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Hello. My name is Alfredo Quinones-Hinojosa. I'm an associate professor
of Neurosurgery, Oncology, Cellular and Molecular Medicine, and Neuroscience
at Johns Hopkins University.
Today, I have the pleasure of speaking to you about the issue of stem cells and brain tumors.
The question is whether or not there is a cell
that has given rise to a brain cancer,
and whether or not that cell was a normal neural stem cell
that has given rise to a brain tumor,
or whether or not there was a brain tumor stem cell
population within brain cancer.
And we're going to go ahead and explore this.
I'll tell you a little bit about history.
In my opinion, the most beautiful organ in the body is the brain,
and this is a 3-dimensional reconstruction of an MRI.
And what you do not see right here
is that, in the left side of the brain, which is this area right here --
remember the image is flipped.
We have done this, right here, you know, in this case label L for left
and you're looking, yourself, at this brain, face-to-face,
like this. This is the left side. Underlying this,
is a very dangerous brain tumor,
also known as a glioblastoma multiforme.
Over 100 years ago, Santiago Ramon y Cajal won the Nobel Prize in Physiology and Medicine
because he was able to contribute greatly to the field of neuroscience
by looking at the different types of cells that make the brain.
He said that we had a harsh decree
that everything may die, and nothing may be regenerated in the adult central nervous system.
He left the possibility open for pathological diseases.
He was a pathologist by training and became one of the great neuroscientists in the world.
Over the next 30-40 minutes, I will take you over some of the history
of the controversy over the adult neurogenesis,
the difference between the human vs. the rodent stem cell niche --
more specifically, the sub-ventricular zone.
I will take you over whether or not there are stem cells that are found
in the human brain, and whether or not
there are stem cells found in human cancer.
The question that still remains is whether or not a neural stem cell or a stem cell
may give rise to a brain tumor stem cell, and that is work in progress,
and I will elaborate on a little bit of that.
What about this issue of the controversy of adult neurogenesis?
Well, it turns out that dating back to the early 1900s,
Santiago Ramon y Cajal knew that we had limitations in technology.
He said once that none of the methods used by these investigators
are capable of distinguishing absolutely a multiplying neuroglia cell
from a small mitotic neuron.
So, the question is whether or not there were new neurons
being formed int he adult mammalian brain.
By the 1960s, Joseph Altman, by doing a study with thymidine autoradiographic evidence,
he was able to find new neurons in the adult rat and cat.
But, it would take a few years later, by the 1970s,
when Michael Kaplan, he combined thymidine labeling
and electron microscopy. This is the early research and the early utilization
of electron microscopy that confirmed Altman's claims
by showing mitotic neuronal precursors lining the lateral ventricle.
But, people still were very skeptical.
It was kind of hard to understand and to believe
that indeed, new neurons are being produced in the lateral ventricle of the mammalian brain.
By the 1980s, Nottebohm and colleagues, out of New York,
were, by doing some really eloquent studies
in the avian brain, they were able, through a series of experiments, to show several things.
The production of new cells with thymidine labeling.
They were able to show that these new cells were not only neurons,
but they were also receiving synapses.
And most importantly, these neurons that were receiving synapses
responded to sound with action potential.
Yet, people were very skeptical about the issue
of neurogenesis in the avian brain
because they said that it was not the mammalian brain.
So, by the 1990s, work in the rodent,
out of several laboratories around the world, they began to unravel
and teach us more about this issue about the controversy of neurogenesis.
By 1997, a group of investigators led by Dr. Arturo Alvarez-Buylla,
also out of New York and subsequently out of California,
was able to elucidate the organization and cytoarchitecture
of the rodent sub-ventricular zone.
But 1999, a very important discovery that was also controversial --
several groups believing there were probably ependymal cells
that were responsible for neurons. And Arturo and his group,
believing that it was actually astrocytes, were able to show
that adult neural stem cells come actually from astrocytes in the rodent brain.
What was not known at this point
was whether or not this was also true for the human brain.
It was even less known the difference in this stem cell niche --
the sub-ventricular zone -- between the human and the rodent.
And that's the next portion of this discussion.
Several years later, by the year 2003-2004,
as Arturo and his group, and myself involved in some of these studies,
we began to unravel some of the mysteries of the organization
of the human sub-ventricular zone.
We began to look at markers of migration, in this case,
PSANCAM and markers of proliferation, KI67,
and markers of neuronal precursors, TuJ1.
And we looked at several parts of the brain.
This is anterior horn, the body of the ventricle,
the other parts of the ventricle, as you can see,
all the way to the temporal horn, right here on the bottom.
And what you can see, actually, not only the organization
was different -- there was evidence of proliferation, migration,
but also we found evidence of some neurons struggling
into this area of the sub-ventricular zone,
going up and down.
So, after many years of looking at the organization
of the rodent sub-ventricular zone, done by many groups around the United States
and the world,
we began to look at the organization of the human sub-ventricular zone.
And you can actually see here... in red
is GFAP, and in blue is DAPI.
For the rodent sub-ventricular zone, you can actually see
GFAP in green, double cortin in red, and DAPI in blue.
And then there's a cartoon illustration between the human sub-ventricular zone
and the rodent sub-ventricular zone.
In the rodent sub-ventricular zone, not only is the brain a little bit more
lissencephalic in sculpture. That means that it has less grooves
as compared to the human brain. But, in the area of the sub-ventricular zone --
that is the lateral wall of the ventricle right here.
This is an amplification. You can actually see a group of cells
just coming out. Also known as a rostral migratory stream.
And these astrocytes are completely welded against the epyndemal zone.
Yet, in the human brain, there is the epyndemal zone right here,
which is called layer 1. Then layer 2 is a hypocellular gap.
Then layer 3 is a ribbon of astrocytes.
And then there is a transition to the parenchyma.
Striking differences between the organization of the human sub-ventricular zone
and the rodent sub-ventricular zone.
So, we got interested in our laboratory to further elucidate
the organization of the human sub-ventricular zone.
We went on to do these very similar studies in the fetal human brain.
And indeed, it appears to be that the lateral ventricle may be connected also
to the rostral migratory stream, all the way down
against the olfactory bulb. This is a so-called
sagittal section along this region that is slightly oblique right here
and a little bit inclined, as you can see
right here, and we were able to study different regions of this part of the brain,
trying to understand how is the fetal brain and the human brain organized
as compared to the rodent brain.
Is there a population of cells that are migrating?
It turns out that we found some evidence that indeed
there are some cells that appear to be migrating outside of this region.
Not necessarily the same way that they appear to be migrating in the rodent brain,
from the ventricle down to the olfactory bulb,
but indeed some of these cells appear to be migrating in groups,
and they're surrounded by these glial cells. This is a confocal microscopy
of such a region in the human fetal brain,
and unfortunately, our group, has not been able to find evidence
of a similar type of migration in the human brain
per se, in the human adult brain,
but other groups around the world have claimed that this does indeed exist.
So the question is, if migration exists in the human brain,
could we learn something about that migration
that will help us understand why brain tumors are so migratory?
This is an image that I will show you of a small piece of human fetal brain
that is put on Matrigel, in which you're going to see evidence of migrating cells
surrounded by glial sheets.
And this is the picture, right here. This is the movie.
You can actually see -- this is from own our laboratory -- cells migrating,
and this is human fetal brain.
They're migrating out, and they are surrounded.
And we've done all these immunostainings to be able to see that, indeed,
the human fetal brain may have some similarities in regard to migration
as compared to the rodent brain.
We have done similar studies, as you can imagine, with human brain tumors
and we are in the process of trying to unravel some of the mysteries
that human brain tumors use for migration.
I'll take you. So, we have gone over the controversy of adult neurogenesis
and I think it's clear that the mammalian brain does have the ability
to generate some neurons, at least in the rodent brain.
And similar evidence may actually be becoming more clear
in the adult human brain.
We know that the stem cell niche -- in this case the sub-ventricular zone --
is different between the human and the rodent.
The question is whether or not there are stem cells
in the human brain, and whether or not there are stem cells in human cancer.
So, could it be that there are human adult neural stem cells?
By the 1990s, Steve Goldman and his colleagues, they did several studies
and published a paper about finding adult neurogenesis
from the temporal horn sub-ventricular zone in vitro.
This was from temporal horn surgeries, in which they did temporal lobe resections
and were able to culture this tissue and create neurons
for the first time, showing that indeed, at least in the petri dish,
it's possible.
By the 1990s, Gage and his colleagues, showed a study
by looking at brains post-mortem -- they call it in vivo, but post-mortem --
in which they showed neurogenesis in the adult human hippocampus.
And I am in the right side of the hippocampus right now
I'm showing one specific picture, and what you can actually see
right here, in this particular cell right here that is marked
with two markers -- red is NeuN as you can see right here
and green is BrdU, so this appears to be the formation of a neuron.
These patients had actually received BrdU for the treatment of other diseases.
They passed away, and this group was able to obtain their brains
and do these studies. For the first time,
freezing these tissues in time, they were able to find evidence of new neurons
being formed in the adult human hippocampus.
So, we ask ourselves the same questions.
By 2004, we asked -- we published a paper in which we asked ourselves the questions:
The adult human sub-ventricular zone could potentially have neural stem cells?
We actually obtained the specimens from the operating room.
We looked at specifically specimens from the sub-ventricular zone,
and we were able to form these beautiful, small spheres.
We called them neurospheres.
We purified these sub-ventricular zone astrocytes.
That means, through a procedure, we shake the cells,
and at the end, in the petri dish,
we only leave astrocytes. And we were able to create even more
neurospheres as compared to those episodes in which we did not purify the astrocytes.
And we obtained astrocytes from the cortex and the striatum.
We had absolutely no evidence of sphere formation,
telling us for the first time that indeed there's got to be something peculiar
in the astrocytes that are found in the sub-ventricular zone,
because not only when we purify them
we find more neurospheres, but
also when we have the same astrocytes or similar astrocytes from the cortex and striatum,
we found no evidence of neurospheres.
This is the picture of one of such neurosphere.
And not only that, when we put them in a special media, we were able to form
the 3 lineages: in this case, neurons,
in this case oligodendrocytes, as you can see right here,
and astrocytes. So, from one of these spheres,
from the sub-ventricular zone,
we found the capability of these cells --
this is a sphere that came from a single cell that was an astrocyte.
It was a GFAP-positive cell,
and we differentiated the cells, we were able to produce 3 lineages. Once again,
astrocytes, neurons, and oligodendrocytes.
But that wasn't enough. The question was can
human sub-ventricular zone astrocytes
produce neurons without the effect of growth factors?
So, what we did right here, is we provide an environment
in which we use astrocytes from the human brain,
and then we got one astrocyte from the sub-ventricular zone,
and we dropped it in here
to see whether or not, without putting exogenous growth factors,
we could actually produce a neuron.
And I'm inside of one of these experiments, and you can actually see
in red, a beautiful neuron that has been formed from an astrocyte
that was derived from the sub-ventricular zone
without the use of growth factors.
We tried to do the same from cortical and striatal astrocytes,
and we were unable to produce a neuron.
Once again, telling us that there is something about the sub-ventricular astrocytes
that makes those cells peculiar and able
to produce neurons in the actual human brain.
Now, all of this was done in the petri dish,
and yet we were able to find, for the first time,
that in the region of the lateral ventricle,
as you can actually see right here, there are
several regions -- this was actually after studying a lot of specimens,
not only from the operating room, but also from post-mortem tissue,
we were able to find that in the lateral ventricle
of the human brain, there is a specific population of astrocytes
that can give rise, not only to those beautiful spheres,
but also to neurons, oligodendrocytes, and astrocytes,
and most importantly, even without growth factors, we can actually
produce a single neuron.
So, indeed, there is a possibility, up until the present,
what we didn't know before, that the human brain
in the petri dish, has the ability to produce neurons.
So, there's probably a cell population that we call stem cells
within this area of the sub-ventricular zone.
Other groups have continued to look at this cell population within the hippocampus,
and they're continuing to move this field forward.
What about the issue of stem cells in human cancer?
That is something that has been obviously one of the greatest challenges
that we have had, because cancer is still the most devestating disease
known to humans. In my opinion, the most beautiful organ
affected by the most devestating cancer.
One of the problems that we have, first of all,
is that we are dealing with tumors that come from the brain,
the so-called intraaxial tumors.
We can have all the way from low-grade gliomas (LGGs)
to high-grade gliomas (HGGs).
Low-grades to high-grades. They're all in the same family of cancers.
All the way from the low-grade to the high-grade.
I believe the lower the grade, the longer the survival of the patient.
The higher the grade, the more devestating it is for the patient.
The limitations are that we can potentially in surgery be able to resect
this mass that we can see in the operating room.
The problem is that by the time these patients get the diagnosis of brain cancer,
specifically if it is a high-grade glioma,
many cells have actually migrated all the way to the contralateral part of the brain.
And I tell you this is how devestating -- this is a mass ***.
Approximately 20,000 people in the United States are newly diagnosed
with the primary brain cancer.
That is the cancer that I just showed you.
That is a cancer that comes specifically from the brain.
And about half of those patients die every year
as a result of these malignant tumors.
That is specifically the gliomas -- that's the kind of tumor that we're discussing.
We have looked at this issue in our laboratory.
You know, I was interested to try to understand whether or not there was a relationship
between glioblastoma multiforme and the lateral ventricle.
So, remember, we study the lateral ventricles in the normal human brain,
and we know that there is a potential population of
cells that we call the stem cells.
So, can these potential stem cells give rise to these tumors?
Well, we've got to take one step at a time.
So, what we did, is we went back to our database at Johns Hopkins,
and we looked at the percent survival
and the months, right here, and we looked at two types of patients:
those that involved the sub-ventricular zone,
which is the dark line right here,
as you can actually see.
And then we looked at those patients that had no involvement of the sub-ventricular zone.
And this is this dotted line right here.
And we looked at the survival. What we noticed is those patients
that have tumors that is involved in the sub-ventricular zone,
which is this right here, which is this type of tumor right here,
survived only 8 months.
Versus those patients that had no involvement of the sub-ventricular zone,
which is this type of tumor, right here, not near the ventricle right here.
Those patients tend to survive 11 months. And this is the Kaplan-Meier curve.
So, for the first time, giving us an idea that potentially within this area,
there may be a population that is either contributing
or potentially originating some of these tumors.
That is still a working hypothesis.
The working hypothesis is straight forward.
How do tumors get formed?
Do tumors come in from the transformation of a neural stem cell
or a neural progenitor cell that is then transformed
into a brain tumor stem cell and then gives rise to these devestating tumors?
Are there epigenetic and genetic alterations that give rise to this transformation?
Is there a potential de-differentiation
between a neuron, an astrocyte, or an oligodendrocyte giving rise
to these neural progenitor cells, which in turn gives rise
to a brain tumor stem cell?
That is all work in progress.
The bottom line is that these tumors right here
are devestating, and the way that we're looking now at the potential
etiology by transformation of either neural stem cells or progenitor cells
giving rise to these brain tumor stem cells is absolutely
giving us the hope that we need that potentially one day
we'll be able to manipulate the system and be able to have a positive effect on our patients.
I show you, what around 2004, when we published our paper
in Nature,also showing that humans do have in the lateral ventricle
these neural stem cells that give rise to neurons
contributing to our understanding of the human sub-ventricular zone.
Around the same time, a group from Italy, and a group from Canada,
they showed that, indeed, when we obtain tissue from
the operating room, and we obtain the tumors, and then we grow them.
Tumors, either as little spheres, the so-called neurospheres,
they look exactly like the neurospheres, but they're called tumorspheres.
Or we isolate these cells based on CD133.
And we go ahead and put either the spheres or the cells back
in the rodent brain, these rodent brains produce tumors
that look exactly like the tumor of the patient.
And since then, other groups (ourselves included)
have actually duplicated some of those studies.
So, for the first time, giving us the hope that indeed
in the end, potentially these tumors are actually being able to be studied
when we obtain the tissue from the patient and bring it into the laboratory.
So, for the first time, beginning to make our patients part of not only the diagnosis,
but also the potential treatment
and making them part of history.
These tumors look much better, and they resemble much better
the parent tumor than some of the cell lines that we have used
for several decades now.
Now, there are similarities between the neural stem cells
and the brain tumor stem cells,
all the way from the migratory capabilities to the signaling.
And you can actually see some of these similarities right here.
What makes the brain tumor stem cells peculiar
is the fact that they seem to have a much more long-term propagation as well as
dual phenotypic labeling. That means that
they can actually mark with several markers.
Now, this is potentially what is making us think that
maybe neural stem cells, they do have to do something with brain tumor stem cells.
Once again, this is a working hypothesis that I think that potentially in the future,
we will be able to systematically begin to understand a little bit more.
What we do know today is that from the sub-ventricular zone,
from these astrocytes, we can form these spheres, and these spheres can in turn
give rise to the three lineages: neurons, oligodendrocytes, and astrocytes,
that you can actually see right here.
What we do know today is that if we obtain tissue from the operating room
from our patients, as we can grow one single cell
into a sphere, and that sphere can ultimately go back into the animal,
those tumors resemble the tumor that the patient originally had.
So, potentially giving us an opportunity to have a much better
model to study brain cancer, and that's exactly what we have done
in our collaborations, some of our work.
And I'll go over a couple of these studies.
For instance, this is a paper that we just published not too long ago,
actually in 2009, where we looked at the Delta/Notch-like
epidermal growth factor-related receptor.
These are tumors that were derived from glioblastoma multiforme neurospheres.
And, as you can see right here, this is the tumor
with no treatment. This is the tumor with treatment.
In this case, right here, this is the tumor value, which you can actually see right here.
These are the two different groups, and you can actually
see significant differences.
Once again, illustrating the principle that we can dream of manipulating the system.
and very systematically begin to understand what are the growth factors, receptors,
that have an influence on this tumor formation.
I'll take you through another study that we have done
that is the Kruppel-like Family of Transcription Factor 9,
a differentiation-associated transcription factor that
suppresses Notch I signaling and inhibits glioblastoma-initiating stem cells.
This is the actual image right here. This is the control.
You can see the massive tumor, similar to the tumor of the patient,
and when we treat these tumors,
for this factor specifically, you can see much less tumor formation
in the animal, in the rodent model.
And you can actually see right here, in this axis
is nothing else, but the actual significant difference between the control
and those treated in regards to the tumor size right here.
And right here is the Kaplan-Meier for the survival of these two groups.
That is the control and the animals that are treated.
And it's a significant difference, once again giving us
the hope that we can learn from this disease, that we can learn from
these cells, that what we learn from these cells or from these deceased
can potentially one day be applied to our patients,
giving us this view.
And I'll leave you with this last paper from PNAS
that we just published a few months ago, in which we look at
c-Met signaling inducing a reprogramming network
and supporting a glioblastoma stem-like phenotype.
And you can actually see the control -- very large tumor
and those treated right here, giving us the tumor area.
The difference between these two right here.
Once again, for the first time, giving us the ability to manipulate the system.
I think, now through the studies that not only our group, but other groups around the world
are doing on these spheres... on these tumor spheres,
that when they are put back into animals, those tumors look like the parent tumor,
we will be able to unravel the mysteries, the signaling,
the characteristics that these tumors have, and one day
we will be able to produce new therapeutics that will have an effect in our patients.
So, I conclude by telling you that there is
adult neurogenesis in the mammalian brain.
There is a stem cell niche, specifically the sub-ventricular zone,
which is different between the human and the rodent mammalian brain.
And that's actually a very important difference, because
throughout the years... throughout decades,
we have continued to study formation of tumors
in the rodent brain -- formation of rodent tumors in the rodent brain.
And I think that it's still a necessity to do so
to continue to unravel some of the common mechanisms
that the rodent brain may have with the human brain.
But I do also think that it is very, very important
that we continue to do human tissue research,
and that is because there are striking differences between the human and the rodent,
and we have to try to understand from both simultaneously.
They do exist. Stem cells do exist
in the human brain. We know that. We have done that ourselves.
Other groups. They exist in the sub-ventricular zone, at least in the petri dish.
They exist in the hippocampus, and that is something very, very important to consider
for us to understand and to use this capability
for future regenerative medicine.
Stem cells also do exist in human cancer,
and we are beginning to unravel the mysteries
and the ways to manipulate this system presently.
Whether neural stem cells give rise to tumors is still not known.
I think this is a working hypothesis.
What is known is of a population of cells within tumors
that behave like stem cells.
And this is actually very, very important for us to be able to understand
not only the etiology, but the potential progression of brain cancer.
I think that the future is bright,
and we will continue with the quest to find the etiology
of brain tumors, so potentially one day,
we will be able to find better ways to treat our patients.
I thank you very much. I thank all the people that have supported our work
throughout the years, including the NIH, Howard Hughes,
the Maryland State Stem Cell Research Fund.
And most importantly, I thank my group.
This is a group of young scientists that have allowed
me to learn from them and allowed me to teach them a little bit of what I know,
and together, we're trying to find hope for our patients.
I have the most beautiful job in the world.
Every day I work not only as a brain surgeon,
giving my patients hope in the operating room,
but hopefully giving them hope also outside of the operating room.
Thank you very much.