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- Thank you so much for this opportunity
to share some of our work on lung stem cells
and how we're using them to study the effects
of cigarette smoking on COVID-19.
I'm a physician scientist at UCLA
and my lab has been studying lung repair
and regeneration from lung stem cells for many years.
But when the COVID-19 pandemic hit in March,
we decided to repurpose a lot of our lung stem cell models
to study the virus.
And so today, I'm really happy to share with you
some of the work that we've been doing
on looking at the direct effects
of cigarette smoke on COVID-19.
So, the lungs are complex and really vital organs
and I wanted to tell you a little bit about their structure
and function and why some of the cells are so important
in the airway.
The upper part of the airway,
which goes all the way from the nose,
down through the trachea and bronchi,
is directly in contact with the environment
and so it plays a very important role in defending the body.
And this is through this process of mucociliary clearance,
that we'll talk a lot more about today.
The other part of the lungs
that's important to note,
is the very lowest part of the lungs,
which are these air sacs.
And this is where gas exchange occurs,
where oxygen enters our body
and carbon dioxide leaves our body.
And it's important to also note that the SARS-COV-2 virus
that causes COVID-19, can be involved
and can affect any part of the airway,
all the way from the nose, down to the distal air sacs.
And we're studying all of these different regions
in the lab in different ways,
but today, what I wanna share with you,
is some of the work that we've been doing
on these upper airways here.
So, mucociliary clearance is really vital
in defending the body.
And the way that this works,
is that we have this very highly-specialized lining
of our upper airways.
And one of the specialized cells is called a goblet cell
or a mucus-producing cell that makes mucus
that we're all familiar with.
And this mucus is very important because it traps viruses,
bacteria, pollution, any kinds of toxins
that we may be breathing in.
And then there are these other highly-specialized cells
called ciliated cells,
which have these tiny hair-like projections on the top.
And these hair-like cells beat unidirectionally
and they beat to allow this mucus to be moved up
and out of the body,
where it can be coughed up or swallowed.
And in that way,
the effects of these harmful particles is abrogated.
And so, there's one other really important cell type
within this lining layer of the airways,
which is called a basal cell.
And this is really a true adult stem cell in a way,
because it's capable of replicating
and making more of itself.
And it's also capable of making more cells,
these differentiated cells,
including these goblet mucous cells
and these ciliated cells.
And the way that this works, is shown in this movie,
where these cilia beat unidirectionally
and they're moving these particles up
and out of the body
and in that way, preventing infection
or other kinds of harmful things to happen.
So, we're fortunate that we have had, for many years now,
a really good system
where we can actually grow these airway cells in the lab
and we can have all of these features
of this mucociliary airway.
And again, I'm showing you a schematic here
with these basal stem cells, the goblet mucous cells,
the ciliated cells and club cells,
which also contribute to mucus.
And I'm showing you where they look in the actual tissue,
this is actual tissue from a patient
showing you what the lining epithelium
of these upper airways looks like.
And we're able, in the lab, to get these patient samples
and we are able to digest
and strip away this upper lining of cells
and then we can digest this upper lining into single cells.
And when we do that,
all of these differentiated cells will die,
but these airway basal stem cells will persist.
And then we're able to put them into this culture system,
which essentially is what we call
a transwell culture system.
And that just means that we put these stem cells
onto this membrane, which has got very, very tiny pores
that will allow all the nutrients to come through,
but won't allow the cells to crawl out.
And then we grow them in this liquid, as I said,
which has got all these nutrients,
which is called media.
And these are primary human bronchial epithelium cells,
or HBECs that are grown on this transwell membrane.
And over a period of about five days,
they divide, they proliferate
and eventually they form this very, very tight layer
of cells on the bottom here, resting on the transwell.
And then what we're able to do at that point,
is to actually remove the liquid media
from the surface of the cultures
and just have the media in the bottom of the cultures
and in this way, we create this air-liquid interface
and this provides the cue to allow these stem cells to go on
and differentiate and make these mucus cells
and ciliated cells.
So, this is a lovely system for us to study a large number
of different processes in the lab.
And the way that this looks,
is that we can actually go in and use fluorescent markers
to stain particular kinds of cell types.
So here in green,
I'm showing you the ciliated cells
and then in red are the mucus cells
and then in white, are the stem cells,
these airway basal stem cells.
And I want to mention that this work is being done
by a really talented group of scientists in my lab,
Arunima Purkayastha, Chandani Sen and Abdo Durra.
And so, what we are able to do now,
is to take these air-liquid interface cultures
and we can expose them directly to cigarette smoke.
And we have this very cool setup in the lab,
which is really this airtight chamber
and we put the cells inside the chamber,
inside their culture plate
and then we seal everything off,
we light one of these research-grade cigarettes
and then through a series of vacuum pumps,
we're able to draw the cigarette smoke
into the chamber, directly exposing the cells.
And then we have a separate vacuum pump
that will then draw the cigarette smoke out of the chamber.
And in this way, we're able to replicate
what smoking really looks like
when people puff on a cigarette.
And so, this is the schematic of how we see the stem cells.
And then once they are confluent
and the cultures look good,
we take them to the air-liquid interface.
We allow the mucus and ciliated cells
to differentiate over about 19 days
and then we expose, once a day,
to smoking for about three minutes, for four days.
And then on the day after the smoking exposures,
we add the live virus
and this is through a collaboration
with Vaithi Aumugaswami here at UCLA.
After three days, he deactivates the virus.
And of course, all of this viral infection
is done in our high-containment BSL-3 conditions,
so under very, very strict conditions.
And then after the virus is deactivated,
we're able to get these cultures back
so that we can analyze them
and look at the effects of smoking on the airway.
And so, this is an example of what the cultures look like.
And here I'm showing you in green,
the SARS COV-2 virus
and in blue, is the DAPI which is staining the nuclei
of the cells.
And the first culture here on the left,
is showing you where there's no cigarette smoke
and just the virus.
And I hope you can appreciate, in green,
you can see a cluster of cells that's been infected
by the virus.
And then on the right panel here,
I'm showing you where cigarette smoke exposure was done
before viral exposure.
And we've done this many times and we consistently see
that there's many more clusters of cells
and the clusters tend to be larger.
So, we see a lot more infected cells,
if the cultures are exposed first to cigarette smoke.
And this is also shown here on the right,
with the viral load PCR reaction,
which was done by Gustavo Garcia.
And what we appreciate,
is that there's a lot more virus around
when there is cigarette smoke exposure first.
And so, we then decided to look
at the different kinds of cell types
with each of these different exposure conditions,
to get a sense of what the virus is doing
to the different cell types
and what cigarette smoke is doing
to the different cell types.
And so, on the left-most panel,
we have here, no cigarette smoke, no virus
and this is this is just a baseline
in the air-liquid interface cultures,
what the ciliated cells look like
and they're shown here in white.
And then, you can appreciate, I'm sure,
that when we add cigarette smoke, but no virus,
we get a loss of these ciliated cells.
But what was really interesting to us,
is that in the setting of viral infection,
with no cigarette smoke exposure,
we don't see a loss of ciliated cells,
the virus doesn't seem to be harming the ciliated cells
and they are still present.
And then, in the setting where there's cigarette smoke
and virus, we see a loss of ciliated cells,
but this wasn't statistically significant.
Now, when we have the situation
of looking at the mucus cells,
this is the baseline number of mucus cells
that we typically see when there's no cigarette smoke
and no virus.
Here, when we see cigarette smoke,
we see a lot of induction of mucus and of course,
this is probably no surprise to anybody
'cause we know that cigarette smoke will induce mucus.
In the setting of no cigarette smoke with virus,
we see that there's really not a large increase
in the number of mucus cells,
maybe a slight increase,
but not statistically significant.
And again, we did not see
a statistically significant increase
when there was cigarette smoke and virus.
So, given these interesting effects of the virus
on the different cell types,
we decided to look at the basal stem cells.
And we look at this in two ways,
one with a marker of stem cells,
which is KRT-5 or keratin five.
And here, we see that with cigarette smoke,
there's actually a large increase
in the number of basal stem cells
in the ALI cultures.
And this is because the cigarette smoke is perceived
by the cells as an injury
and so they respond to injury,
by dividing and making more stem cells.
However, the virus alone, without cigarette smoke,
does not induce that same proliferation of the stem cells.
And we see a little bit more proliferation
with cigarette smoke,
but not a lot in the setting of viral infection.
And then we're also looking, in another way,
at the stem cells by looking at the dividing
and proliferating stem cells,
which is shown here by these pink dots in the nucleus.
And here, we see that with cigarette smoke,
we see a lot more of these dividing cells.
But in viral infection,
we don't see that the stem cells are dividing.
And in the setting of cigarette smoke,
plus the virus, we saw sort of an in-between phenotype.
And so, the virus seems to be doing something
very interesting in these air-liquid interface cultures.
And then there doesn't seem to be much
in the way of an injury being perceived
and so we decided to look at the single-cell level
by using single-cell RNA sequencing,
at exactly what might be going on within the cultures.
And so, this is work done in collaboration
with Katherine Plath and Justin Langerman.
And what we noted in these red boxes,
is that the number of viral transcripts,
the gene expression within the cells,
is markedly reduced when virus is introduced
into the cultures,
whereas smoking seems to have very little effect
on the number of transcripts.
And looking at this in a different way,
we looked at these heat maps
and really what these do,
is show us patterns of gene expression
across these four different groups.
and whether gene expression goes up or down.
And the prevailing pattern that we saw,
was that in the setting of virus infection,
either with or without cigarette smoke,
it looked very similar,
but that we see that most of the genes,
are down-regulated in expression,
whether there's no cigarette smoke,
or there is cigarette smoke.
Although, of course, other patents exist, for example,
we do see that some genes are increased
in the setting of viral infection.
And this particular group over here
was very interesting to us.
This is genes that are increased in expression
in the setting of just viral infection,
but are decreased
when there is viral infection with smoking.
And what was really interesting to us,
is that in this particular group,
we found that the interferon response genes,
which are very important in immune function
within the airway epithelium,
that they were decreased in the setting of cigarette smoke.
And this imply to us that cigarette smoke
reduces the interferon response.
and therefore, this may be at least one of the mechanisms
whereby the virus is able to get into the cells
and why we see so many more infected cells
in the setting of cigarette smoke.
And so to look at this further,
we performed another experiment.
And this time, what we did,
is to think about adding interferon beta to the cultures,
in comparison to remdesivir, which is used clinically
and has a response in patients,
to see whether interferon beta might have an effect
on the cultures,
especially in the setting of cigarette smoke exposure.
And so here's the data
and I hope you can appreciate this first column is no drug,
I'm just showing you the viral infection
and how much more there is with cigarette smoke.
With interferon beta-1,
we found a complete abrogation of infection,
so we completely prevented infection,
even in the setting of cigarette smoke.
And with remdesivir, we saw a good effect as well,
although there was slightly more infection
than what we saw with interferon beta
and this is quantified by these graphs.
So in summary, what I've shown today,
is that we really believe that cigarette smoke
has a large impact on the airways
and that at least acute cigarette smoke exposure,
makes people more at risk
for having more virally infected cells
and therefore, we believe more severe viral infection.
And so, I really want to acknowledge my wonderful lab,
who despite the pandemic, have worked really hard
to generate what we think is very important data.
And I also want to acknowledge Vaithi Aumugaswami,
without whom we would not be able to do the viral infections
and Gus Garcia, who's a very talented technician in his lab,
has also been instrumental in all of this work.
And I want to also want to acknowledge,
Catherine Plath, Justin Langerman
and the UCLA molecular shared screening resource
and Robert Damoiseaux for all their work.
And I also want to thank CIRM very much for their funding,
because without this funding,
we would really not be able to do any of this COVID-19 work.
And we're hoping that this will lead
to greater understanding of the disease
and also ultimately, hopefully, to a therapy.
Thank you so much for your attention
and I'm happy to take any questions.
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