are transient
amplifying cells in the sense that even though they're committed to
create progeny of one sort or another they themselves are not yet
fully differentiated. Keep in mind in the context of the
crypt these transient amplifying cells are on the way to becoming
fully differentiated, but only at the bottom of this
exponential expansion of cells do we have cells that are fully entered
into a highly differentiated state. Here we see that these two cells are
also stem cells in the sense that the can self-renew.
They have a limited self-renewal capacity, but they can self-renew.
And then they begin to create progeny which themselves can
undertake several distinct alternative differentiation paths.
So, the lymphoid cells can become the progenitors of the T lymphocytes
and the B lymphocytes. And, in fact, if you recall our
discussion of immunology, there's actually several different
kinds of T lymphocytes and B lymphocytes. So,
this pathway has further radiations further down. Here,
alternatively, these are the myeloid cells. And myeloid refers to the
bone marrow. And the myeloid cells can become these kinds of cells up
here, eosinophils and basophils and neutrophils and monocytes,
and this class of cells is largely involved in gobbling up infectious
agents and as agents which are able to defend us largely against
bacterial infections. Here's the macrophage.
We talked about the macrophage. Remember the macrophage was this
glutton, this pig which wandered around our tissues and gobbled up
whatever kind of material it could find and presented it to the immune
system. And here are several other of the lineages.
Here is a megakaryocytic and this is an erythrocyte.
What's a megakariocyte? Well, it has a very large nucleus.
That's what the term implies. And what happens to the
megakariocyte is it buds off little chunks of cytoplasm lacking nuclei.
And these little chunks of cytoplasm become the blood platelets.
Blood platelets lack nuclei. They're enucleate because they're
just little bags of material which are sent out into the circulation.
And, as we said also earlier in the semester, once the platelets are in
the circulation they are there ready to help should there be any kind of
wounding, any kind of hemorrhaging occurring.
And the platelets can release upon being activated in a site of wound
coagulation factors and growth factors for the regeneration and
reconstruction of wound sites. And, finally, here are our friends
the erythrocytes. So, here we have a whole sequence
of different kinds of differentiation commitments which
are going on at an enormous rate. How do we know that there actually
is a pluripotent stem cell? What evidence can I provide you that
this actually exists or it's just a figment of my normally florid
imagination? And the most direct demonstration of that is,
in fact, the use of bone marrow transplantation.
So, when we talk about a bone marrow transplantation,
or BMT as it's called in the trade, --
-- one can do a relatively simple
experiment. You can take a mouse or even a human and you can irradiate
it rather heavily. And if you irradiate it under the
right conditions you'll actually be able to kill off all the cells in
the bone marrow without killing off the mouse or the human being.
In fact, there are drugs you can also use in human beings to
eliminate virtually all the cells in the bone marrow.
And then what you can do is you can take bone marrow from another
organism, from another mouse or another human,
and you inject it into the blood of the irradiated mouse or human.
And the bone marrow cells, many of them will home to the bone
marrow. In other words, you're injecting the bone marrow
cells in the general circulation, but within a couple hours they'll
all end up in the bone marrow, in the space in the middle of the
bone because there are many kinds of cells which have this homing
capacity. They go to the right place in the body.
So, they can home. The injected cells can home to bone
marrow. And then, if things are going well,
these injected bone marrow cells will begin to proliferate and they
will ultimately regenerate this entire cascade of differentiation
decisions, as is indicated here. And, therefore, that individual or
that mouse will actually be rescued. Because in the absence of such a
rescue an individual will rapidly die.
You can't live for very long in the absence of an active bone marrow
because these cells here are rapidly depleted. They turn over with some
speed. The red blood cells hang around for 120 days we said and,
therefore, you don't need to make them immediately because there's a
whole bunch around that have a rather slow turnover.
But the platelets only have a lifetime of several days before
they're lost, they're turned over. And if you don't have platelets
you're in very bad shape because you start hemorrhaging all over the body
because, remember, the platelets are there to stop up
all the holes in the dike to prevent bleeding.
These cells here are very important, the eosinophils, basophils,
neutrophils, and even macrophages in preventing bacterial infections.
And in the absence of having these on site one can readily succumb to
overwhelming infections. Keep in mind that the reason why
we're not constantly dying from bacterial infections is not because
each of us takes an antibiotic pill every day, it's because these cells
are on watch to kill any bacteria that happen to be in the wrong place
in the body outside of the lumen of the gut.
And consequently the question is always can one rescue a mouse or a
human rapidly enough? Can one replace its bone marrow
rapidly enough so that this disaster from losing all ones bone marrow
doesn't overtake one and the organism dies before the bone marrow
has had a chance to become reconstituted, regenerated,
reconstructed. Still how do we know from all this
that, in fact, there is a pluripotent stem cell?
If you listened to everything I said correctly you could say,
well, there isn't such a thing as a pluripotent stem cell.
There are these other kinds of stem cells, this one and this one,
or these might all be stem cells. And when I'm injecting the bone
marrow of a donor animal into the recipient, I'm injecting a whole
mixture of different kinds of stem cells here each of which then goes
on and populates a specialized compartment in the bone marrow
or in the blood. So how do we know there's one
pluripotent stem cell? One way to prove this is the
following. Let's say we take the bone marrow from the donor,
that is the bone marrow that we're going to inject into the irradiated
recipient, and we irradiate that bone marrow very lightly,
not to kill the bone marrow cells but to introduce random chromosomal
breaks, a very small number of random chromosomal breaks in the
donor bone marrow. So, the purpose now of irradiation
is quite different from what I said before. Before we wanted to give a
heavy dose of radiation to wipe out the recipient bone marrow.
Now we're going to just give a wee bit of radiation to the donor bone
marrow. What's the purpose of that? The purpose of that small amount of
radiation is to create chromosomal abnormalities.
So, for example, if here are two homologous
chromosomes in the donor cells. Since the radiation, the very weak
dose of radiation is acting randomly it will create all kinds of
abnormalities including, for example, a very specific
chromosomal translocation so that what might happen after this is that
a whole chunk of this chromosome is translocated over to this chromosome
here. This is called a chromosomal translocation.
And now here's the donor,
these are donor bone marrow cells. And keep in mind that every donor
bone marrow cell that gets a little bit of this radiation will get its
own very specific randomly occurring translocation just because radiation
is able to break chromosomes and then they will rejoin in
unpredictable ways. What that means is that if we take
the donor bone marrow and irradiate it very lightly so that we don't
kill the cells but we do induce these translocations,
one donor cell will have this translocation and another donor cell
over here will have a totally different translocation from a
different chromosome also induced randomly by these stochastic
processes. So the karyotype which is the whole
array of chromosomes of a cell, which can be viewed at the metaphase
of mitosis when all the chromosomes condense, the karyotype of each of
these donor bone marrow cells will be messed up slightly.
And it will have recognizable abnormalities,
but they're all different, one after the other. And after
we've done this, after we've marked millions of bone
marrow cells in the donor with these random low-dose radiations,
we can then inject a small number of bone marrow cells into
the recipient. And what we can sometimes find on
occasion is if we look at the recipient after that recipient has
been rescued, i. ., after the donor bone marrow has
established itself within the recipient, is that the donor bone
marrow is established in the recipient and populates all of these
different lineages. And if we're able to look at the
karyotype of these different kinds of cells in the recipient organism,
we can find that in some mice all of these cells have the same very
peculiar translocation. They have either this one or this
one or they have yet a third translocation,
any one of a whole series of randomly occurring mutations,
a very peculiar idiosyncratic unusual translocation induced by the
low-dose radiation. They all have it.
The T cells and the B cells and the monocytes and the basophiles,
they all have exactly the same translocation.
Obviously, we can't do that experiment with the platelets.
Why? Because they don't have nuclei. And we can't do that with
the erythrocytes either, the red blood cells. Why can't we
do that? Because in mammals, when the red blood cells are formed,
the nuclei are spit out. Our red blood cells don't have nuclei in
them anymore. They've become enucleate or, to put it another way,
they have been enucleated. That is to say they've been deprived
of their nuclei. Why? Because they're post-mitotic.
Obviously, a cell which lacks nuclei is by definition post-mitotic.
And our cells don't really need, red blood cells don't need nuclei.
We know that ancestral organisms, for instance chickens,
their red blood cells are nucleated, but our red blood cells are not
because they're just not necessary.
How is translocation different from crossing over?
Crossing over occurs between two homologous chromosomes.
So if we have a chromosome here, here's chromosome 13 and here's
another chromosome 13, they're both chromosomes 13.
This one came from Ma. This one came from Pa. All right?
Each of us has a pair of homologous chromosomes.
Here's the maternal one. Here's the paternal one.
When we talk about crossing over we're talking about a process of
homologous recombination.
And when that happens we have a situation like this.
This chunk is exchanged with this chunk over here.
It's an equal exchange, absolutely equal down to the
nucleotide, so that after this flipping has occurred we have two
fully intact chromosomes. It's just that both of these
chromosomes are fully normal. It's just that there's been a
switching, an exchange between the two homologous chromosomes,
the two paired chromosomes 13. When we talk about translocation
there a chunk of chromosome 15 can go onto chromosome 7 or a chunk of
chromosome 2 can go on chromosome 8. It's totally random, it's
non-homologous, and it creates aberrant chromosomes.
Neither of these recombined chromosomes is abnormal.
It's just changed it allelic configuration.
So, it's an important distinction. And the fact is you can pick out
these translocated chromosomes because one even has specific dyes
that can be able to tell you which chromosome this came from and which
chromosome this came from. So, there is a profound difference.
Translocations are, invariably, pathologic. When I say pathologic,
I mean they're really sick. They're not the proper course of
things that happens in a healthy cell. The fact,
the very fact that we're able to generate an entire array of cells in
the blood indicated, by necessity, that if all these
cells have the same chromosomal translocation that they descend from
a donor cell that originally was lightly irradiated and happened to
receive that translocation. If we never get this array of common
translocations in all the cells in a bone marrow recipient then we can't
prove this, but the fact is this has been proven time and again over the
years. And this indicates to us that this cell which is genetically
slightly altered can, therefore, generate all these other
cells in the body. Again, keep in mind that the
irradiation of the donor marrow is simply to create these chromosomal
markings. They're not necessarily good for the organism,
but they don't compromise the viability of the cell.
They just reshuffle the chromosomal structure. Now,
in principle the levels of each one of these kinds of end-stage cells
need to be carefully regulated. And, by the way, let me just note
here, you see the T cells have the arrow going back on themselves,
as do the B cells. That indicates that they have not become
post-mitotic. Remember we talked about these embraces between helper
T cells and B cells where they're walking down the allies and they get
excited and they start multiplying? The fact that the T cells and the B
cells are able to proliferate in response to certain antigenic
stimuli implies that they're not post-mitotic. They still have the
ability to proliferate, and that retained ability to
proliferate like that of hepatocytes is indicated by these arrows that
are looping back on themselves. Conversely, these cells are all
essentially, as I've said before, post-mitotic. So how do we insure
that's there proper concentrations of all of these different
cells in the blood? And the fact is the concentrations
of many of these cells in the blood are maintained to concentrations of
plus or minus 10%. And this is, itself,
a stunning testimonial to the successes of human physiology.
We're talking here about a process which is sometimes called
homeostasis. Homeostasis means that somehow there is a balance,
an equilibrium that is achieved, and that there aren't profound
fluctuations so that we always have roughly the equal level of red blood
cells, a proper level of lymphocytes in our blood.
And I want to get into the homeostasis which results in the
formation of red blood cells, the RBCs, the erythrocytes in the
blood. In fact, it happens to be the case that the
red blood cells are one of the cell types that could actually vary quite
profoundly in response to environment.
To the extent that lymphocytes change, they go up and down,
that might be due in response to an infection. So,
if we have a serious bacterial infection we might have increased in
the lymphocytes in the blood that have been mobilized in order to
attack the infecting bacteria. But what about the RBCs? What
about the red blood cells? What causes them to change?
Well, if you move from here to Denver, Colorado or you go up skiing
in the Rockies, you're going up to ten or twelve
thousand feet. And within a matter of three or four
days the concentration of your red blood cells increases very
substantially. Why? Because obviously the oxygen
tension at high altitude is down. And in order that your peripheral
tissues become adequately oxygenated, the oxygen carrying capacity of the
blood must be increased. And the way that is increased is in
part to increase the concentration of red blood cells.
So how does that happen? How is it possible that we can
rapidly modulate the concentration of red blood cells?
And the way we can do that is in part through a hormone called
erythropoietin, EPO. We're going to talk about
erythropoietin at the beginning of the lecture next time,
but the homeostasis which maintains the appropriate number of red blood
cells in our circulation is dictated in no small part by the levels of
EPO that are in their blood. To anticipate some of the things
we're going to say next time, when you are in a low-oxygen
environment the levels of erythropoietin shoot up.
And when they shoot up they insure that there is shortly thereafter a
rapid increase in the level of circulating red blood cells which in
turn enables the oxygen coming into your lungs to be transported more
efficiently, more effectively into the peripheral tissues.
You've heard about athletes perhaps who are able to dope themselves with
erythropoietin. This is a rather devious strategy
because it means that if they do so, they inject themselves with a little
erythropoietin, the oxygen carrying capacity of
their blood is temporarily increased and as a consequence they might be
able to run further or jump higher. This, by the way,
has its dangers. Because if you're injecting erythropoietin not in
response to certain physiologic signals but just because you want to
win a marathon or something, you're violating the normal
physiologic mechanisms in the body which very carefully control the
levels of erythropoietin. And if you inject too much
erythropoietin you get in a very bad situation because the bone marrow
makes more and more red blood cells. And then what happens? You start
clotting up everywhere all over the body, and this isn't good.
In fact, you can die. So this is not a warning against
erythropoietin in the way that I warned you against cigarettes.
This is just to tell you these kinds of drugs,
or these kinds of growth factors, which they are, are maintained at
very precise levels as we'll discuss in more detail next time.
See you on Wednesday.