Well welcome

welcome welcome Reverend Love.

It's great pleasure to be here.

I've looked forward for

a long time there to visit the institute
to meet with you all know that you're

not talking about surfing in Australia
today but I can talk about it at

the end of my talk if you like but
more than happy to what I'm going

to talk about those the work we've been
doing over the last five or so years.

My lab is broken up into two we we work
half on human embryonic stem cells and

we'll learn trying to learn
how to differentiate them into

specific cell types it can be used for
cell therapy and the other half of the lab

is devoted to looking at biology of early
embryonic development in the mouse there

is like pre-implantation and early post
implantation development trying to

look at how cells in the embryo commit to
become one of the three germ wires and

of course to do by chemistry
molecular biology.

It's important to have in vitro models and
in-vitro model we used to model

early developmental advance or embryonic
stem cells and they're differentiated

derivatives and what I want to talk to you
today are some of the what I consider to

be some of the most remarkable
properties about embryonic stem cells.

We can talk about the link
between messiest cells and

human ear cells at the end if if
you're interested but I want to

want to talk specifically about that
the mass embryonic stem cell system but

of course there will be
overlap between human as well.

Now for me the remarkable things about

embryonic stem cells that they have
this unlimited polyphony of Tench will

they have the capacity you've grown
under the appropriate conditions.

To divide indefinitely so they're immortal
and I express things like Tom arise

if you look at look at the profile of
Uncle James and Jim suppressor genes

are expressed in embryonic stem cells they
very much would like to Masel embryonic

stem cells are in fact originate when
placed into or hatred towards environment.

If injected into a compromise
mass of form tumors and

as cells differentiate they
lose this children potential.

So I'm going to talk about so cycle
control mechanisms are we talking about

molecular mechanisms of soap
liberation and cell division and

embryonic stem cells is fundamentally
different to all other so types.

And then I'll talk about
maintenance employee part and say.

Now the this is the other thing that's
important about embryonic stem cells.

It's not that they can just divide
indefinitely with their But

their immortal.

They have the capacity to differentiate
to practically any cell type in the body.

So they have this amazing
developmental plasticity so

embryonic stem cells can divide
indefinitely while at the same time

retiming their potential to differentiate
to virtually any cell type.

And that that those two properties
together known as cell for

your are not talked today about the
molecular regulation of so for Neural have

a stem cell divides indefinitely and have
it retains its developmental potential.

Now there are two broad classes of stem
cells which will be aware of course

the adult stem cells which are found
in organs in the body in the adult and

these cells typically divide through as
a matrix cell divisions of my ptosis which

generate two daughter cells one
which retain stem cell identity and

the other one which doesn't
retain still a stem cell identity

which will then differentiate to
generate more specialised cell types.

So we have as symmetric cell division in
adult stem cells embryonic stem cells

divide to generate two equivalent
daughter cells so we have a symmetric

mottos of both of the daughter cells at
my toes has retained stem cell identity.

So that's what we're dealing with when.

We're talking about cell
division of embryonic stem cells.

Now embryonic stem cells have this amazing

capacity to differentiate into
basically any cell type in the body.

I mentioned previously the stem cells
are also quite sure regenerate.

And as part of this differentiation
program they lose chip margin is it saying

we're very interested in understanding
what the link was between sofa Newall

immortalization of stem cells and
cheer merging history and

one of the things we focused on initially
was so-so who control mechanisms because

many of the regulators of soul
divisions are who controls or

effect on the genes
Juma suppressive genes.

So it could be related to
that your merging properties.

Now what got us interested in this general
question of developmental context was that

the pluripotent cells in the embryo.

Divide a very unusually rapid right so
this is a summary of work done

around about twenty five years ago now
by snow and basically it summarizes

the number of pluripotent cells in
a specific size of a brown development.

Now this is a picture of a mouse
embryo and what I'm going to be

talking about of just these purple cells
here these are the pluripotent cells

which will generate the embryo proper
all of the other tissues and cell

types that we have here extremely onic and
really supportive tissues for the embryo.

So the purple cells are pluripotent cells.

Here we have days post code and
so days after fertilization and

we here we have cell number of
the pluripotent cells in the embryo so

these are the cells which
are the embryonic epa blast.

And here at the bottom.

We have average so cycle time which really
is deduced by just counting the number

of cells pluripotent cells in
the embryo in stage embryos.

So by counting the number
of cells at different

stages you can deduce the average so
cycle time.

And what's quite remarkable
about this stage development

around about by six point five
is that the average size.

Cycle times in the order of four and
a half hours.

Some of the cells are dividing
faster some of dividing slower but

this is a very rich is a remarkable
writer so play for Asian for a minute.

So this one impressed the soft xenophobes

developmental biologist because they
undergo their own unique rapid mode Isolde

vision in the early cleavage division's
issue since it will cycles but

from a million cell this is a very
rapid rate of sober vision.

So we go from a brisk ride a sort of
vision to an incredibly rapid rate

of salvation and as cells differentiate
at the gastrulation our talk about

gastrulation the minute the soul
division cycle slows down again.

So we have this political burst
followed by slowing down of so

politicization when the pluripotent
cells in the embryo differentiate.

Now differentiation of the pluripotent
cells in the embryo occurs

during a process known as gastrulation and
gastrulation is the point

in development the recurs around about
by six point five to day seven where

the pluripotent cells which previously are
uncommitted to form any of the three germ

lies become committed to form other
ectoderm into them or may see them.

So that's the process known as
Guest Relations a key event in development

because the pluripotent cells become
committed to become a specified cell type.

Now coming back to model systems in vitro.

If you actually look at the number of
cells in the embryo the pluripotent cells

in the embryo at this
stage of development.

We're looking in the order
of several hundred.

You simply can't do biochemistry
on this number of cells.

What we can do however is surgically
dissect that the pluripotent cells from

the embryo so these at this stage
where we're post implantation so

you can surgically dissect that the embryo
from the deciduous then you can dissect

away the extra Briana tissues and
then pillow why they pluripotent cells

from the extreme around again to get
pure pluripotent so populations but

on a per embryo basis.

You're only going to have several hundred.

So you can't do serious
biochemistry molecular biology.

On this sort of tissue because the demand
of materials is not large enough so

we have to use in vitro systems
in the richer systems we use or

embryonic stem cells which are the in
vitro because we've learned

of the in a cell mass stage and
we have a blast embryo.

And we use another cell type known as
A.P.L. cells which are a slightly later

stage after report development which are
equivalent to the primitive ectoderm And

if you actually look at the one
of things we're interested

in doing was trying to understand
why these cells divide so

rapidly and it's not just in the embryo
the pluripotent cells divide rapidly

the pluripotent cells in
vitro divide rapidly as well.

Embryonic stem cells divide a new order of
ten hours the primitive ectoderm that we

can make which is still pretty potent
divide in the order of eight hours.

This is very rapid So division and
then jury in their differentiation these

pluripotent cells cycle are much
slower right now this is some

private property a modified Stein
profiles of embryonic stem cells and

these A.P.L. cells which will still apply
reported and so population and what you'll

notice you've ever seen a before is that
we have a very high proportion of cells

in a space and a very low percentage of
cells in the G one phase the cell cycle.

Now in Mosul type cell spend most of
the timing G one G one's a very important

part of the source cycle because
that's when so volume increases so

growth increases and the cells respond
to margining signal light signals which

cue them to either undergo another round
of replication or to enter quiescent So

the G one phase is normally
very important and

much longer than most of the other phases
pluripotent cells both in vitro and

in the embryo have a very unusual cell
cycle structure and part of the reason for

this is that they truncate their G one
face and basically come out of my toes and

they go straight into S. buys and this
all in all a cancer rapid writes the cell

division and then you can differentiate
pluripotent cells into Ambroid bodies by

growing them in suspensions in
the absence of light in a spy.

Better such as Live.

And then the soul division
writes will slow down and

then the sociological completely
remodels and starts to look more like

my he lost soul or in I at st e three or
mass embryo father blast.

We have this very high percentage
of cells in the G one face.

Of course the cell division cycle we
are interested in understanding what

the molecular basis was behind this
very rapid mode of cell division

in the pluripotent cells and
how that changes as pluripotent cells

differentiate into one of the three germ
lines and the molecular basis behind so

Division is that so Division cycles
phase transitions are driven by a family

of protein kindnesses known as stock
independent prodding kinases which foster

projects required for
the difference Osaka transitions for it.

So for each specific so cycle phase.

There are sock independent kindnesses
which are activated the process points of

the Soul Cycle and their job is to
phosphor light proteins required for

things like dinner reputation and motto
says OK so we have different flavors of

stock independent candidate is required
for the different so cycle transitions and

a stock independent kindnesses consist
of the catalytic subunit a city.

And regulatory subunit
known as the cycling and

it's only when the cycling bonds the.

Catalytic C.D.K. that the complex
becomes active The important thing is

that cycling protein levels are in fact so
cycle regulated so that cycling proteins

a periodic we synthesised at specific
points of the cell cycle and then degraded

at specific points of the soul cycle so
that cycling levels are in fact so

cycle regulated force allowing for
cycle regulation of the Conners activity.

Now if we look at the SO cycle structure.

This is now we've taken twenty days
six point two five day embryos and

this is an embryo has been dissected
out of a deciduous home and in situ.

And we stain in the embryo with an empty
of four and since our and I probe.

So this is lighting up the pluripotent
cells in six point two five day embryos.

We took a total of twenty
embryos surgically done.

Thank you.
That these pluripotent cells here might

single cell suspensions and

Stein an appropriate model and we get this
sort of so subtle structure which is very

similar to what we see the pluripotent
cells in vitro so the so

psycho structure of pluripotent cells in
the embryo is very similar to the in-vitro

ple part in cells that we work with and
this is just an example of two cell lines.

In a it's really three Farber's and
there's embryo father parts which have

a totally different type
of saw cycle structure.

Now what's the basis underpinning
this rapid rate of cell division and

unusual cell cycle structure.

Now we know the stock independent
kindnesses of the orchestrators

of cell division.

So we asked well could it be that suck
independent candidate is a very active in

in pluripotent cells and

I drive rapid rites of social interaction
what we did was to essentially perform

kind eyes as eyes on embryonic
stem cells so we made whole.

So life sites and then immune or
precipitated either the catalytic subunits

of the Connery C.D.K. or the siphon
waitress subunits and then to the miller

precipitates So we then added histone
which one is an in vitro substrate and

P thirty two and then read the can
as I say I don't want Joe and

then put it down on film and this is
represents a thirty two libel history and

one which has been fast forwarded by the
stock independent canonizing the E.S.L. I

say and I just want to draw your attention
to the levels relative levels of stock in

the Pentagon is activities employee part
and sells compared to differentiated so

and we're looking at least ten
fold greater than any other so

on this being characterized previously so
pluripotent cells.

Embryonic stem cells have very high levels
of sight in the Pentagon as activities and

in a number of a number of cases now it's
been shown in the up regulation of stock

in the Pentagon as activity in German so
lines is responsible for

driving rapid writes of liberation.

So we think that the killer basis
behind rapid rates are so division and

this very unusual so cycle structure
is the very high levels of stock in

the pending client as activities in
the pluripotent cells are not going.

Talk much about the source cycle
words dogs that's published but

just to summarize what we know about so
far who control in embryonic stem cells

the time they divide it very rapid
writes that short get faces.

City connectivity years in is unusually
high I just showed you on the previous

slide and importantly the sock independent
candidate to release that you have

an embryonic stem cells
are not cells like a regulated

one of the one of the the principal
features of source cycle regulation

is that you switch off site independent
canisters a perspective it points the SO

cycle and then switch on
other stuff in the Pentagon.

It says you have this oscillation where
can is a switched on and switched off just

defining the difference or
cycle transitions in embryonic stem cells.

There's no so psycho regulation of these
can I say to these With the exception

of the Matar it can I see the.

So these cells are very unusual and
effect if you would if you want

to look at the molecular profile in
terms of their political regime.

You would say they're incredibly
Cierra genic based on the activity of

the political machinery.

Many of the chimneys suppress the
functions that normally existing normal

cells which restrained so

the vision are effect not functional
embryonic stem cells the retina blastoma

chim a suppressive family are inactive
in embryonic stem cells because they're

held in a by chemically inactive state
by phosphorylation through city K.

activity so suppress the proteins
can be regulated by sucking

depending canisters when they're phosphor
lighted the germ a suppressive protein

family is switched off in the embryonic
stem cells because we have such high

levels of C Co It's a really that active
throughout the SO cycle the R.B.A. family

are held in a by chemically inactive state
and we also have a distinct absence of

inhibitors of independent Conner's which
also a republic has a German progression.

So basically the whole political
machinery of embryonic

stem cells is wired to divide in more or
less an uncontrolled rapid manner.

But what happens during differentiation
some of very significant.

Changes the cells go from looking
at the molecular level at least

from looking like a chimera
genic cell which divided rapidly

in an uncontrolled manner
to a soul that now.

Starts to respond to its environment and
has very strictly controlled so

psycho regulatory mechanisms and you can
differentiate embryonic stem cells in

broad bodies by growing them in suspension
in the absence of live for example and

then what happens to the C.D.K.
two releases they start to collapse and

decrease during differentiation so
both the Conners activity and

the protein levels of the sirens
decrease during different generation and

this occurs to the point around about
where differentiation starts to occur so

this is a normal blood just showing you
a number of are and I probably are.

And I markers which come up during
differentiation Preki year is a mark of

nice and nice of them formation This is so
this is indicative that in

differentiation is starting to occur die
three and this coincides with the down

legalization of these sort of
impending can I say to these so

the loss of these these precociously
character of these correlates with

the activation of markers which correlate
with specific differentiation programs.

So during differentiation
the cell cycle length increases.

You have a restructuring of the Soul Cycle
So where you go from a so-so ocular lacks

G. one phase control you go to a so-so arc
which has a long G. one phase control.

You have establishment of so psycho
regulated City character with these and

importantly we can talk about
this later you start to have.

Signaling from the outside
the cell controlling the cells

like machinery service so
now is more responsive to its environment.

So this sort of summarizes
the political control and the AS cells.

We have these very high levels of
can stitch deeply active C.D.K.

activities which are very similar to can
control city caregivers Juma cells and

then as part of
the differentiation program.

This is part of the natural
differentiation program the records in

the embryo as world.

You get a decrease in city corrective ease
and then they start to become so psycho

regularly just as it's as it's all written
about enough to graduate textbooks.

OK So we have a very unusual
situation in the embryonic stem

cells which differs markedly from
the molecular control of liberation.

In different child cells.

So I think that goes a long way to explain
the properties of embryonic stem cells

the bar chemical properties
the biochemical mechanisms underpinning

this so division is much more like
a tumor solve on a normal cell.

But what happens during differentiation is
that these chimera genic features are lost

and then so

cycle starts to become cells Arkell
regulated like like other cell types.

So once or more about molecular
mechanisms the stem cell might and

it's one of the mechanisms
which maintain the stem cell

to allow it to divide indefinitely and to
return its full developmental potential.

And you'll know in mass embryonic stem
cells the way you grow cells is typically

in the presence of our six family
members start a kind such as Live

in here which we factor which binds to
heterodyne Merrick receptor G B G P one

thirty live receptor this is them
recruits latent in active step three

transcription factor which isn't the sort
of pleasant to the nucleus where

it becomes phosphor lighted by
receptor tars in kind as activities.

Now flowing phosphorylation the receptor
Step three then transfer case to

the nucleus and the idea is that step
three then activates genes required for

stem cell maintenance.

Now that the genetic program required for
STEM so maintenance and stem cells so

for Neil has not been defined
previously it's a big black box.

We know that lyfe is involved in docking
to the receptor would activate step three.

But we don't know what
happens after the step.

An interesting experiment is you can
actually grow cells in the absence of live

topically expressing
an active form of step three.

So we know that active
step three under most

conditions is sufficient to maintain so
for Neil.

So the everything then downstream
a Step three is of course going.

Be crucial to understanding
the stove in your mechanisms.

So we answered this question by trying to
asked while one of the targets of step

three has Step Three working on embryonic
stem cells to maintain itself in your.

Now just stopping for a minute there are
a number of transcription factors which

could be perceived to be downstream
targets of step three at the transcription

or level four which is the powder
main transcription factor

expressed not only in the pluripotent
cells in the embryo but also the germ line

actually started to occur in many
adult stem cell populations as well.

Nano go home.

You're done.
Main transcription factor.

Last year by two groups.

Sucks to U.T. if one of the three theories
are all transcription factors which have

some important role in development of
the three part cells in the embryo.

But us want to say these are not
the targets of step three.

If you express any one of these it
topically they will not maintain

embryonic stem cells in
the way the step three will.

And the important point there is want to
mention is that there's an alternative way

of maintaining human enmasse embryonic
stem cells has been reported on

I'm not totally convinced that this
story holds together in its entirety.

But let me just mention this much.

It's been reported by Britain with
his group that when signalling can

maintain embryonic stem cells in a sofa
nearing States this is both mass and.

Human embryonic stem cells and more
importantly that the wind signalling acts

to suppress G S K three activity and
it's argued that suppression of G.'s K.

three activity is important for
promotion of the softening state.

So you need Logies code three
activity in order to be a stem cell.

So just remember that
because of become better

later in terms of trying to provide
a mechanism to explain this.

So we have two potential pathways that
can maintain most embryonic stem cells.

We have we have the live.

Step three pathway which activates So

if we knew all genes the genetic
programming transcription.

But this can be replaced.

We can.
Take a while if I'm a minute

window when condition media and we work
through basic training to see if left and

activates target change presumably which

the sign target genes that maybe live
in step three are acting for it.

So one of the questions we asked was
how to separate signalling pathways

independently promote self for New York
is a very different signalling pathways.

But they can substitute for each other.

You can take away with and give the cells
lift you maintain so for New York and

vice versa so we reasoned that perhaps
these two signaling pathways although very

different were focusing in on
the same set of target genes.

I'm promoting So for you in that way.

I'll come back to that in a minute
though there are not another

another several other
levels of complexity.

When you have when you consider so
for new embryonic stem cells.

What a stem so
basically is the way it's starting to look

is a sofa new wing stem cell is a so
that's blocked in its ability to

differentiate into the three germ liners
and in order to maintain the stem cell.

You need to provide multiple lineage
specific differentiation blocks and

there's evidence now that factors in serum
for example in conjunction with live

are required for so if we knew all of
medicine we don't extend cells on the why

this works is the billion Pais which is
the key component of serum suppress neural

differentiation and other factors in
the culture conditions suppress other in

the demo me System differentiation so
these are the three main.

Germ line the germ germline
specific differentiation pathways

that any yourself can go down in
order to maintain surfer new and

to maintain pluripotency you need to block
differentiation then these pathways.

OK now coming back to the lift.

Step three story.

We were very interested I've taught
already about the soul cycle and

we're very interested in sort of trying to
establish links between soso who control

mechanisms that mention
the very unusual mode of so-so

who control the embryonic stem cells.

So we're important.

We are interested in order to
find links between these cells

our control mechanisms.

And stemcell so from New York.

So for example could start could be
unusual motorcycle The Pentagon has a duty

actually be intrinsically involved in so
for new mechanisms.

Another important point is we know that
live in winter paid to substitute for

each other in their role in
promoting self and your so

we reasoned that they could be working
by targeting common downstream factors.

So the hypothesis we came up with
trying to pull in so psycho control and

listen when together with Saudi
there's a common downstream target for

the little pathway is that make family
members could be involved in so

often you'll because make Simic
in particular is a known target

of step three dependent and will depend
signalling pathways in other cell types.

So there are precedents to the link.

So you make regulation into lift and
we depend signaling.

And say make is a a helix
loop Elix basically

extra scription factor which Dharm arises
with its obligatory bonding partner Max.

Goes to the nucleus and
can either activate or

press target genes in the nucleus.

And it has a very many plot Trebek
functions that Nick has been the most

elusive target gene in terms
of trying to identify.

So the functions for
because it regulates so

many different genes climate immune him
in a possible Taishan our size showing

that Miki's banter around about ten
percent of all active genes in the cell.

So trying to translate so that Mick
is involved in political process or

differentiation is a bit naïve what
I think Mick is it's a crime which in

remodelling factor which binds to
many print promoters and recruits.

Other components of the carnage in
remodelling machinery and then allows

those genes to respond to signals that
are coming in from outside of the cells.

So in different types Mick will regulate
very different repertoires of genes.

So you can't predict what
effect make over expression or

make under expression
will have on a given so.

It will vary depending on
the sort of you dealing with.

There is a sort of a short list of the
different genes it's been involved with

but make make has.

It's hand in in a lot of pars

differentiation optos
Chima Genesis elevation.

So by just saying what what would you
expect me to be doing in an embryonic stem

cell is very difficult because
it has certainly defined roles.

Of course there are over twenty
marker write papers at least

probably more near there.

Of try to identify me target genes and

no can no clear consensus in terms of
target genes have been established through

their extensive transcription
of profiling studies.

So the hypothesis was that maybe
step three in winter fading in and

preprogramed promoting so
often you'll throw a mic

dependent mechanism maybe mix in both
because we know that both downstream.

Is there but in the case downstream
both step three and live.

So in order for me to be a regulator so
for new we will expect that MC will

be expressed in we don't extend cells and
that perhaps

it's transcription Profar will become
down regulated during differentiation.

So this is some Q. and some R.T.
P.C.R. analysis just looking M.R.I.

levels in embryonic stem cells and
then in differentiating in broad bodies.

And we start off with a panel of a small
panel of differentiation markers

Rex One is a classic E.S.L. market
which is also expressed in the Unisom

as in the embryo opt for
is more representative

of the broad spectrum of pluripotent
cells not just the inner So mass is so.

Group after five is a mark
of a primitive ectoderm and

brekky Ury is a mark of a nice and
Mr Berman effect may send to them.

And then when we hear we can
sort of stage where we are in

differentiation has a mic say Micky's high
in embryonic stem cells and it gets down

regulate rapidly during differentiation
of low levels there throughout but

it's very high in embryonic stem cells and
then down regulated and remember when when

these cells are differentiating
they're not going to her middle.

They're not going to post my
target they cells divide.

And form specialists types for
several for several weeks.

So this is this is not just the fact.

The cells are exiting the source a lot
of the cells are still dividing So

the first part of the idea was Yes Mick
is there an embryonic stem cells and

it gets down regulated your
differentiation which would

perhaps point to a role for
Mick in stem cell state.

This is an experiment.

Showing that Mickey is a direct
It is directly regulated by live.

Step three signalling.

So what we've done is to grow embryonic
stem cells in the absence of live.

So you go from high sing make M.R.I.
you take a while if it goes down and

then you give lift back and
see me camera and I always go back up so

see me transcript levels are responsive
to lift dependent signalling.

Now the next question is is so you make
a direct transcription or target of step

three in vivo So we need to show is step
three actually activating the same gene in

an embryonic stem cell and we need to show
this in order to validate the hypothesis.

So what we've done is to perform even of
our size climate in human observer Taisha

nurseries where we grow the cells and then
of the other cross-linking agents the SO

culture which cross links
transcription factors to the D.N.I.

So you cross-link and then you can
basically harvest the cells share

the crime returned with a sonic cage or
up into fragments around about one K.B.

in length and then immuno precipitate
the step three protein from the SO

life side and if the step three is cross
linked to a promoter in vivo it will pull

down the climate in fragment within the
day and I promote a fragment with it and

then you can simply go into the e-mail or
precipitate and

then use primer specific for
the same promoter to ask has

the same amount of been precipitated with
the step three transcription factor.

And this will be evidence that scenic is
bound to the same promote in river so

what you see in embryonic stem cell.

Step three is banned.

When you take a while if and differentiate
the cells this bonding of step three to

assume it provides decreases.

And this is totally consistent with
the inactivation of step three following

the food roll.

So when you take away live.

So as to.

French right step because Step
three becomes inactive and so

we have a nice correlation there
between step three and the same chain.

The next question is well can step
three actually trans activate the same

gene in vivo and what we've done
there is to make a cell line

will be stable express a fusion
protein where we express Step three

fused to the steroid binding done
mind of the estrogen receptor and

what this is it's basically an e juice of
a system whereby in the absence of added

estrogen and load for hard drugs it's
a marksman we can express the extremists.

Step three fusion protein in
an interactive form that essentially

accumulates in the side of Plasm However
when we had for her drugs it to Mark seven

it binds to the estrogen receptor and then
the step three are fusion transfer case to

the nucleus and activates genes that are
presumably involved in sort of a new all.

So it's an enjoyable system whereby
we reload collides the the light and

form of the protein and
sort of Plasm into the nucleus and

this works very nicely for
transcription factors.

So he's the experiment cells
grown in the presence of live

of course have high levels of scenic and
high levels of what for.

We take away lift the thirty six hours and
so you make transcription goes down and

then to this this pool of cells we
can then either add an estrogen and

load for hard drugs or to modify which
activates the step three protein.

Or we can add live back and in both cases
you get rescue of scenic transcription.

So what this shows us is the scenic chain.

It can be directly
regulated by by step three.

So we have three lines of evidence to link
the the live Step three signaling pathway

to say make first of all see make is
responsive to live signaling in cells.

Secondly Step three binds
assuming promoter and

thirdly Step three trans
activate sing with Jane lever.

OK.

So Soumik is the direct target of lift
dependent Step three signaling in

the answer so so far this is consistent
with the hypothesis that Nick

is part of the self in your mechanism.

Being downstream of step three.

And I just want to talk for
a few minutes about.

Mick and Mick regulation and
embryonic stem cells.

If you actually look at the MacPro so
far talked about make M.R.I. levels and

I want to talk about MC protein because
there are two levels of control which

control make it through a transcription or
mechanism which involves step three.

And by controlling protein levels.

So this is a Western blot looking at
the same produce levels in a cells and

then during differentiation
you see that the levels are so

you make collapse following leaf
withdraw as is cells differentiate.

Now we noticed in several experiments that
say meat protein is actually disappearing.

This was disappearing before
Sumeet message hour and

I was going down which in which indicated
there was some post transcription levels

of control involved in controlling mic
levels in the AS cells and one of the best

characterized mechanisms which regulates
the meat protein levels and in the cell

is through protein stability and probably
instability of so you make is often

controlled through its phosphorylation
on a residue three hundred fifty eight.

So when three hundred fifty.

Ideas phosphor lighted seamy becomes
ubiquitous night in the gets rapidly

degraded in the UN phosphor like state
on a fifty eight scenic is stable.

So we look to see whether phosphorylation
on three hundred fifty at car lights with

a dam regulation of say Mick Jagger and
differentiation and

you'll see straight away that in embryonic
stem cells see Mick is not first far ahead

on three hundred fifty eight which would
indicate the protein is not going to be

targeted for destruction but following
lift would draw you start to get to fifty

eight phosphorylation which is coincident
with the down regulation of say Mick.

Now the kind eyes that Fosse rollers
throwing in fifty eight is is as

being characterized in quite amount
of detail and H.G.'s K three beta and

remembering back to earlier on I said to
have been published reports now saying if

you use inhibitors of G S K three B.
directivity it can promote a so for new.

Of both mass and
human embryonic stem cells.

So what I'm trying to lead you towards
a possible mechanism whereby G.'s K.

three activity is important for
so for Neil.

Through controlling say Mick.

So the question would be well if G.
escaped three beta is important.

Controlling so you make proteins ability
then you would expect that cheesecake for

a baby would be in activity
embryonic stem cells and

then perhaps will become active bited
very early age and differentiation.

So we assessed years K.
three beta activity using a connoisseur.

Whereby we might ha so
license from embryonic stem cells and

then differentiated cells and then him in
a precipitated G S K three for mouse or Y.

sites and then use modern basic protein
is the reason we tried substrate to

evaluate G S K three beta activity and
this is a radioactive US A where we have

preferred to libeled Marling by supporting
what we see in embryonic stem cells

is no three better activity but
within one DI of the draw of life as

start to differentiate G.'s code for
a better activity is elevated around about

fifteen fold which ties in nicely with
the kinetics of phosphorylation on T.V..

Importantly the mutation of T.

fifty eight pops up in very many
cases where MC functions gene

in Burkett's lymphoma for
example mutation of c Make a residue T.

fifty eight contributes to the
transforming orchid Genet capacity of c

MC Because essentially what the transfer
what the mutation does is to stabilise

same we can elevate same levels in
the tumour so so as opposed to having.

A gene duplication or Komsomol
translocations a point mutation of say

MC can in heart seem probably Knoebels
by increasing its stability by the same

residue that we see here being crucial for

regulation of scenic levels
in embryonic stem cells.

I was just pointing out
the key residue here.

Lies in the trans activation domain of C.
MC.

Now what I'm told you about so
far is is is C.

Make actually really a stable
protein embryonic stem cells.

The implication is that it would be
because it's not fossil it's ninety fifty

eight and we have some anecdotal
evidence suggests that

because the Promoting there was going to
happen for transcription then there is

different to a protein stability
following with withdrawal.

So this is actually an experiment
addressing the question of well

as stable is so
you make an embryonic stem cells.

Now the most stable form of same link
that's been reported in the literature

is in so winds with Burkett's lymphoma.

And that's in the order
of ninety minutes so

Mick is generally a very unstable protein.

He has a half life in the order of
fifteen minutes in most so types and

that shown here in a night
show eighty three cells for

example this is a soccer as much as
experiment where we've done my extracts

in three cells truly was stuck in his
mind for various amounts of time and

then we're looking at the disappearance of
the preexisting sing mix so we're blocking

new synthesis and it's looking how quickly
the existing protein disappears and

you can see that the half life of c milk
in a three cells is very short less than

fifteen minutes and this is typical of
basically every cell type this being

known Chima so long it's been
characterized in the literature.

If you look at the half life of say milk
in embryonic stem cells are we comparing

the authentic saying make
the indulging the same make and the.

This is a trans Gene
expressing a Mickey fusion.

The stability of milk in embryonic
stem cells is extraordinary.

You're actually exceeds that what
you see in work is an environment.

So it's at least ninety minutes and
probably one hundred twenty minutes.

So we have a very
unusually stable form a C.

mic in embryonic stem cells and
we think part of the basis for

this could be is that G S K
three activity is suppressed.

So you don't get phosphorylation it.

So I make doesn't get turned over.

This is another experiment just showing
how this is a Mickey are fusion

the mickey are fusion Express from a trans
Gene behaves indistinguishably from

the invasion as authentic
saying make you take

a while if by day to the proteins gone.

So you have these very
very tight regulate.

Of seeming protein in
embryonic stem cells and

that as part of differentiation
Samy protein levels can collapse.

If you actually mutate the two fifty
eight residue do the same experiment and

allow the cells to differentiate
the MC protein persists.

So this demonstrates that it
is the safety it was due.

That's crucial for mixed ability in
the AS cells and again implies or of G.

as CO three between
controlling stability and

again this just reinforces even more that
the the significance of stability of Mick.

In embryonic stem cells here we have
an experiment where we have ear cells and

then embroidered bodies so

this is differentiating cells
they want two three four and

then what you normally see is a collapse
of make a diet so is what you see up here.

So normally a diet so
you make levels of disappeared.

But if you have produce arm inhibitor
in G one three two you can rescue.

Meat protein levels at a day four.

So this is due to the fact you've got
a little bit of residual transcription.

But because the protein is so unstable.

You don't see it on the normal conditions.

If you have produce an inhibitor
you can actually see

the protein through it's through
blocking its degradation.

So this is good evidence that the protein
goes from a stable form to a very liable

for Journey differentiation and we think
this is because you get G S K three beta.

Activated very soon in differentiation.

Now to make to try and tie in make
function as part of a new mechanism.

We would have to be
downstream of winds as well.

So we asked whether Mick was regulated
in similar why in winter time cells as

it was in lift nineteen cells of course if
it wasn't it would be a leaf specific type

of regulation but for it to be fundamental
and core to so for new all we would have

expected that Mick would have behaved in
the sign why in both both nearing systems.

So we've done the same experiments
in will maintain cells.

Make is not foster Are you take away
wins by day once phosphor lighted and

Dear Sky three beta is not active in when
one thousand cells take away when cells

differentiate and J.F.K. becomes rapidly
elevated so that the if you have milk and

the regulation of J.F.K. three is
the sign in one thousand cells and

you live nineteen cells which sort of
gives extra support to the idea that

the MC has some fundamental role in
general aspects of stem cells so for you.

So just to summarize where I've
mentioned regard to control of C. make.

C. Make Use control of
the transcription or

level by lifting pendant mechanism which
involves activation by step three.

So the main role of step three in
promoting So for new all of try and

demonstrate to you is through
transcription or control of seeming.

It's also important not just to have Step
three activity you need to suppress J.F.K.

three beta activity in order to elevate.

Or have seen the protein
levels elevated and

you need to have an absence of two
fifty eight phosphor lation OK So

that's that's sort of just saying well
is expressed in your souls it disappears

in differentiation and here are two
mechanisms by which it's regulated one by

step three dependent mechanism another
by suppressing G. is quite three which

impacts on protein stability but of course
the burning question is well just so

you make actually promote So from your
We've we've sort of we've got some sort of

interesting evidence correlates
with self in your mechanisms but

it does seem a control so
from your so the expression.

We did was to express Simic from a trans
gene either from a cat promoter or

an E.F. one of the promoter.

Hoped up to one IRA as in the drug
selectable market so his style.

So was expressing high levels of MC and

then we asked that we asked the question
Can elevated levels of seeming promote so

for neural stem cells
in the absence of lift.

So it's a.

Actually you do this experiment you
might so longe cycle while if and

in the presence of
enforcing MC transcription.

Can you still retain stemcell identity and
OK so

that the system the system would we use
was again this estrogen receptor fusion

system whereby we might stable Solon's
expressing make it our fusions which gets

expressed in statistically in the sort
of pleasant in an interactive form and

then by adding the steroid analogue for
hydroxy to mobs of on the tomatoes from

Barnsley estrogen receptor stored bonding
domain displaces heat shot proteins which

are bad to the extra receptor and then
our master nuclear localization signal

which then allows MC to rely
claws into the nucleus and

it does so in a very tightly controlled
concentration dependent manner.

The motor marks from us.

The more Mickey our Fusion protein
you can look lost or nucleus and

it's totally reversible.

So you have a concentration dependent
reversible Gene induction system so we can

switch make on switch me cough and then
asked Does that correlate with seventy or.

So we perform the number of policemen or
ASOS to try and see whether

expression of milk could maintain So
if you knew me absence of live.

So we we do flow cytometry looking at
subsurface markers one of the classic for

medicine we don't stem
cells is a year one which

is the carbohydrate epitope
on the cell surface.

Now we did this two ways we made
Solon's expressing wild type make.

So just normal authentic make and then we
expressed the mutant form of meat which

was the stable form of milk that shouldn't
be forced follow him by G S K three.

OK And what we found when we
expressed while tightening is that so

we are taking the cells they are now in
the absence of live for up to twelve days

and then asking the question do
the cells still retain high S.S.E.

I once dining and essentially what
happens is these cells expressing milk

don't resign as a so you're once training
indefinitely but they do lie the last.

Of S.S.E. one stunning.

In contrast few expressed
the stable form of make

you retain as they say
once training definitely.

So the difference between
the normal MC and the.

The stable MC normal MC
delays differentiation.

But the stable MC blocks at
least as judged by this market.

And then if you look at alkaline
phosphatase activity another read out for

stem cell markers.

In the stable form of C. MC can maintain

alkaline phosphatase levels completely in
the absence of life after fifteen days.

If we express what type form of
make it allies differentiation.

OK So this is consistent with what we see
up here and this is just showing that some

of the transcripts associated with stem
cell stage are returned in the MC.

Fifty eight.

Fusions we can keep on genes
associated with the stem cell state

by expressing stable scenic but
not normal seeming.

So the logic is is that you need
two levels of control to maintain

dependence of a needle you not.

You don't just need to express make you
need to express or have a stable form of

MC and if you think what was going on in
the normal case in embryonic stem cells.

You have Step three dependent up
regulation of the transcription

which is one requirement.

And secondly you need suppression of G S K
three activity which blocks its degree

rotation so in the absence of lift if you
expressed normal so you make will just

be degraded because J.F.K. will get up
regulated but by expressing a mutant form

of MC That can't be phosphor lighted
that very sufficient to maintain so for

Neil so it comes back to these
two levels of control required.

And this is the mickey are so
once and is the parental So

winds go in the presence of live
nice dumb shit round colonies.

You take a while if in the absence to
Mach's for both so lines flatten out

become more hitter genius in terms of the
morphology indicative of differentiation.

If you switch on into marksman
the parent will cells

in the presence to Moxon different
church as they did before.

But the mickey are so orange we tie
in this nice uniform random shirt

collar in morphology so Bob morphological
criteria make can maintain is so

morphology and this is just an experiment
just showing that for the stem so

people that the proportion of
stem cells in the pop culture

in one thousand cells is equivalent
to that in live nineteen cells.

So essentially what you do is you
grow souls in the presence of live or

you grow them in the presence to mobs
of and in the absence of live and

then pull it and
then do that for thirty days.

And then take the cells at a single so
suspension placement low density and ask

how many of those cells in the pop culture
can give rise to stem cell colonies and

the upshot is that the ability to
maintain stem cells in the box

is indistinguishable between live nineteen
cells and make one thousand cells

in multiple years so and so
this is not just restricted to one.

Cell line it's it's a characteristic of
different so ones that we've worked with.

So do you make one thousand cells return
very potent you have shown you if you keep

elevated levels of milk.

On In the absence of Lith you can
time stem cell markers such as

this is a one alkaline phosphatase
a very strange scripts and

the cells look like stem cells and they
have a similar clone applied to a fish and

see so little one thousand stem cells but
are they really still truly very potent

that the best test the stem cell
is then to test with those cells

can differentiate in a normal
setting into the three germ layers.

So we wanted to address this question.

By asking well OK we have maintained
cells do they still have the ability

to differentiate we could be and you know
this may be on a carcinoma work that

over time some so once use the ability to
differentiate into certain cell types or

even lose the ability to
differentiate completely

We want to show these cells are still
capable of differentiating So we took so

they're being grown in
the absence of live in the.

Residence to marksman.

And then split them into two and then grow
without smokes from the with a marksman

And last question do you get down
regulation of stem cell markers when you

take a white marksman and the answer is
yes you get down regulation along for

up regulation of rec Ury if you keep
some Oxford on I.E. if you keep make

activity elevated you don't down regulate
use them so markers and you don't turn on

brekky Yury So that shows that the MC
dependent serve renewal is reversible.

If you take a Whites a marksman and switch
make of the cells will differentiate at

least as judged by those markers.

But the best as I to actually demonstrate
this is to show the cells are being

maintained with making culture for
extended periods of time

can still differentiate into free germ
line isn't the best way to do that is to

take those stem cells and inject them
back in the blast staging areas and

ask can I contribute to the three
germ lies in come your cannibals.

And that's what this experiment is with
essentially grown make in our cells

in the absence of life for at least thirty
days and this is what the cells look like.

If you plant them down in the absence of
leaf they flatten out in the absence of to

Moxon The flatten out and differentiate
which again is consistent with them

being capable of differentiating
when you switch make off.

And then what we've done the American
phosphorus positive and then what we've

done is take the cells which are being
grown into Waksman haven't seen live for

over a month and injected them into
blasters stage embryos and the trick.

We've done is to use a green fluorescent
protein Martin bionic stem cell line

expressing make.

So these are G.F.P. libeled ear cells.

From A N S We want to know and genetic
background which has a sandy colored

college and these cells which have been
timed with between one time with make

a been injected into a black
six blasts a stage embryo.

And so
we can easily see that the jail cells.

Integrate widely into all
of the tissues by this.

This is the where about
a die thirteen die embryo.

This is a Mockingjay.

In the show.
This is not all of for Essence and

this is just higher higher magnification
zooms of different tissues so we can see

not contribution to muscle got heart
kidney and cut color we can see communism

there so we got ectoderm lineage is being
generated from these maintained cells.

So you can see the sandy color which.

Which is generated from the.

Injected G. of P S cells in
the black host Black six color.

So we can say now that these make
maintained cells can be maintained

the stem cells that look like stem
cells or retain all of the so

markers of stem cells and we jetted
back into a blastocyst I generally or

they can differ it in differentiate into
the three Germans in a in a manner that is

indistinguishable from
LIVE FROM one thousand cells.

This is this is you need
to use the stable make.

OK.

No it's not going to grade but when you.

When you put we when you when you inject
into the blastocyst you don't have a need

to Mark's from there.

So it just the levels
just decline over time.

Yeah.

So.

That establishes that make
nineteen cells three times for

Republicans and we wanted to sort of
a time some more evidence that mic was

downstream of step three and one of
the one of the tricks we've used is is

that if you express a mutant
form of step three.

This was seventy five F. mutant it gets
recorded to the receptor but it doesn't

get phosphor about it so it acts as
a dominant negative and essentially what

this does is to block all downstream Step
three signaling and under those conditions

when you express the dominant negative
form of step three the surface

will differentiate much in the same way
as the soles would if you took away lift.

Now what we want to do is ask the question
well if we express MC Can this override

dominant negative step three.

And that's exactly what you'd predict if
Mick was downstream of step three and

the major target of.

That three stick like so you could bypass
all upstream signalling if you if you

activate the the mind downstream target.

And this experiment was done by
making double double transfer act and

stole and
we have the mickey on a pure America.

Wow type step three and
on the other so as we have.

And the dominant negative and

essentially under conditions where
souls would normally differentia in

the presence of dominant negative step
three activities make can maintain.

So for Neural as shown by this
alkaline phosphatase activity.

OK So this actually gets around
another potential problem in that some

differentiated cells can might live and

then feed back onto the existing
stem cells and help.

Retain surfin you'll what we're doing
here is bypassing all forms of life.

Step three signaling by expressing
a dominant negative and then asking can

mimic maintain sort of a new owner think
this but this demonstrates that nicely.

So we've shown that milk can
maintain surfer Newall But

is it is it is it necessary for so
for new under normal conditions.

Is it normally involved in promoting
sort of renewal in normal cells.

So the experiment we did to address
this was to make cell lines expressing

a Mickey R. fusion but rather than having
the mc two fifty eight of the water

make fused the estrogen receptor
we had a mutant version which like

the transit division to a mind of MC and
in a number of so

types has been shown to act
as a dominant negative.

So essentially when you make this form of
MC It will be expressed with dawn arrives

with its bonding partner Max it will bind
to all of the most of the regular proteins

promoters that normally regulates but
won't activate transcription and

therefore will compete with
the indulgent A C. MC.

And will block normal seeming function and
then body juice will expressing this

dominant negative you asked the question
is this compatible with my timing so

for me or not.

So here's the outline Foster
ties are size we start

off in the population we switch
on the dominant negative for

six days we start to see a loss
of alkaline phosphatase that.

Typically in a clear morphological
change of cells which is indicative of

differentiation and then if we look
at Jane's the gene expression.

Markers off or is on to start with but
we switch on the dominant negative make

it disappears as there is none of which
are both stem cell markers and we start at

the opera Galatians of differentiation
markers such brekky Yury and socks one.

So in activity make function ears cells.

We can promote differentiation.

So we have evidence now that Nick is
both sufficient and necessary for self.

So this is where we're rap at the moment
with less embryonic stem cells.

We have lift which binds to the receptor
which activate step three and

the beginning of the study we didn't
know what was downstream of step three.

We can now firmly placement downstream of
step three as it's both a direct a direct

target of step three transcription or
regulation and we've shown that it's

sufficient and necessary to maintain
itself when you're in a number of Chima

systems it's quite obvious that
step three just doesn't activate.

C Make It.

It works in collaboration with other genes
make has a known program popped out of

activity and what you do tend to see when
you express high levels of making it so

if you do get some appetizers.

So we think that there are other
target genes which are involved

in suppressing up houses and
the PM one pm to colonise as we think.

Step three can arise which suppress
the seeming target function.

So here's the here's the sort of
the model at the moment we have

lifting it to step three and

step three works principly through
the transcription regulation of say Mick.

Now under normal conditions when so for
new is promoted you have low levels

of G S K three B. directivity now we don't
know what suppresses G S K three beta

it could be went because it has a known
role in suppressing G S K three beta.

It could be lift lift
is known to promote P R

three kinase depend signalling or
it could be.

Actors in serum such as R.T.F.

so insulin which also can be involved
in suppressing G.'s K. three activity

where when you when you when you remove
little went the the road block on G S K

three suppression is lifted and then this
is the last MC to be fossilized on T

fifty it becomes ubiquitous knighted and
then presumably you do so

for new and we've shown if you
lose make functionally yourselves.

So as differentiate.

Now in terms of how
winter only fit together.

There's a drug known as bio and in fact
many three baiter inhibitors will do this.

It's been reported that.

G S K three better inhibitors
will maintain human M.S.M.

bionic stem cells so for Neil and we think
we have a mechanism is to have this works.

We think it works if you talk
about when dependence signaling

it could work through
suppression of G S K three.

Which regulate seem transcription
through basic training or

it could work through suppression
of G S K three which is involved in

phosphorylation of two fifty eight.

So the the two main levels
of control with regard to

surf renewal which which we identified
as being important with regard to mic.

Dependence of when you're
a step three dependent or

with dependent transcription liked about
action and also the suppression of G.'s

create three directivity which allows for
a stable form of me to persist.

So I guess that there are two
questions which sort of.

Persist after the study.

How is Mick actually imposing sort of
a new zero nine time in the pluripotent

state so what is Mick working on and
we sort of try and do some transcription

profiling some by chemical analysis in
terms of what Mick is partnering with.

BLOCK differentiation of course make

as well defined roles in
driving soap liberation.

So obviously we think there are links
between seeing the console cycle studies.

I began talking about and

the other issue is well we know that step
three and lift are not important for us.

So if we knew all of human.

Cells.

So are there any is there
any overlap between so

from you all of human embryonic stem cells
and there's embryonic stem cells and

I love thing that's that's that's an issue
that we're interested in addressing.

People did the work paid a cart right.

Cameron McLean did most of the mic work
align Steadman out of fast Joe White and

one rivet attribute to
the source cycle work.

Thanks.

Thank you.

Yeah I just want to clarify
something before I see question.

The levels of mic that we're
expressing from a trans chain in fact

are equivalent to what you'd normally
get in the years for anyone.

So we're.
This

is this is not an over
expression artifact where it.

You have a whole levels
of making the stem so on.

We're expressing equivalent or
actually lower levels.

So in terms of reversibility well though
there are other things required for

self renewal as opposed to mimic and
we focused on the lift.

We pathway what could be
downstream of lift and went.

We're going to be up at the other
factors involved and so

from the all that we
haven't identified yet.

And clearly make isn't switching on me.

Is not sufficient to restore a more
primitive pluripotent state so

I guess what years.

You're suggesting is well if you
express making a different show itself.

Can it do differentiate and
the answer is well not to my knowledge but

it could break could be part of
the mechanism that's involved in

differentiation in
addition to other things.

So we would actually be interesting to
look to see what happens to make when when

cells in India differ.

Church and systems.

Really.

Well yeah there's a two to two
key changes in the plot for

action in the embryo and
in vitro you have this.

So cycle time around about fourteen
hours which is fairly fairly fast then

it accelerates dance for about four
five hours and then it slows down so

you sort of Excel or item and they sell
right as part of a developmental program.

So what you saw with the ears
cells in the E.P. also the E.P.L.

cells represent the very fast most mice
fastly dividing cells in the embryo

that that they're that they're like
the father cells in the embryo.

It's a good question and
you know why why why do you need to

divide quickly Well you know it could be
due to developmental timelines you know.

But.

Another obvious question is What do you
think human cells an embryonic context

divide is quickly and
I don't know the answer to that question.

There's a great deficiency of human
embryologists who we can interact with and

we would be really good if we could go and
talk to someone who would say yes this is

the developmental profile in
a human versus a mouse but

I really don't know how the politicization
of one cells in the human embryo goes.

I think.

I think.

You know I think in the mistake the stem
cell situation where I think Nick is doing

it's bonding to have perhaps hundreds
of Janes could be a hundred.

Could be to him but
what I think it's doing it's just

it's mocking James require for
a stable genetic study to.

Well you it binds to promoters
in a certain specific manner.

I think if that's what you mean so what I
what I think is happening in the stem so

it's bonding to a certain set of
genes are required for a stable

genetic state which is step establishes
cell type of entity in another context

it will bind to different sets of Jayne's
which establish another cell of entity so

make is not just involved in
differentiation or controlling self in

the Olivia's cells has been implicated in
another sort of differentiation paradigms.

But it's a very I mean look inside

is the No ten percent of of genes
are marked with MC credibly large number.

And that just makes it a very
difficult problem to solve.

Like I

was thinking of thank you.