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- Hello, my name is Aileen Anderson.
I'm really pleased to be here today to be able to talk
about some of the work that we've been doing in my lab
over the last eight or 10 years of funding through CIRM.
I wanna just thank CIRM for providing the funding
to support this meeting, and also all of the folks,
the boots on the ground,
and that had participated in organizing.
It's an enormous effort to put together
a virtual conference like this in the middle of a pandemic.
And so a real shout out there.
I think one thing that's quite critical in that regard
is conveying this kind of information,
both to our colleagues at conferences like this one,
and others that are more scientific is critical,
but also having this conversation with the public
is something that's really important
about the grant holders meeting.
And so I hope that I'm able to convey that line
in this talk.
So speaking both to the public and conveying
some useful information, and my colleagues
have to bear with me a little bit,
if I'm a bit less technical and less detailed
than you might find in a normal talk.
So the title of my talk today is
Human Neural Stem Cell Efficacy and Repair,
but actually I kinda struggled with this.
What I really wanted to call it is, The Way Stem Cells Work.
Why, because this is a great book.
If none of you have read it, it talks about the background
of the physics and the design parameters
of how things around us in everyday life,
and, you know, even going back to the middle ages, function,
and that that's really important.
So if we were going to try and build a mousetrap,
or a trebuchet, we wouldn't just put all the pieces
next to one another and hope that they would self-assemble.
And we can't do that with stem cells either.
We need to understand some very basic aspects
of these cells, which were really discovered very recently.
And this is a field that has launched forward incredibly
quickly over the last 15 years,
but we have to remember that we need to be able
to understand the basic biology in order to move forward.
So in that context, with that background very early on,
when I was starting out my lab
at the University of California, Irvine,
I worked on spinal cord injury, and trying to understand
mechanisms of damage, and how we could get to repair..
And we had the opportunity to work with
human Neural Stem Cell transplantation to see
if we could produce repair after injury,
by transplanting these cells.
So in our animal models, in fact,
we found this to be awfully successful.
So shown in the panel to one side, is vehicle,
where we have an animal that has not received any cells,
and she's not capable of doing,
stepping really with her hind limbs, only very occasionally.
On the other side, though, the animal that received
human neural cell transplant is consistently
able to move her hind limbs.
She's stepping very well, she's moving with coordination.
It's a large difference in terms of functional performance
between those two animals.
Very exciting from our initial studies.
In fact, that work was so exciting
that over the next eight years or so from 2002 to 2010,
we worked and did a lot of preclinical work
with our collaborators to try and enable that
to go forward into testing in a human clinical trial,
and that happened.
In 2010 this study was launched
with Armin Curt and Mike Fehlings
as the principle investigators.
And in fact, what it found was that transplantation
of human Neural Stem Cells in chronic
thoracic spinal cord injury.
So individuals that were not just injured,
but really the most challenging thing
that we can think about doing for people
that might live with a lifetime of disability.
And there were some preliminary efficacy measures,
mainly in terms of sensation,
which is what shown in the diagram panel.
But in fact, this was sufficient that even after
a six-year retrospective, which was just published
from Dr. Curt and his group,
the conclusion was that spinal cord injury,
and the damaged spinal cord may still be a suitable target
for stem cell transplantation.
That preliminary efficacy measures held up,
and that there's promise here in terms of moving forward
to the clinic.
But what we know from other work in my lab
is this isn't always the case, it's not uniform.
In fact, in my lab using preclinical studies
with the different cell line,
we found that those sometimes fail, and we don't know why.
That really is the essence of what
I wanna talk about today, trying to understand,
and the need to understand how the interplay
of basic science and knowing how stem cells work
interacts with our facility,
our ability to go forward with an effective
sub stem cell population into the clinic.
So understanding why stem cells behave in specific ways
is important, at both the basic science level,
and in terms of clinical translation.
So before we go on with that, and we need to know
a little bit in terms of terms and basic science terms.
All stem cells occupy a place in the environment,
and in Stem cell biology, we call this a niche.
So in this diagram, we show a basic niche.
That's good enough for our purposes.
The idea is that it's a place in three dimensional space
where a cell sits and basic stem cell
that's sitting within it.
So an important concept to understand from that analogy
is that niche can be different,
it can vary, or it can change over time.
So if we look at different types of niches,
you can get the idea.
There might be different components within them,
in terms of the basic biology of how cells work,
this would be different molecules,
different parts of infrastructure,
so different types of substrates that the cells
could come into contact with.
And that a niche could be subject to different states
of either damage, or repair, or being built.
And those all present a different environment,
and different signals that are conveyed to cells
that are gonna affect how they behave.
By that same token no two cell lines are identical.
Cells themselves have properties that define them,
even if they're in the same category of cells.
So we work with human Neural Stem Cells in my lab,
but one line and another line can't be assumed
to be the same, even if they're in the same category,
they're going to be intrinsic variation between them.
And so let's introduce those two terms.
There are extrinsic factors that are a part of the niche.
The niche itself, and how different it might be,
the state of repair, or damage, or regeneration
that it's in, and intrinsic properties
that go with the cells themselves.
Those extrinsic and intrinsic signals
control stem cell behavior.
And those are really important aspects
of some key stem cell behaviors, including maintenance,
and self-renewal, whether those cells,
the stem cells themselves are able to make more stem cells,
and maintain themselves over time,
whether they survive in their normal niche,
or after we transplant them.
Lineage restriction and cell differentiation and fates.
In other words, what those cells can become
and whether it's productive potentially in terms of repair,
so in the case of a neural stem cell,
the three things a neural stem cell can become,
is a neuron, an oligodendrocyte, or an astrocyte,
and cues around it, and the intrinsic properties
of the cells are gonna determine what proportion
of those three different lineages are generated,
and also the migration of the cells.
In some cases, certainly in the central nervous system,
we may need a therapeutic Stem cell population
to be capable of migrating quite a long distance.
And if it's not,
if it's not getting the right extrinsic signals,
or if it doesn't have the proper intrinsic programming
and state to receive those signals,
it may not be capable of migration.
And then we may end up with a cell that fails
in the context of repair.
So an example of this is shown in this slide.
So there's three panels that say,
zero days post-injury, nine days post-injury,
30 days post-injury, one on top of one another.
This is all the same cell line transplanted
into the same spinal cord injury model
into the same location in the spinal cord.
But at different times after spinal cord injury.
Everything in those panels,
that's brown is a human Neural Stem Cell.
And if you look at where the brown is distributed,
you can see pretty obviously that it's not the same.
It doesn't take an educated eye to view that
in the very top panel where it says
zero days post-injury, that's acute.
The cells that we transplanted are migrating.
They're moving in towards the injury epicenter
at the very center.
And they form a dense plexus of cells that is behaving
very differently than the cells that we see
in the lower panels.
And the only thing that's different because the donor cells
are the same is the niche that they're seeing
because of timing.
So there are altered extrinsic signals
that are in that niche, and those are affecting
the behavior of the cells.
And in fact, that has consequences for whether or not
we see repair enabled by those cells.
In the top panel, the cells failed to guild repair
in the bottom two where we're transplanting
at a delayed time point.
Now those cells are successful.
Why, so understanding those extrinsic cues
becomes really important.
I'm gonna thin slice through a lot of data
from my lab over the years,
where we've tried to focus on understanding
the difference between those environments.
One thing that we knew and became clear early on
is that there's a tremendous amount of inflammation
that's influencing the central nervous system
after a traumatic injury, and that is changing over time.
And so we went on to investigate a number
of the molecules that are involved in that.
And one of them that we became really interested in
was called C1q.
So without giving a lot of detail about C1q,
I just wanna highlight a take-home that we found,
and just was published about a week ago,
in fact in Elife, showing that C1q is an immune molecule.
In that early acute niche,
where cells might fail is the extrinsic signal
that is dictating how those stem cells behave,
and whether they're capable of repair or not.
So how do we figure that out?
Well, in science, we did something called
an unbiased forward screen to identify
transmembrane receptor proteins that could be
partners for C1q.
What that means in English is that we went
on a fishing expedition.
And really the idea here is we used C1q
as a protein, as bait.
And we looked for what things neural stem cells expressed
on their cell surface that were capable of binding it
with high affinity.
And we showed that there were five proteins
of particular interest.
I'm gonna tell you about just one of them today.
And we looked at those proteins,
showed that they could signal using C1q
to neural stem cells,
that if we disrupted that signaling,
that we would alter the behavior
of those neural stem cells in a dish.
We showed that C1q and those receptors here
has shown CD44, bound to one another at the surface
of the cell membrane.
That's what you see is the little red dots.
And then we asked the question of what happens
if we, instead of allowing those interactions to happen,
if we blocked either at the level of the receptor,
by taking it away, call the knockout, or we blocked the C1q,
the extrinsic signal that was present in the environment?
And so we wanted to test what the functional role
for these extrinsic signaling C1q to these receptors here,
CD44 would be when we tested in vivo, and not just in vitro.
So here blocking again at the level of the receptor,
we see this same type of cell behavior
where they're clustering in towards the center.
That's alleviated when we knock out the receptor,
and in fact, we convert to repair, when we look at
functional locomotor recovery.
We do the opposite experiment,
and block the extrinsic signal itself, C1q,
Here it looks the same way
that I showed previously in terms of the wild type,
but blocking the C1q,
in fact, we block that accumulation at the center
of the spinal cord,
and again, we convert to the potential for repair.
So this tells us that in a very meaningful way,
extrinsic signals can influence capacity for a stem cell
to mediate repair after transplantation,
at least in the case of spinal cord injury.
So understanding those signaling pathways tells us
that we have a new path to be able to think about
how to augment the capacity of these cells
to behave in not just an in vivo setting,
but in a clinical translation setting.
And I mentioned also the other parameter
that we're interested in, and I think is important,
are intrinsic factors that affect the ability
of human Neural Stem Cells to enable repair and recovery.
So, one question that underlies that is how different
are different human Neural Stem Cell lines.
We'll actually, while we wanted to look at this,
this turned out to be really difficult to tell,
because there are very few tissue-derived
human Neural Stem Cell lines,
or comparisons that have been made between them.
So with CIRM's support we developed
additional new lines, first to set in our lab,
and then using cGMP compatible protocols,
and cGMP techniques to enable testing,
so those would be clinically relevant going forward.
So how much variation is there?
I mentioned a number of things about extrinsic,
and intrinsic signaling that were important.
One included stem cell self-renewal or stemness,
and these are six different lines
compared in terms of their capacity,
and you can see that they're widely variant
Differentiation/fate is another factor
that we can look at.
Again, all you need to do is look at these bars
to get the sense of how different
these six different cell lines are,
and the same in terms of migration.
So is there also that same variation in terms
of what the repair capacity of these cells is
for spinal cord injury?
And so we transplanted all six lines
and controls in parallel, and the answer is yes.
In fact, out of these six lines, only two cell lines,
this one and this one, met criteria
for consistently demonstrating locomotor recovery
of function in a chronic cervical spinal cord injury model.
So what do we do with that?
Well, we can't go forward very effectively in terms
of a clinical setting.
If we don't know how the lines that we're using
are gonna behave.
So we set as our goal for this particular project
to use these lines and the new ones we developed
to make a profile that would help us to be able
to predict whether a candidate cell line
would be efficacious for a given function,
in our case, chronic spinal cord injury.
And the way we did that was to generate these lines
in parallel using the same protocols, cGMP compliant.
Grow them up again in parallel,
testing them under growth conditions,
differentiation conditions, or after in vivo transplantation
to see if they were efficacious.
Surprisingly, just comparing growth conditions
and differentiation conditions,
in terms of the gene expression profiles, we saw some cases,
which is exactly what we had hoped for
where there were just incredible switches in cell lines
that didn't work, almost no expression,
and cell lines that did work, very high expression.
Or the opposite, in cell lines that failed
on efficacy testing, very high expression.
Or in cell lines that were successful in efficacy testing,
very low expression.
And this differential gene expression actually gave us
a shockingly small number of genes,
at least under growth conditions,
and even under differentiation conditions.
But 24 is a good number to be able to work with
where we thought we could develop a profile
to be able to help us understand
and predict in advance
whether or not a given cell line would yield repair.
And so we tested this.
We applied this protocol to a new set of lines
that we developed again with CIRM funding,
and made those predictive calls
about efficacy in advance of actually doing
behavioral testing in vivo.
So these are the four lines we tested,
182, 183, UCI184, UCI191.
And here was our threshold for prediction of efficacy.
We predicted two would be positive.
In fact, those same two lines in our in vivo testing
were able to yield quite robust efficacy
in chronic cervical spinal cord injury.
Whereas the other two lines where we predicted
a negative call failed.
That, we hope, will give us some new tools moving forward
in terms of helping to understand intrinsic variation.
Because now we have a whole new set of lines to look at,
and be able to dig into that in a better way.
It also gives us potentially a tool
that we can really think about applying
from a practical perspective in terms of
monitoring cell lines,
in terms of their expansion for clinical use,
where we might be able to say with much more assurance
moving forward into clinical trial,
that there's a good chance versus
a not good chance of success.
So I hope I've convinced you that understanding extrinsic
and intrinsic control of stem cell behavior is important.
It's important, not just at the basic science level,
but because it has very real implications
in terms of our potential for success
in moving forward, ultimately, to clinical translation.
So with that, I'd like to thank a few folks,
for the DISC2 Project,
which I just described on intrinsic factors.
This was an enormous project.
I can't emphasize how much work that was
in a 30-month period of time.
And I'd like to highlight a couple of people,
in particular Katja Piltti, shown here who developed
our protocols for being able to develop
cGMP compliant lines, manage the whole project,
start to finish, and has been
a tremendous collaborator at UCI.
Anita Lakatos, who is our gene jockey,
and has dealt with everything to do with
RNA seq analysis, and really the very sophisticated methods
that are needed to develop the profile.
All the other folks who did the boots-on-the-ground work.
And my longtime collaborator here, Brian Cummings,
but Josh David, Krystal, Rebecca Nishi, Chris Nelson,
Javier Lepe, and Joseph Requejo,
and at UC Davis, Gerhard Bauer and Brian Fury,
without whom this really would not have been possible.
And then a much larger group of people who over time
have contributed to all of our Neural Stem Cell work.
And in fact, looking at the extrinsic factors
that I mentioned, I'm very grateful to them,
and it's been a wonderful experience to have them
as our team.
Last of all, our support, CIRM, NIH,
Wings for Life, and The Craig Nielson Foundation,
in terms of support for the specific work.
Thank you very much for your attention.
And I look forward to the panel discussion.
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