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- Hello, everyone,
my name is Rob Signer and I'm an assistant professor
at UC San Diego in the Division of Regenerative Medicine.
And I'd like to start out today just by thanking CIRM
for everything that they've done over the past 16 years
or so,
and to thank the organizers of this meeting
and for inviting me and giving me an opportunity
to speak today.
So my role here today is to tell a story of how CIRM success
isn't only quantified by how quickly and effectively
it has accelerated the development of stem cell treatments
to patients with unmet clinical needs,
but also how it's grown and shaped the field
of Regenerative Medicine by investing in trainees.
As you all know, following the passage of Prop 71 in 2004,
and the subsequent establishment of CIRM.
A first round of funding was issued in 2006,
and included a series of training grants
that provided funding to pre doctoral students,
as well as postdoctoral and clinical fellows.
And as a third year graduate student at the time at UCLA.
I was a proud recipient of CIRM pre doctoral fellowship
during this inaugural round of funding.
The funding I received from CIRM
was instrumental in my career development.
It helped propel me through my PhD,
and helped me land a postdoctoral position
in stem cell biology in Sean Morrison's lab,
which ultimately led me back to California,
where I established my own stem cell lab at UC San Diego.
Now, the reason I included these pictures on this slide
wasn't just to embarrass myself,
but because it reflects a central focus of research
throughout my career.
Aging.
So what I'll share with you today
is how that investment by CIRM
in a graduate student 14 years ago,
has contributed to the development of a new aspect
of basic stem cell biology, that has some exciting potential
for impacting treatments in patients with degenerative
and malignant diseases.
So the work in my laboratory focuses on how hematopoietic
or blood forming stem and progenitor cells
regulate proteostasis.
So what is proteostasis?
Well as a word proteostasis is a portmanteau
of the words protein and homeostasis.
And the idea that there is a network of biological pathways
that regulate both the content and quality of the proteome.
These pathways include physiological mechanisms
that control protein synthesis, protein folding,
protein trafficking, and protein degradation.
So why is proteostasis important?
Well, the maintenance of proteostasis is really key
to ensuring normal development,
resistance to environmental stress,
coping with infection,
and most notably healthy aging and lifespan.
So quite amazingly, genetic or environmental interventions
that enhance the capacity to maintain proteostasis
will actually extend organismal lifespan
in an evolutionarily conserved manner.
That is, if you can improve an organism's ability
to maintain protein quality,
it will actually live a longer and healthier life.
In addition, the loss of proteostasis
is considered one of the hallmarks of aging.
And the accumulation of misfolded proteins
is associated with a number of degenerative diseases.
Most notably neurodegenerative diseases.
And the reason that neurons
are thought to be particularly susceptible
to misfolded proteins, is that they're long live cells
that can no longer divide.
And it turns out, the best way to eliminate
misfolded proteins is by dilution
that occurs through cell division.
So hematopoietic stem cells are also very long lived,
and they divide very infrequently.
So a central hypothesis that drives our research
is that stem cells are particularly dependent
on the maintenance of proteostasis.
And that disruptions in proteostasis
contribute to stem cell dysfunction in aging.
And overall, we favor the idea that mechanisms that confer
organismal longevity, are conserved at the cellular level
to maintain healthy, long lived stem cells.
And one of the most fascinating things
that I hope you take away from this talk today
is that many of these mechanisms,
which are highly conserved and essential,
actually function differently in stem cells
than they do in restricted progenitors
or other types of cells.
So my foray into this arena really began
during my postdoctoral fellowship,
where I adapted new technology
that allowed us to quantify rates of protein synthesis
within single cells in vivo.
And when we applied this to the hematopoietic system,
we found a couple of striking things.
The first was contrary to what most people thought
that protein synthesis is a housekeeping function
that's performed similarly by most cells,
turns out not to be true,
and that different cell types
really establish very different rates of protein synthesis
that are each maintained at their own homeostatic level.
The other striking thing that we observed
is that hematopoietic stem cells,
have unusually low rates of protein synthesis,
much lower than many of these restricted progenitors
and differentiated cells.
And subsequent to our work,
low protein synthesis has now been shown to be a conserved
feature of stem cells present as far as I know
in every type of stem cell that people have looked at
within adult tissues.
So low protein synthesis isn't just a feature of stem cells,
but it's actually really important for their function.
And the way that we figured this out,
was by using a set of genetic mouse models.
The first is where we used a mutation in a ribosomal gene
RPL 24, that reduced protein synthesis by between 30
and 40% in stem cells.
We also use the mouse where we conditionally deleted
the tumor suppressor Pten,
which is a negative regulator of the PI three kinase
and mTOR signaling pathways,
which are known to promote protein synthesis.
And then we cross these two mutations together
and found that they can mutually rescue each other.
So the ribosomal mutation prevents the increase in protein
synthesis that we see with the Pten deletion.
And the Pten deletion partially rescues the reduction
in protein synthesis that we see with the RPL 24 mutation.
And when we tested stem cell function within these mice,
what we found was that even these small changes in protein
synthesis had dramatic effects on stem cell function.
So the way we measure stem cell function
is by measuring their ability to reconstitute
the blood forming system of an irradiated mouse.
And you can see when we transplant just 10 stem cells,
the level of reconstitution we get from wild type cells
in the black line.
The ribosomal mutant with reduced protein synthesis
impairs the regenerator function of stem cells.
The Pten deletion, which increases protein synthesis
leads to a total loss of stem cell function.
But incredibly, when we put these two mutations together,
they mutually rescue each other.
And you can see that the level of stem cell activity
that we get is comparable to the wild type.
And so when I started my lab,
we really wanted to understand why these changes in protein
synthesis were so detrimental for stem cells.
And we went back to considering protein homeostasis,
and that we had the idea that increased protein synthesis
might increase the generation of misfolded,
or unfolded proteins.
And so we needed a way to figure out how to actually measure
these things within small numbers of hematopoietic
stem cells.
And so we did this using two different approaches.
The first is where we measured levels
of ubiquitylated protein.
So ubiquitylated protein are proteins that are targeted
for degradation and consist mostly of misfolded proteins.
And you can see here when we compare the level
of ubiquitylated, or misfolded proteins in stem cells,
they have much fewer of these as compared to restricted
progenitors.
We then also adapted a cell permeable dye
that allowed us to quantify unfolded proteins in the cells.
And similarly, the stem cells have fewer unfolded proteins
than these restricted progenitors.
Importantly, this was directly tied
to the rate of protein synthesis.
So going back to our genetic mouse models,
we found that in our Pten deficient mice
that have increased protein synthesis,
well, they have increased misfolded and unfolded proteins.
And when we block this with the ribosomal mutation,
we block the increase in protein synthesis,
we also block the accumulation of these misfolded
and unfolded proteins.
And so we really wanted to understand what happens
in a stem cell when you accumulate misfolded proteins.
And I'm gonna tell you one of those small stories right now.
And that is that you can activate a stress response pathway
of which there are several in the stem cells.
And we focused on this heat shock response,
which is actually the central pathway
that regulates cytoplasmic proteostasis within cells.
So, under normal conditions, the heat shock pathway
is relatively inactive
because it's regulated by a master regulator transcription
factor called heat shock factor one or HSF 1.
And under steady state conditions HSF 1
is sequestered in the cytoplasm,
where it binds to chaperones
that assist with protein folding, things like HSP 90
or trick.
Now under conditions of protein stress,
these chaperones will actually bind unfolded proteins
and allow HSF 1 to translocate to the nucleus,
where it functions as a transcription factor
to drive the expression of things like heat shock proteins
that can help to restore proteostasis.
So in stem cells,
where there are very few misfolded proteins,
we actually don't really see activation of HSF 1.
So when we look in the nucleus of young adult
hematopoietic stem cells, HSF 1 is basically absent.
But when we start to look in aging stem cells,
we can see HSF 1 begin to accumulate within the nucleus
of the older hematopoietic stem cells.
And this is telling us that aging stem cells
are starting to experience protein stress while they age.
And surprisingly, we found that one of the functions
of HSF 1 was to actually suppress increases in protein
synthesis and aging stem cells.
You can see that here that when we delete HSF 1,
the older stem cells show about a 50% increase
in the rate of protein synthesis.
So is HSF 1 actually important for stem cell function?
Well, when we look at young adult stem cells,
remember, these cells have very few misfolded proteins,
and they don't show activation of HSF 1,
we can conditionally delete HSF 1 from our stem cells,
and see that there's no effect on stem cell
regenerative activity, either in a primary transplant
or a secondary transplant.
But if we aged these mice out,
when HSF 1 becomes activated,
now we can see that HSF 1 is in fact important
to maintain the regenerative function of aging stem cells.
Now, in addition to this role in aging,
we found an important role for HSF 1 elsewhere
that we think has important therapeutic relevance
as it comes to stem cells and stem cell transplantation.
So one of the biggest limitations
in the hematopoietic stem cell field for many years
is that we've been unable to effectively culture these cells
outside of the body.
And we really don't have a good explanation as to why.
And one of the things that we found,
is that a pathway that gets particularly disrupted
when we put these stem cells into culture
is protein synthesis.
So you can see that within four hours in culture,
hematopoietic stem cells have about a 700% increase
in protein synthesis.
And within 18 hours, that's about a 2,000% increase.
Which if you remember, just a 30% increase in vivo
is sufficient to impair stem cells.
In consistent with what we saw in aging stem cells,
our stem cells in culture also begin to start accumulating
HSF 1 within their nucleus.
So what we did was to test if HSF 1 is actually important
for maintaining stem cells in culture.
And we did this by culturing wild type, or HSF 1
deleted stem cells for 10 days.
And the way that we do this is to plate just 10
purified stem cells,
culture them for a period of 10 days,
and then transplant them with wild type bone marrow
into irradiated mice.
And you can see here that HSF 1 deficiency
doesn't impair proliferation of cells and culture.
But when we transplant those cells,
we see that the HSF 1 deficient cells
have a severe reduction in their reconstituting ability.
Telling us that HSF 1 activation
is actually promoting stem cell maintenance in culture.
And so now that we knew that HSF 1 is getting activated
and is important for stem cell maintenance,
we wondered if we could further enhance the activation
of HSF 1 to support stem cell growth and culture.
And the way that we tried to do this,
was using small molecule inhibitors of HSP 90 or trick
which remember, these bind to HSF 1,
and sequester it in the cytoplasm.
And we show here that within cultured
hematopoietic stem cells,
that addition of these inhibitors
does indeed lead to a significant enhancement of HSF 1
present within the nucleus of these cultured stem cells.
And so we did the key experiment
where we culture purified stem cells
in the presence of either our vehicle
or either the HSP 90 inhibitor or the trick inhibitor,
and then we perform serial transplantation experiments.
And you can see in primary transplants,
the addition of an HSP 90 or trick inhibitor
leads to about a two fold increase
in the regenerative activity of the stem cells.
This is not actually due to stem cell expansion,
but we think better maintenance of the stem cells.
And the really big difference
comes upon secondary transplant
where we now see a three fold
or greater increase in the reconstituting activity
of these previously cultured stem cells.
Indicating to us that the HSP 90 inhibitor
or trick inhibitor are really enhancing
the maintenance of stem cell fitness in culture.
Finally, I'll show you that the effect
of these small molecules is in fact due to HSF 1
activation.
So if we repeat all of these experiments, using HSF 1
deficient cells, now all of a sudden,
the HSP 90 inhibitor no longer has any effect
on stem cell regenerative activity.
And the same is true for the trick inhibitor.
And the last piece of data that I'll show you today
is that many of these mechanisms that we've observed
initially with mouse stem cells
appear to be conserved with human stem cells.
So when we culture human cord blood derived hematopoietic
stem and progenitor cells,
we see that they also exhibit a rapid and massive increase
in protein synthesis and they activate HSF 1
And I can tell you also that these small molecules
have the same effect on human stem cells
to enhance HSF 1 activation.
And we're excited to continue studying this
in the context of human cells.
So with that, let me just thank all the people
that did this work.
And in particular, leading our protein quality study
was Lorena Hidalgo, a former postdoc,
and leading our HSF 1 study is a former postdoc,
Miriama Kruta.
And thanks to everyone, all our collaborators,
all our funders and to CIRM for everything
that they've done.
And thank you all for your attention.
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