[MUSIC PLAYING]
We now start the panel
talks, four short talks
from Stanford Medicine.
The first speaker
is Hiro Nakauchi.
Hiro is a pioneer and
a leader in the field
of hematopoietic stem cells
and, as you will hear,
also tissue stem
cells, including liver.
He was the director of the
Stem Cell Regenerative Medicine
Institute at Tokyo
University, and we
have been very fortunate to have
him back at Stanford this year.
So he's now a member
and a professor
at Stanford Medicine at the
Institute for Stem Cell Biology
and Regenerative Medicine.
Hiro, thank you for being here.
So I start with some
background slides.
As you know,
hematopoietic stem cells
reside in a very special
bone marrow microenvironment
called stem cell niche.
And their fate, such as
self-renewal, differentiation,
hibernation, or
proliferation, are regulated
by this special environment
called hematopoietic
stem cell niche.
So it's very important
to understand
what are the components
of this niche
and what is the mechanism
to regulate HSC.
So I've been interested
in this hematopoietic
[INAUDIBLE] for a
number of years.
Several years ago, we
encountered a very interesting
paper published in
Science in 1946,
so it's almost 70 years ago.
The first author was Arthur
Kornberg, Professor Emeritus
and Nobel Prize Laureate.
And what he found
in this paper was
that when the rats were fed
with a low-protein diet,
they developed very
severe granulocyte anemia.
When he gives a mixture
of purified amino acid,
that corrected this disease.
So the first demonstration of
relations between amino acids
and hematopoiesis.
And since then, no paper
has been published.
And luckily to us, also
Kornberg didn't go any farther.
I think he was more interested
in folic acids and DNA
synthesis, which led to his
Nobel Prize later years.
So we decided to study this
amino acid, amino acids as one
of environmental niche factors
crucial for the function
of HSCs.
The first experiment we did
was an in vitro experiment.
We cultured highly
enriched stem cells first,
with complete media containing
all the 20 amino acids,
and also 20
different media, each
of which depleted
one amino acid, so
single amino acid deficient
media, 20 of them.
And also the medium
without any amino acids.
After one week of
culture in the presence
of SCF TPO and the
serum three condition,
we counted the cell
numbers in each one.
So this is the result, which
was quite surprising to us.
Actually, in two worlds we
did see severely retarded cell
growth.
One was depletion
of amino acid X,
and the other was amino acid
Y. Because these are all
new and unpublished
data, I am not
able to disclose what
they are at this moment.
Sorry about that.
But anyway, this is
quite surprising.
So we went on to see
its effect in vivo.
So I put C57BL/6 mice on
either amino acid X-depleted
or amino acid Y-depleted
diet for four weeks.
And we examined the
bone marrow cells
for the frequency of
hematopoietic stem cells.
And as you can see here,
we didn't see much changes
with the diet depleted
with amino acid Y.
But with amino acid X, there
was significant decrease
in the hematopoietic
stem cell number.
And on the right hand side,
you can see, almost as early
as one week after initiation
of this special diet,
we did see a decline
in HSC numbers.
This slide shows bone
marrow morphology.
And only the mouse bone marrow
from the X-depleted diet
showed a significantly decreased
cellularity in bone marrow.
The influence of
this depletion of X
is also found in
lymphoid organs.
For example, in the spleen
in the right hand corner,
we could observe destruction
of the lymphoid follicle
structure, and
spleen is very small.
And thymus was
almost undetectable.
So it is very significant
in vivo effect as well
just four weeks of depletion
of amino acid X from the diet.
To further validate
these findings,
we performed competitive
repopulation assay.
So here we put B6 mice
on X-depleted diet
for three weeks, and then
we transplanted a whole bone
marrow cell into the lethally
irradiated congenital mice.
Along with the control, equal
number of control whole bone
marrow there.
And three months later, we
used these genetic markers
to discriminate competitor
and donor cells,
to calculate the
contribution of these cells.
And this is the results.
Again, up to two months post
transplant, both populations,
both bone marrow cells
contributed almost equally
for the reconstitution.
But at three months
post transplant,
we could see a very
sharp, dramatic decrease
in the contribution.
So these data indicate
that influence
of this X-depletion
diet is rather
restricted to long-term
HSCs but not so much
on progenitor populations.
This, I think, is a very
important observation.
So I have to explain
that HSC niche is also
critical for successful
hematopoietic stem-cell
transplantation.
As you know, we have to do
myeloablative pretreatment
to vacate, to
amputate HSC niche.
But this myeloablative
treatment,
such as high dose irradiation
or high dose chemotherapy,
it's a major complication
of this treatment,
and this limits the
indication of this therapy.
So I've been looking
for a way to avoid
this myeloablative
preconditioning regimen.
So if you think about the
fact that dietary depletion
of amino acid X reduces
the number of HSCs,
we thought that it might be able
to replace the myeloablation
in the setting of the
hematopoietic stem-cell
transplantation.
So we designed a mouse
model of allogeneic HSCT
to immunodeficient
patients, because they
are the patients who really
needs nonablative HSCT.
So we put now the
SCID mice, they
are the immunodeficient
mice, on the X-depleted diet
only for two weeks.
And then we transplanted
allogeneic stem progenitor
cells into this SCID mice.
And then we follow them
after switching the complete,
through the complete diet.
As expected, we could
see long-term engraftment
by the donor cells.
As you can see, essentially
100% survived recipient,
so engraftment was relatively
high donor chimerism,
whereas the control mice
recipients didn't show
any engraftment, as expected.
And I'm not showing the data,
but all the lymphoid organs
also repopulated very nicely
after the transplantation,
but without any myeloablation.
So lastly and most
importantly, we
tried to see if
the same amino acid
X influence on the human HSCs.
So first we prepared
humanized mice
by injecting cord
blood [INAUDIBLE]
into the NSG
immunodeficient mice.
And after confirming
the engraftment,
we put them on two weeks
of X-depleted diet,
and then we analyzed preferred
blood mononuclear cells.
And on the left hand
side, the [INAUDIBLE] plot
show that actually
there were not
much difference in the human
to mouse ratio proportionally,
but on the left hand
side we observed
a significant reduction in
white blood cell numbers.
So we were able to recapitulate
the observation made by Arthur
Kornberg almost 70 years
ago just by depleting
single amino acids.
So in conclusion, so
amino acid X and Y
are critically important for
proliferation survival of HSCs.
And dietary restriction
of amino acid X
alone enabled non-myeloablative
HSCT in a mouse model.
And human stem progenitor
cells are also dependent
on amino acid X for are
proliferation and survival.
So the future direction
is to elucidate
the mechanism of amino acid
X dependency unique to HSCs.
And also search for
a similar amino acid
dependency in leukemic or
solid tumor stem cells.
So the work I presented today is
done mostly in my lap in Tokyo
by the two students of mine,
Yuki Taya and Satoshi Yamazaki.
So I stop here.
Thank you for your attention.
