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stem cells impact factor

stem cells impact factor

>> well, thank you very much indeed, it's an honor to come and give this talk about the recent work going on in this my lab. this is being funded by the heart lung and blood institute. so as we've been walking around in the beautiful air looking at

the cherry blossoms and breathing the warm spripg air, i'm really happy that--spring air, i'm really happy that my lungs are in good shape but i realize that for millions of people throughout the world this is not the case. and our researchers are trying

to understand what cells are maintaining the lung and how it's--how it helps to repair them after certain kinds of injuries and what can go wrong. so the lung is a very complex system, like many it's made up of 2 cell types, epithelial tubes that branch finer, finer,

finer and end in these multiple millions of little air sacks where the gas exchange takes place. and these are highly vascularized for the gas exchange and of course there is a lot of mesodermal cell types around which contribute not only

to the vasculature but to the smooth muscle and lymphatic and cyber blasts so we've been particularly interested in the stem and progenitor cells of the epithelium and 1 of the important things is to remember there's not just 1 stem cell type for this system because the

epithelium is very different in this the different parts of the lung. down here in the alvermen infectedole i of the exchange, these thin walls are lined by type 2 cells and type 1 cells place. but up in the air ways, the air

coming in has to be hydrated, particles have to be removed and this is effected by a mouso epithelium and this is made up of mostly secreteatory cells and a few endocrine cells. so there's very different cell types and they have very different cell biologies and

physiologys. so the work from many different labs has led to an understanding of the kind of stem and progenitor cells which are in the human lung and in the mouse lung. now there's a big difference in the size, between the mouse lung

and human lung, but the basic idea is very much the same. so up in the mouse trachea and main bronch i, there's the sealium in these cells and are maintained by basal cells and like basal cells in the tissue and skin and prostate and the mammary gland are characterized

by a p63 and keratin 5 expression. and they do not--they're next close to the basal lamina but don't reach up into the lumen. and these are present in the human lung, throughout the air ways inside the lung and right down to the very smallest air

ways. so the very small air ways where a lot of disease happens as i will show you in a minute is very similar in organization to the trachea and main stem bronch i of the mouse lung. so we often use this as a model for understanding what's going

on in the small air ways of human lung. --type 2 cells and the type 1 cells. so this is just summarizing a little bit about what we know at present about these stem cell populations and i will say that it's quite a complicated story

because in steady state in the lung there's rather little cell turnover and only 1 can really see what the potential of these cell types is, is to do various injury ask infection models all of the mouse lung and then we see the cells is damaged, resident of a cells can change

their behavior and start to proliferate and each change their phenotype. so, what has been found is that these basal cells here will self-renew and overtime, very slowly give rise to the multifilliated cells and secreteatory cells by lineage

tracing but recently, a colleague at harvard showed if you have an injury model where you genetically ablat many of those basal cells, some of the differentiated secreteatory cells can change their phenotype and can go back and behave like basal cells.

they will express p63, they self-renew over a long period of time and behave as bona fide stem cells. so this was quite surprising plasticity which now has been seen in other organ systems as well. and then we showed these club

cells can self-renew over a long period of time and can give rise to multiciliated cells. but a number of labs have shown in certain models injurying the lung with chemo micein with a chemo therapy agent that can lead in lung damage in humans when it's gib for cancer

treatments. under those conditions, some are bona fide club a cells expressing markers of differentiated cells can start proliferating and will actually give rise to type 2 cells and to type 1 cells. again sort of fairly unexpected

plasticity which has now been seen in different injury models. and then not long ago we showed that type 2 cells can self-renew over a long period of time and can give rise to type 1 cells both in steady state slowly and then after partial newellennectomy when there's

regrowth and development of new alvermen infectedole i in the remaining lung but under those conditions when there's a lot of change and remodeling in the ment even some of these type 1 cells which are very flat and thin and cover a large surface area, some of them by lineage

tracing can round up and give rise to type 2 cells. again, quite an unexpected finding, rather a small population, we're doing it but it's been repeated in different labs. and then most recently of all, people had been looking at lungs

after influenza virus infection, in particular a pr8 strain of influenza virus which causes a lot of damage and a lot of inflammation and under those conditions, some cells which are still rather unpoorly characterized in these small air ways which have been called

lineage negative epithelial cells or distant air way stem cells can start proliferating and turn on p63 and keratin 5 and actually migrate out of the air ways which some strains of influenza do this, others don't but they become migratory and they give rise to what's called

keratin 5 positive pods of epithelial cells and it's still a bit controversial but under certain conditions after chapman's lab in ucsf has found that after putting these cells into ted trans--transporting them into the lung and inhibiting notch signaling, some

of them can give rise to type 2 so again very unexpected but really generating a lot of data that's quite fascinating and is pointing towards mechanisms for plasticity of epithelial cells that had been rather unexpected. so what we're trying to do, is to understand the basic biology

of these different populations and to understand the mechanisms driving both their steady state self-renewal and differentiation and thou they respond to these different models and inflammatory appearance of macrophages and cytokines and so on.

so i'm going to focus actually on these basal cells of the mouse tracheal area and how we've been translating this to studies with a primary human basal cell in the lung, so this is work of 2 post doctoral fellows. the work has now actually been

published in the work on tomomi, tadokoro, on the role of signaling in the basal cells. she did this wo, and it's now being published in development but she got her own job in the city university and is now running her lab in japan and this is what was published in

the journal of cell biology and she's still continuing this but will be on the job market soon. so what--just very briefly these basal cells as i said there in the pseudostratified mesothelium, they express p63, unlike the basal cells of the skin which is a continuous layer

and in the larger human air ways it's almost a continuous layer, these are sort of individual cells here and they express keratin 5 and some of them express keratin 14 but fortunately they express nerve growth factor receptor on the surface which has made it, we

don't quite now know they express this receptor, they don't seem to respond to culture in the neurotropic molecules but there must clearly be a biological reason, we haven't picked it up yet but it means it's easy to isolate these by facts sorting.

now i said this is a similar kind of organization in this the human lung and in the mouse there are a few submucosal glands in this region and in the human these submucosal glands again extend throughout the lung and i want to include 2 slides just showing why the relevance,

sort of studying these basal cells to human pathology because here is the normal human air way taken from a lung sample showing here we've got the p63 positive cells, the secreteatory cells and the expression of the nerve growth factor receptor so you see there are many more

aboppeddance of these basal cells in the humans air way, but in this this condition of copd, which is frequently found in heavy smokers but in people with lung pathology, there is squeamous metaplasia where these basal cells will overproliferate for multiple layers and they

will lose the affiliated and secreteatory epithelium and of course then, it can lead to obstruction of the air ways. so that's a pretty serious condition and then also in asthma, i said that in a normal air way these basal cells will differentiate into multiciliated

cells and secreteatory cells and this is a highly regulated process to keep the balance of these 2 cell types correct. but in asthma there's over production of the secretattory cells producing glucose and you can see here this is a serious condition of asthma in which

there are many of these cells packed with secreteatory materials, globlet hyper plassia, then there's another condition called bronchioliteis oblighter ans, which is almost fast--there are far fewer epithelial cells, they're not well differentiated but there

are definitely p63 positive keratin 5 positive basal cells in this region. and the possibility of this epithelium can lead to an overgrowth of the urpd lining and there's an exchange of information between the stroma of which we have revealed in

some of our injury models. so this is why we want to understand these basal cells and we want to understand the mechanism by which they give rise to a balanced proportion of ciliated and secreteatory cells. so what do we know about this mechanism of self-renewal and

differentiation. felt well, this schematic is just to summarize what is known at the moment: these cells and they have the markers but are they a homogeanious population of cells or is there heterogeneous activity among them.

some are quiescent and some which are on the way to differentiating and even though they express these markers, are somehow got a different genetic program going inside them. well, some very beautiful paper came out last year from [indiscernible] lab and she

teamed up with ben simons who is a theoretical chemist who's become interested in the population virology in the stem cells in different systems and looking at cell turnover in the normal steady state lung and looking at lineage behavior of the cells, they came to the

conclusion that actually about half of the cells which we were calling basal cells are are in fact committed progenitors, they call them committed or lumen progenitors and have got a different gene expression profile and are more likely have a higher probability of

differentiating than the other 50%: so that's quite interesting and as i will tell you at the end, i think we have identified another component of the genetic differences between the basal cells actually in the human lung which is predicting those which might start to

differentiate toward the filliated lineage. --not ohm in the lung but also in the xenipost skin and other multiciliated epithelia and this is the ciliogenesis program and this is the rfx2, and foxj1. and any of these mutated will effect the differentiation of

these multiciliated cells. fox j1 mutants for example had very short malformed cilia, they do start the program but the multisillian is at the top of this hierarchy and then others believe that [indiscernible] lab thinks that cmib might be earlier in the progenitors

giving rise to the multiciliated and then the other program is going towards the secreteatory cells forming the goblet cells or the club cells they're called in the 1s which are making these a secretat globins and we know now from work in this my lab or others that high notch signaling

promotes the differentiation of secreteatory lineages but in addition to these lineage choices this is very, very interesting cell biology which is going on here which we try how the change in the behavior of the cells is integrated with their lineage choice.

and that is because these basal cells really don't have a clear apical membrane. they're attached to the basal lamina through integrins and they don't have a clear apical domain, i will show you that we've been interested in how these cells start to

differentiate into either the multiciliated program or the secreteatory program now become polarized much more clearly and then form junctions between the cells to form a barrier. and it's this barrier which is crucially important for lung function because it's keeping

out all the microbiome that we're beginning to learn about what's in there is keeping out the viruses, keeping out the particles and if this barrier becomes leaky, it's thought that it allows infiltration of things and can then lead to inflammation and abnormal lung

function. so we become interested in how you coordinate this morph o genesis of this epithelium. now i said right early on, there's very little normal turnover, the lung is not like the gut and the skin where the stem cells are turning over and

replace the epithelium every few days. people have looked at this, turnover and it's very, very slow indeed, there's very little incorporation, edu into basal cells normally. so we are really challenged to give some kind of stimulus to

the lung to get the remodeling going and this can either be infection with bacterial or with viruses or 1 model that we've used quite a lot is to have the mice to breathe in sulfur dioxide, which is sort of mimicking exposure to toxic things in the air.

now i've been realizing there's a push to include both male and female animals in injury models or studies at nih. i have to say we do this with male mice because the females don't injury very well i've never really quite know why. i think the females all huddle

together and hide theirinoses in each other's fur. they cuddle up where the males say [sigh, ] and take brig breaths of the so2, i haven't tested that. but it dissolves in the liquid, in the acid, the exposure and the lumnacelles sluff off

leaving behind the basal cells and the then most of them seem to survive and they will start proliferating, you see here are the p63--here are nonspecific staining of the dead cell and by 24 hours most of them have incorporated brdu and start to proliferate so there's a special

population, most of them proliferate and then by 2 weeks this epithelium has been restored, the silliary are beating again and the mice go back to normal. so this has been a model we've looked at over the years and this is what we conclude is that

the surviving basal cells as i will show you, their first response is to spread. actually they change their behavior so that now they didn't have any junctions between them but i will show them that they do assemble junctions between them to act as a barrier as

quickly as possible. let then we have the undifferentiated progenitor produced and then they start to express a lineage marker like fox j1 and we get back to having the right proportion of ciliated secreteatory cells. there are also changes in the

stroma which include changes in the angiogenesis or leakiness of the blood vessels, influx of nutrifills and macrophages and changes in the stromal population of fibroblasts that are in here. now just very briefly we did have funding from the counteract

program to see what would happen if mice were exposed to chlorine gas. and in there, the response is a bit different because the basal cells are damaged. this was done in collaboration with pete koren at duke and his studies show that the base at

cells die in this condition. and then in about 50% of the mice, the stromal cells overgrow, they're not--the proliferation is not suppressed by the proliferation and behavior of the basal cells and in this case, there is stenosis of the air ways.

we think this could be an ablight rans type situation in the air cells if they're defective in being able to proliferate. so what we wanted to do was part of our general program is to find them factors that will promote the proliferation of

basal cells so that you might give this as a therapeutic agent after exposure to toxic chemicalling or just in general to say what is promoting the proliferation and what is controlling their differentiation into ciliated verses secreteatory cells.

so we set up a post doc in nigh lab this assay, before organoids it was about the same time that people were doing intestinal organoid 3 dimensional culture system and we set it up as a way of screening for molecules that would promote proliferation and sophisticatedy we took the mouse

air way, large air way, disassociated them and it's a simple fact sort based on nerve growth factor expression and they're about 90% basal cells. we feed them into mate rigel in 3 dimensions in these little inserts and then put them into a culture medium here and over the

next 2 weeks, single cells, we did a clonal analysis, single cells will give rise to a little sphere which we call bronco sphere or tracheal sphere and then--if i can show this movie, okay the single cell, mostly the cluster and eventually you form a central lumen that will pump

full of fluid and you will see a basal cells around in contact with the mate rigel and the keratin 8 positive cells inside and then they will differentiate into secreteatory cells which are making this protein called plunk and the multiciliated cells which the cilia will beat

functional and they're marked acetalated tube lynn, and something was published that tomomi did with the screen is that we isolated basal cells from mice which are transgenic for a reporter gfp driven by the fox j1 promoter. so all the ciliated cells would

be green and she could screen then for spears or for wells which had more green cells in them and thal way she identified il6, the cytokine il6 as a molecule which would promote the differentiation of secreted and this assay was used by no artis by aaron jaffe who had

been very interested in polarity of epithelial cells. they use this as a very, very high through put screen, for new drugs and drugs that would inhibit or promote the differentiation of secreteatory cells as new possible and antiasthma drugs.

so we're looking for things which promote the cilia genesis, they were looking for things that were inhibiting secreteatory differentiation, but you can see the utility of this kind of primary cell, organoid assay. so, what tamomi did was then

screen another users assay but then rather than look at affiliated verses secreteatory cell types was just to look at compounds, promote colonning forming efficiency and cell number because what you can do is you can take the spheres in 1 of these wells and you can

disassociate them and count--fact sort them again and base all those secreteatory cell number. so if you did a screen, of course there are many more molecules than this, i just put on here those that were particularly good at forming

colony efficiency or cell and top of this list was a molecule called ldm, 193189, and we will call it ldn, there's other molecules in here, there's sonic hedge hog agonist, tgf beta inhibitors but they were not as effective as the ldn which is an inhibitor of the bmp

signaling, through bmp receptors and through smads, so as many of you know of course, the bmp cononicle path bay is going through phosphorylation of smad 15 and 8 which links up the smad 4 and goes and promotes or represses certain down stream genes, and ldn was an inhibitor

of this cononicle pathway. now there's a little bit, a little bit of inhibition of the veg f pathway of this drug, so we were very fortunate to have help--i'm sorry, collaborative from vanderbilt who had made other molecules which you can now buy commercially called

dorsal morphins, and dorsal morphin is very similar to ldm and it inhibits the bmp pathway much more specifically, so some of the studies i will show you were done with dorsal morphin as but this has exactly the same effect. so what we found was that dorsal

morphin would actually promote--oh i think i left that slide out. no, it's here, can you see them here with dorsal position o morphin 1 and 2 and ldn, there were more spears and they were--spheres and they were bigger.

but we sectioned them we found they had the same proportion of silliatory and secreted cells. so unlike il6, it wasn't effecting the fate decision, it was just effecting proliferation of the cells. and then we found, if you're not on here but a bit later if if we

added bmp then we got fewer spheres and they were smaller. so then, tomomi, looked to see, was this effecting proliferation and was it effecting differentiation? and so the spheres with added bmp were much smaller and much less proliferation and in this

case, all of the cells remained p63 positive. so they stayed as stem cells and they didn't differentiate, but with added dorsal position o morphin, there was more proliferation in here, more edu, incorporation, the spheres were bigger but they had luminal

cells and they had the same proportion of ciliated and secreteatory cells and they had basal cells around the outside. so it seems that the bmp is inhibiting cell proliferation and maybe then, we're looking into this now whether it's slowing down the cell cycle and

therefore it's slowing the differentiation of these p63 positive cells, into committed progenitors. and then, the dorsal morphin is speeding up proliferation. so now what happens in the injury model, this was our screen, so now we said is there

any relevance of this to the in vivo repair of the epithelium after damage by so2. so we went in and collected both whole trachea and luminal cells and basal cells and epithelial cells and stroma and did a whole lot of studies that i'm just showing you a key finding here

is this is in the uninjured and this is the level by pc r of certain components of the bmp signaling pathway. and tomomi, found that early on after the injury, it was down regulation of many components of the signaling pathway. down regulation and these are

significant of ligands down regulation of receptor levels but what was striking was that there was an upregulation of sali stat--follistatin and it is normally made in annal maland it's an inhibitor of the bmp thal case, we think that it's effecting the bmp signaling

pathway and by gene expression studies we showed it was upregulated both in the epithelial cells and in the stroma and also the bmp ligands, bmp 4 which we had good reporters for, are also normally expressed in both the epithelium and in the stroma.

so then we asked, we showed that there was--upregulation of the follistatin and we think that this was promoting the proliferation of the basal cells during the wound repair process. so then we asked, well if we give the issues ldn or the dorsal position o morphin to the

mice after the injury, can we promote the repair? would this be a potential way of treating people to promote proliferation of basal cells after exposure to something toxic or people who are at risk for bronchialiteis oblight rans. so what we did was to first of

all count the number of cells that were present in this the tracheal epithelium, different stages after the exexposure to so2. and this led to quite a surprising findings. i think we looked at the repair process and drawn these little

diagrams but we hadn't looked really, really careful and what we recognized was that when it started off there was this cell number per unit of basal lamina or this total cell number in the tracheal epithelium and obviously after the injury, the total number fell, but then as

it repaired there was an increase over, about so the cells became more dense, became transiently, there were about twice as many cells per unit and wasn't just everything that contracted because the total number also changed. and then gradually, it went back

to being the steady state level again, so if we looked at this peak time, about 5-6 days after the exposure you can see that there's a piling up of the epithelial cells in the trachea, so this is what it normally looks like and this is what it goes back to after 2 weeks.

but there is a transient piling up. so when we gave the ldn, it did indeed speed up the repair process, we got more cells after 4 days but by 2 weeks it had fortunately gone back to being the normal steady state level, so that didn't lead to a

continuous hyper proliferation of the--of the epithelium. so this increeing egged us and we wondered it was how this epithelium goes back to being the normal density and there had been reports in cell culture and in a few model systems by jodi rosen blat and others about a

process of active cell extryings from epithelial sheeps. this is both active extryings of living cells and extryings of dead cells. we only looked at extryings of dead cells in the modeling we did but this hadn't realee been observed in a kind of wound

of wound repair situation like this. so what we did was look at when we thought the extrusion was starting and look at it through a microscope, the trick was to keep it flat, and then she added a drug that stains the cells with the active caspase 3 and

then take a little movies, well, actually, this was done with fixed tissue is it this is done with [indiscernible] view, which stains the cells in a living tissue and to image the cells and see what was happening and so this is a movie here showing, it's going to repeat itself but

it was just showing, i hope you can see that this is cells which are being--being pushed out of the epithelium, this 1 is going to. q. floating off and then the other 1s will pop off and get floating off and then if you look at the after fixation and

look at different levels, this is a cell coming out and if you go down in different levels you see the cell as it were next to it. that now got floating tight rings here, rings for thinking threr contracting as they're pushing out of the cell.

so obviously this is various stills from the movie and this is still another--[music brick ] --and again we have been interested in this repair process in making these progenitors and how they get assembled into--into something with how these basal cells and

how the daughters become polarized and form tight jurchgzal complexes between them as a barrier. and i said, the basal cells will actually transiently form jurchgzs between them and then as the epithelium remodmodels it's lost again.

so i want to show you this briefly, zone 1 is an expression of tight junctions--most of which are p63 positive, you can now see zone 1 between those cells and terry lechler had made a cell fusion protein and we see translating this to this looking down on the epithelium and you

can see these occluding proteins between the cells. they also upregulate many jurchgzal proteins like claudeine 4 which is highly upregulated and then as the luminal cells are made, the expression 1 goes back from the luminal cells and lost from the

basal cells so there's a lot of interesting cell biology going on here and we had proposed a number of years ago, that maybe the changes in cell behavior and apical base or polarity or junctional complexes was being regulated by a protein called grainy head like 2, a family of

grainy head proteins. which are highly conserved transcription factors that control epithelial cell behavior in a number of organisms highly conserved particularly in drosophila, beautiful work has been done about wound repair in drosophila ectoderm.

these act as dimers so it's possible to make dominant negative versions of the grainy head transcription factor and as i said function had been looked at in drosophila but in humans, there had been the homozygous mutants in mouse are very early on embryonic lethal but in

mutations it was grainy like 2, and it's associate wide deafness, changes in the skin but also with asthma. so maybe there's a hipt here that abnormalities in the leakiness or of the epithelial layer of the air ways might lead to some changes.

so what we had done, i don't have time to go into this but we had made a dominant negative version of grainy head like 2, by replacing the dna binding domain with the gfp fusion protein and using that we had infected primary basal epithelial cells from the human

lung and we had seen changes in the behavior of the basal cells, so that they were no longer able to form tight junctions between the cells and they were no longer able to differentiate into ciliated cells. and in the course of this, we had collaborate wide tim ready

at duke to do chip seq and rnaseq and microarray studies on either the control of primary human epithelial cells or those with a dominant brainy head like number 2. so we found many genes that were changed in expression. the chpseq had shown there were

thousands of grain genes that had brainy head like binding sites. but the rna seq had shown change and hundreds of genes had gone up and hundreds of genes had gone down. but looking at these genes and then testing by pc r for those

which had been down regulated in response to the dominant negative grainy head, we came up with many potential of targets for the grainy head regulation. our hypothesis was that the grainy head which was expressed in the air way epithelium and the basal cells during the

remodeling process would be controlling a whole set of different genes to do with adhesion, polarity and making junctional complexes. and indeed, many of these genes that we found being regulated by the dominant negative grainy head were encoding membrane

associated protein, small gtp as, adhesion proteins, things like claudeins and many components which had been found to change in these epithelial and this is just an example. here is the claudein 4 gene and this is just showing robust grainy head binding sites and

rab25 which had shown to be a target to grainy head in kidney cells had nice robust grainy head binding sites and the their levels were changed after the dom inapt negative grainy head but what we were interested also is this transcription factor that in this s750 had been

implicate indeed the differentiation of epidermal--epidermis from the basal cells and that had the binding sites here so we wanted to test to see whether these were indeed binding sites and my student chris vokley came to me and said you know you could test

of all these in time using crispr technology. we got aulenty krispr vector and put in short guide rnas for 10 of our potential down stream targets and a cas9 expression hire. so we used this lentivirus to knock out, make mutation in

these target genes in primary human basal cells from the lung which are given to us by scott randle in chapel hill. so we seeded these cells, infected them with crispr, and have a short guide rna and after selection we put them in these 2 test systems and we either put

them into the air liquid face culture to say can they make a barrier epithelium. can they make junctions that will enable us to have electrical resistance between this layer and the underlying medium. and then can we--if we put them

into the sphere culture, will they form spheres, will they pump fluid and differentiated sekeithattory cells and say if they form a barrier can they differentiate into secreteatory and affiliated cells. so for proof of principle we put in a grainy head like 2, short

guide r, ina and then you can see that it very well makes little indells in the grainy head like 2 gene. so part of the problem here is that we couldn't select out individual clones because it's not possible to expand clones in the median we had.

it's now possible we could now do this. but at the time we took a population of these cells and tested them. so as proof of principle, the control short guide rna will form barrier function and they would difrepresentiate in this

air liquid interphase but those that had been mutant for grainy head like 2, couldn't form a barrier and i will show you so they didn't form electrical resistance here. but even though they couldn't form electrical resistance, they differentiated into the ciliated

cells but they did differentiate into a mucus producing cell here, so they are able to differentiate but not down the lineage which is what we found with the dominant negative and also they formed spears but they formed fewer spheres and they--those which were mutant

couldn't form a lumen but if there was a case where there was still some protein being made, possibly wasn't a mutant protein, then they could form a lumen and they could differentiate. will so mutant lungs didn't form a lumen so this system was

working. so in the last few slides, i'm going to show you here that we did make mutants in these--i think in the end there were about 8 of them that came through and this, i want you to focus on the znf750. want we were interested in it

because it showed to be a component of a complex that was regulating differentiation genes in the basal cells of the epidermis, both a promoting differentiation gene and inhibiting progenitor genes but its function in the lung had not been seen before.

so just in this last slide, i'm going to show you a knocking out of the 750 in the cells delayed formation of a barrier but electric at barrier but they did, they did finally form 1. but it inhibited the formation of ciliated cells here and again in little patches that

presumingly were coming from cells which hadn't been mutated they did differentiate but mostly they were forks j 1 was down regulated a great deal but the mussine producing cells were not changed. so this led us to say where abouts in the lung where it was

expressed and it anthromorphicized been lookedda the in the lung before. we got sections of normal human lung and stain them for p63 and, zdnf 750 and it's in both the basal cells, some of them and in the affiliated cells, here but the interesting thing was that

not all of the basal cells were expressing zdnf 750. and this here is indicating some which are p63 positive, here, at least 3 of them but they are zdnf 750 negative and then there are others which are p63 positive and zdnf 750 positive. so what we think is and this is

a last slide here is back to our general model of trying to understand the pathways which are promoting these differentiation and morph o genesis. we think that we have identified zdnf 750 as being a key player for the differentiation of the

ciliated lineage here. and we think that grainy head l2 is coordinating and working on these morph o genetic pathways and these are marking all genes which from the chp seq, are binding sites for the grainy head and these other components of the cilia genesis program so

we think the zdnf 750 again is marking population of basal cellsot way to differentiation. so we're now encouraged by this and the fact that we have media in which we can grow these basal cells better, we will go in and test other grainy head potential targets seeing where they fit

in, into this program. so i think this is the potential for using crspr, secnology from the primal basal cells from the so i want to say a particular thank you to nih because they've given me 28 years of support ever since a came to vanderbilt and particularly i want to

acknowledge support of this lung repair regeneration consortium that has funded a lot of the junior investigators, in particular chris has got a junior investigator awards from and they have been tremendous at promoting collaboration between different labs within the united

states. and this is my whole lab. you can see where it's lots of babies, lots of food and lots of fun we're having. so thank you very much indeed. [ applause ] >> please use the microphones we will start over here.

>> hi, bridget, matt hoffman from ncbi, do you know what the fts signal in your basal progenitor cells? is it directly come being from the epithelium, fss binding that or indirect signal, an fgf or something coming from the messentery an kine.

>> we don't know. we're assuming it's egf and fgf but we haven't actually look the at that. >> comment and question, first comment on asthma you spoke of the silliary epitheliumot inside of the bronchus, we have--it's more complex that that we have

thickening and you get fibrosis and you get a progressive rigidity and narrowing of the air ways and you have a lot of things going above and beyond what the semester cells might be doing. the question, bronco pulmonary dysplasia which is common in

neoinates who get oxygen. this seems like the type of thing that would be perfect for investigating that and modes of therapy? do you know of animal models and have you thought about move nothing this direction? >> well, obviously i've

implified everything about asthma. it's been used for looking for new drugs. for displassia, it's really important question that nobody has looked at it very well in human, in human neoinates. it's all based on what's

happening in mice. in this mice we don't have these basal cells down to the small air ways, and nobody knows when basal cells, first appear in the i think i'm correct in seaing this, because that would be important because they are the stem cell for repairing the

small air ways and for the growth of the air ways in afterbirth and i don't think people know when they first appear in the human lung. in mouse they actually appear just at the time of birth the wear ways in the developing human lung are actually all

affiliated to begin with and they're all p63 and then they--i mean p--i'm not even sure now where the ciliated and p63, but after a time, many of the p63 disappear and they're only confined to a basal layer, so those basal p63 keratin 5 cells don't appear to until after

birth in the mouse. when they appear in the human, it's not known. so there's a lot to learn. >> just a comment, there have bye-bye studies in europe but not here, and i think it's in the guidelines of asthma written by the nih and i think the first

changes aren't seen until they're 2 or 3 years of age and sim tomatic infants so that might be correlating with when the stem cell starts appearing, but the stem cells are making their imperative things and the changes in the epithelium and that and that might be a magic

number and might correlate when all this is happening. you might want to look at the literature and look at biopsy and asthma, very few papers doing it because it's unethical. >> we have an interest group at nih that's looking at single cell biology now that 1 can

actually do transcriptomes of individual cells so i couldn't help but think of that being loamacying at the complex types of cells in the human air way, wouldn't it be interesting and look at individual proteins by imue o fluorescence to get a repertoirey of individuals--

>> ohias but it's being done, it's funded by nhlbi, something called the lung map, people isolating single cells from human lungs doing transcriptome, talking about heterogeneity in the basal cells about you it's also in the type 2 cells which are a stem cell population and

in looking at disease lungs with fibrosis, so it's done, funded by nhlbi, cincinnati, a lot of places are just pouring out the data, and it's need but we need bioinformatics people to work on the data. i would would love to tell you about this.

carol is being involved in all this and there's a lot work being done. >> well, there is a reception in the library. please continue the conversation if if you would like to and mean while let's thank professor hogan.

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