Thank you.
Go ahead and get started.

Certainly my pleasure to welcome to

this morning

just a little bit of background so

much actually is a degree from a Ph D.

from Cal Tech I think you probably
all of heard of Cal Tech itself but

there are a couple post-docs wasn't
exactly at the University of Utah but

ended up in the industrial world

when you look at his C.V.
is he has a variety of titles but

basically he's with Victor just
part of Life Technologies and

vice president for regenerative medicine
or something like that is that correct.

That's correct.

So he's certainly in the Horton
leader in this whole

area of the translations
benchtop stem cell biology

to patients to clinical
therapies to commercialization.

So it's great that you could be with us.

I know your schedule is
always very tight so

you flew in this morning home late
tonight but we we haven't for the day and

we're very fortunate to have some
of them to welcome very much.

So it's a pleasure to be here have
actually been to judge a take before but

I have been to me several times and
I had a series of collaboration from

different groups there but
I won't talk about those so tall.

It's nice to come back and specially meet
some of my colleagues from Athens Georgia.

So I want to start off my talk by giving
you an apology when to tell you first.

I confess that I'm not a bio engineer and

have had no bioengineering
training whatsoever.

On the other hand I have dabbled in
bioengineering all throughout my graduate

school and postdoc and faculty days at
one extreme I worked on space biology and

looking at zero gravity micro culture
systems and the other end I looked at

powered acquirements for transmitting
at a physiology multi-unit recordings

to using by less base multi coding
techniques which also means

that since I didn't know what I was doing
most of those things did not work right.

And so might some thesis of
my top today is going to be

the advice I give to a lot
of my students right.

Is that I think a lot of us
when we do by engineering.

As a degree or

as a career start off by trying to solve
the most difficult problems first.

And I think it's pretty important
to learn to walk before you Don.

And so
you'll see that the whole experiment and

what I describe in the next
set of talks is going to be.

About how to do that.

And then secondly one difference between
what bio engineering students do and

what my science students and

biology do is that we don't think
of solving the whole problem.

Engineers like to focus so they focus on
one specific piece of the problem and

once they've solved if they've
declared it as a success but

that success doesn't necessarily
translate it to being

used because it's not part
of a general solution.

So what are you going to see in my talk
today he's taking a simple solution and

applying them in a comprehensive way and

hopefully even find see why
this thesis might be important.

Somebody tell you to do stories
about work that we've been doing

run it's going to be
an mesenchymal stem cells and

there's going to be a new stem
cell embryonic stem cells and you.

Them as a source to do.

I've needed related stuff.

So for those of you don't know
anything about mesenchymal stem cells.

They're an adult type of stem cell and
if you look along the side here

which is outlined in that
you can get mis income and

stem cell like cells from that idea of
sources so you have bone marrow adipose

tissue umbilical cord amniotic
fluid skeletal muscle that sector.

There's also a subclass of mesenchymal
stem cells which the inventors

are discoveries have claimed by
different because their properties in

addition to what M.S.C.

have but they're all similar and
those include things like maps see M.P.L.

see my me says very small out a small can
stem cells from adult tissue at sector.

And then there's the idea that you can use
other sources such as embryonic stem cells

to give you measure and kind of
says there's a process of trying to

differentiate may be able to take cells
and make them mess and damage cells and

then the two important things that we
learned from the field were that the one

class of therapy where people think about
using Mesenchymal and differentiated sets

and that includes things like sort of
basic biology cancer biology anyone can

run subclass of cell therapy very I think
and Gulf Coast disease that section.

And then there's a process where you can
say I'm using mesenchymal stem cells as

a source of differentiated cells and

what you see here is a whole series of
differentiated phenotypes that have been

shown to be did I from as income that
sets both in vitro and in vivo and so

those include condo sites ostracise
adipocytes Maya Blas at sector.

So you can imagine that people
working with cotton age may want to

take a different shape them into
cartilage and use them for therapy.

To keep all of this in mind
because this is the problem right.

How do you devise a process so

that you can scale up culture of these
cells and do it in a complete system

that can be applicable to the largest
number of people using stem cells.

And so the story.

I'm going to tell you in the next.

Slides is looking at
that kind of solution.

So when I first started
thinking about this problem.

You can imagine thinking about this
problem by going from the reverse

sensor taking a medical background and
saying what is the kind of therapy

people are thinking with mess and gamma
cells and using that to work backwards

in terms of what is your scalable issue or
problem in terms of manufacturing sets.

How many cells will I need and
for how many patients.

And so broadly we can divide that into
saying that mesenchymal stem cell therapy

can be broadly classified into
two types one where it's a dollar

a personalized medicine type of therapy
and there this allergenic and for

me it doesn't matter what that means in
terms of regulatory authority studies for

this stock but what it means is if
it's personalized then I'm taking

a small number of cells and I'm amplifying
them for a limited amount of time

if it's added generic then I'm going to
be doing a lot scale manufacturing I'm

treating ten thousand patients
with the same sort of says so

have a much bigger scale of
beliefs you're probably right.

So personalized medicine might
require a different solution that's

killed manufacture will require
a different class of solution.

And I need to worry about both
of those sorts of solutions.

So right away.

That gave me a very simple answer.

If I want to solve this problem.

I need a complete system.

And I need to system which might be
valuable for a small number amplification.

And I need a system which would be
viable for the only truly large

number of applications of sets and
I need a system which is for

growing the says expanding them and
I need a system for differentiating them.

So that seem fairly straightforward
knightly said a kind of down what kind of

problem I was going to try and solve and
to to need a complete system type Let's

see what we can start with first and
where it was really quite useful for

me be talk to a lot of companies which are
already working towards therapy here and

we asked them what their process was so

that I could identify all
the things that I needed to see.

To get to the complete system and
the answer and this is taken

a bottle from a company called that assist
which is based out of Cleveland Ohio.

And they were going to sell which
were first identified by cats and

Buffett and they have a process for their
ideas that they're going to perform and

so you can and
just using it as an example.

It really doesn't matter because I could
have used it from some other company such

as Osiris right.

So it's isolate me.

You take this you process it.

Hopefully you want to do it in a closed
system they needed to expand it and

I'll tell you a little bit about
the kinds of trials they were running so

the numbers that they needed then
ultimately after you've expanded

the cells you need to concentrate
them into some fashion.

You need to create a must.

A bank.
You need to take that and

make a working bank from which
are going to get lots and

lots of plenty could doses and then you
need to ship it to the end user and

they need to do some kind of qualification
tests that are required by the F.D.A.

and then you need to administer cells.

So.

If I can break it down
into these steps then for

each of the steps I need a solution and
that solution in

my mind to Christ by engineering
solutions because I need a device.

I need media.

I need storage solutions I need
temperature controls I need terms of being

able to do it but I need to make
sure that they all work together.

And I knew which number I needed
based on their protocol and

trials and the number of
patients that they wanted and

so we won't go through all of this but
suffice to say they need it.

Much larger numbers and they needed to
treat tens of thousands of patients.

Once the trial was successful but for the
initial trial and manufacture they needed

it and it is really small number because
they were thinking about fifty patients at

a time and they didn't have a big issue
on shipping the cells to multiple sites.

So it skipped that and
they were using undifferentiated cells.

So I'm going to focus then and
just tell you that.

This spot rest pretty
straightforward to address.

It's not anything novel so
I'm not going to tie.

About it.

For today.

It was pretty much what we've been
doing with tissue processing and

tissue Some are saying making cell lines.

That part could be solved peacefully.

Then we had to ready about
tissue processing and

figure out what would be a system
which would work together and

then see whether we could have it in
a close system and what would be there and

then figure out what would be the quality
control that needed to be done and

I'm going to quickly go through all
of this and you don't need to write

it too much about the details on
the slide because as I pointed out.

I'm talking about simple things and
then we putting them all together that

are then something which is technically
million dollar completely challenging.

So the first thing that became clear was
that we needed to fit it figured out and

define a media and
that media need to be able to work with

multiple cell types because there were
multiple sources of isolation but

developed related cell types and then
I needed to figure it out immediately.

It would work at different scale and
this is an important issue that

people need to worry about and
I could have started by doing this.

Empirically But then that's a problem
because empirically do it but

I won't quite understand the basis of
private media and every time I have to

radiate for the new cell type the effort
is going to be about the same.

So is that a way I can do this in some
sort of generalized process at least I did

defy the basic mechanisms by which
one can design a good media.

If I can do that then I
could solve the problem for

most of the cell types and I'll tell
you a little bit about what I did.

And and
that's all summarized in this one slide.

So what I decided to do was to say let's
take advantage of the genomic revolution

that we already have and the way that we
have technology in terms of being able

to look at gene expression profiling and
what we do is take software and

analyze mess and camel selves.

OK And
what we do by this analysis is look for

all signaling face that are active and
mess and time and stem cells.

When they're being grown and expanded.

Because there was a.

Standard sort of seed a containing
media which could be used to grow and

expand the cells.

Once I knew that.

I could also check which growth
factor receptor is a present on

the surface of the cells based on this
sort of gene expression analysis.

I could verify that by proteomics and
then I could go back and check but

that those corresponding growth
factors had an effect on the sets.

So I could compare across
multiple cell types and

I could compare at multiple
stages in their defense.

Ation process was a spell
efficient process.

I should be able to identify the key
parts base that are important for

keeping them probably fairly.

They can do it in a relatively cheap and

quick way then I have a way to design
media which may be delicately generalize.

The answer turned out to be yes we
could do this and this was done within

the image and environment by a research
group so this is the research part not

the manufacturing and production of the
commercial part of invitation and where

people are able to identify and again be
identified several critical part place.

The three that seem to be the most
important of teach if they to signal.

Activation of P.D.F. signaling
an activation effect sheriff pathways

we could look at alternatives such as vege
F. might read through the same P.D.F.

Pop A and B.
had a lot of data on that front.

And you also knew what the addition of
things which could synergize with these

but these were the three that we chose
because they had the biggest bang for

the buck when we did some
come from a tree tests.

So now we knew that we
had a generalized system.

These three partners very important for
most of the missing time and

populations of mesenchymal stem
cell like populations and we should

be able to decide define as enough to
see them free media politically quickly.

So the next part of it was
pretty straightforward.

So I won't talk about it except to say yes
we could do it because validated we could

test it and the first media that I
could produce from all of this stuff.

Reza see them free media so we had found
an opportunity to see them because do it

in a relatively short period of time and
this media will.

With that mouse and human cells and

it worked with bone matter and
rock record blood did.

A mess and common sense and
well put cord tissue.

So that was the first step.

However this is not enough for
the regulator authorities.

You really don't need just to see
them free media you also want it to

be Xena free.

And you don't just want it to be a media
solution a member I said you have to start

with having a whole package
that works together so

we have to figure out
whether you need a substrate.

What would be the passage
of enzymes you would need

whether you would need to alter anything
within those components that might be

important in terms of what would be
nice for manufacturing processes so

it turned out that we needed substrate
that substrate had to be of humanized

proteins to be able to identify something
that looked at that we turned out that

a simple change from blue to mean to
go to Max which doesn't release as much

money into the system was important going
to says we found that been invented.

I'm like triple could work in passage in
the sense we also found I could casework

and so we had a list of enzymes which
could be used for passage of cells and

also a mechanical method
of being able to do it and

we could show that all of
these things work together.

So now we had a combined system which
included media substrate passage and

system as well as a kind of preservation
system which we could use fake

spending the cells.

Because whenever you do this and
this is a really important think that

all of us to keep in mind you always
have to test against some standard.

It's not just that it works.

The question is does it work
in a cost effective way.

And how will people compare
it in terms of whatever

is commercially available otherwise
terms of defining success.

And so this is just a series
of tests that we did and

I won't go through them in a whole lot
of detail but except for to with one.

Important test for us because this is sort
of at least criteria when you're making

your own product says his father
says functionally got you.

Good.

You change the media you need to make
sure that the cells are functionally

appropriate and so
that's what you're looking at and

the right hand side here
is that can differentiate

into the three major lineages that
a de facto standard what's important.

Really.
Here is to consider congress site because

that's the property that's lost with so
that was a good sensitive indicator.

We also want to look at good rates and
other characteristics and

that's what we found that the cells
grew at least as well in this case it's

actually better than grown in see them and
they didn't undergo senescence that early.

You could also maybe you need to measure
any kind of change when you change

conditions when you're growing sets on
a large scale and you have to figure out

of it to monitor that and so we look back
at our whole gene expression analysis and

we just use the simple
correlation coefficient right.

If it's a similar then the whole
pattern of gene expression that

you see should be very
similar if they're different.

You should be able to
distinguish between them and

because you can use an OS great
combination to look at that

you can use that as a number
to define related yourself.

And if they experiment that it published
that this is a very sensitive method and

relation coefficient of point nine nine
then he suggested the samples identical.

If you have a lake.

It says are two different
isolations of the same population.

Generally in the point
nine six nine seven range.

If you culture the cells and
you grow them for

ten passages that's what you will see
between the first intent passage.

That's the overall correlation coefficient
and in this case multiple isolates so you

go to them and see them containing media
you go them in the see them free media.

That's the official.

So what it suggests is that your
media is substituting for serum.

It's not changing the cells
in any significant way and

gene expression Rayna unbiased
with that you can look at it

the function of that is confirming
that and the growth rates are good.

So that you have a good media and
that it works reasonably well.

This is this is required for
regulating requirements if you build

something so skip that except to say that
it all comes from what I just said so.

Skip that.

So the next thing that we had was

remember I said don't run before you can
walk right so that was my first step.

We had to see them free media.

And I had tests by which I could
compare if I wanted to improve this.

So what was the improvement that people
want to write some kind of stem cells and

think that they wanted was not
just to see them free media they

want to design a free media because animal
components require a lot more regulation

that you have to pass but
now I had a process right.

I had media.

I know all the components in that media.

I know which ones come
from an anonymous source.

So I can go back and check all of those
and I can see if I can swap them out.

Maybe that a competence or sort of human
protein source to make it seem afraid

I won't go through the whole story because
it was pretty straightforward in terms of

being able to do it and just tell you the
problems that we had in terms of doing it

but we came up with free media as a web
and what I want to emphasize here

is not that we came up with that media but
the speed with which one can do that.

If you do things from first principles and

you start by making sure that you identify
the basic science and the part ways and

have an understanding of what
went into designing that media.

So we made as you know free media and
when we did it.

We had two problems.

The first problem was that all of
the common proteins didn't work

as effectively as purify
proteins from human sources.

So we don't have a fully defined media.

It certainly works the other problem
that we had when he did this

development of this media was how
long the experiments take and

this is something I want to emphasize
here it's also going to be important for

the second half of my talk as well is
that to test the quality of your cells.

If I use a shot.

Passage ing experiment.

I can see very nice quality results with
a variety of things that I discarded.

So Mike cut off is that
they have to go for

passages with human cells passage
ing his every five to seven days so

doing this experiment for eight
passages and then differentiating which

takes me three more weeks is fifty
six days plus twenty one days.

You know that's a lot of time
especially when you work for

a company which is reporting
results every quarter.

Being on the research side
effect of the heat so.

So that was a big problem but
I have no solution for that.

So this is what I'm going
to recommend to all of you.

It's easy to say success when
you're looking on a short term.

I actually have five different other
growth factor cocktails that will

work really well for
three or four passages.

But when failed by the seventh or
eighth passage right.

And that's a really important thing to
keep in mind because it's easy to get

food and that that was even more
important for embryonic stem cells.

We looked at that.

So now.

The next obvious thing of
course was to save and

then when you change the media's does it
work with all the other components that

you've put together because that's one
thing you learn when you do all of these

things is it's a complete package you
change one thing that might be resulting

changes in other things as well but
in this case we could show that.

And we could show that there was a
complete package and that worked together.

And we were lucky there that the same
components that worked with him fully

worked with Zina free.

These were all the same
tests that I showed and

the only thing I want to highlight here is
that the growth rates with zero three were

not much better than that with serum.

So they met the standard but
they weren't better.

Unlike say them free media was but

that generally a common price that people
pay whenever you go to Zina free and for

large scale manufacture but
that's something to keep in mind.

Because now you need to ready a lot
whenever you're making clinical.

Stuff is the carrot type meant to be
did snip in Alice is to show that

the cells behave the same vein you look
at multiple lots you look to see you know

by facts analysis you look to see
which test would be the right.

Test for reproducible lots in
terms of being able to do it but

these were generally
relatively standard off

the shelf things which we
just simply had to adapt.

OK So but we needed to adapt them so

that we had a complete process
of being able to do this.

So skip all of this in
the interest of time and

the emphasize this because I'm
a strong believer in doing.

Sort of gene expression analysis as a good
sensor you know for being able to do it.

Not necessarily the discovery for
which individual genes may be changed but

just as an overall state of the cells it
turns out to be a very good prices have

come down significantly from being able
to do these sorts of tests and I can

do the same sort of thirty thousand thirty
five thousand gene expression profile that

it would cost me to do for maybe
fifty genes or twenty five genes and.

And what you see below.

Here is just a hierarchical plot string
just to show how sensitive this can be in

terms of being able to
differentiate sense.

So the next question then is
you've got a media that media

works you've shown it works as a complete
package and it run out of the different

defense of the development process will
it work when you isolating the sets.

Can you isolate in this media.

If you can't then you have a problem
because now you have a separate.

Development protocol that
you need to worry about and

if you can then you know you've
saved yourself some time.

So the answer here was that
the bone marrow isolation

component worked pretty bad.

And so
we did this with multiple groups and so

you can do a fresh isolation in this
condition but this complete package.

OK I'll skip the comparisons.

I have to comparisons here is that
you have to be quite rigorous

in testing against standards right or
whatever.

Is there to be able to make sure
whether you will have a basis for

improvement but
even on a development scale.

What would be a competitor if you have to
build something better to be a part of and

how would you be able to know whether
that's true and it's not easy for

me to say I can build something
which will be better then.

Anything unless I know that that's the
cells actually capable of behaving better.

I don't want to push the too much because
I can have to grow faster because if

that's going to cause them to become Cata
typically abnormal then I have a problem.

So I need to know how much I can step
on that Sunday to first read before

I do that.

So it's always important to check
a wide variety of different conditions

in which the cells go to know how you're
going to set up your test experiment.

To take them here was that as fast as

sitting for a meeting but
the difference was about ten percent and

that ten percent was my range and
I didn't think I could use that range as

a good sensitive test as I read out to
be able to develop a better system and

so it seemed like in the time
period that I had so

this seemed like the best thing I
could do with the time period I had.

So now that I had one leg to stand on.

So I knew that I had a solution for
the first two parts of the isolation

of the cells and to expand the cells and
I had a solution to qualify them and

I didn't show you all the data but just
as you that I do because we tested it.

We have a storage solution as well it's
a storage solution which takes D.M.S.O.

but work seamlessly with the cells
grown in this particular media and

we have different ways of monitoring
the cells and you can ignore the rest of

the picture just basically to
give you an idea of the various

kinds of tests that you use and the
options that we had to be able to do it.

So I'll be done.

You know that's the.

Next important question to ask oneself and

the answer is No you're not it done right
in terms of being able to say that you

have succeeded in providing
a complete solution.

You can isolate you can expand but
I haven't shown you what is the size and

the number we can expand in and I haven't
shown you that's all compatible with close

systems as well in terms
of being able to do it and

those are the next sort of important
things to be able to do and I need to show

that I can have to says in a selfish and
state that I can take the cells and

I can do all the appropriate
testing that's needed.

I just showed you some ways that we can
do the testing so I'll skip that part and

I just focus on some of the close
systems that are there and

what we need to do to evaluate them and
what issues that I would size and so

I won't go through all the details but
just highlight some of these things and

say we've been working with different
collaborators here two times

trying to figure out that we
can have close systems and

we can walk from one place system to
another through the whole process and

this is actually pretty close it's
not like we have a nice single device

that works all of a Together they
can just feed in and start a cell

population on the matter aspect and then
get to manufacture cells at the other end.

But what we can do is do
the following big film.

What people have done with close systems
from harvesting bone marrow blood.

The bags have been ripped out the size
of the bags has been worked out

the steadily T. of the new percent.

You know the plunger and

of ads has been rubbed out and
I can just figure back on that.

So I can take a spade and
collect in the same kind of backs.

That's been met.

It's worked out to harvest mononucleosis
actions from bone marrow or

from blood in a close system they actually
twenty six such devices that I'm of

it off to be able to do that.

So we've tested some of those systems
with media and components and

sector to make sure that we can link these
two pieces together then taking that and

look at various close culture systems and.

We've done more than this one but this
is a standard sort of system which has

to be able to expand the cells and so

this is the more personalized medicine
this is certainly not at a large scale.

And then look at whether there's a close
system in which we can harvest and

collect says and
how we can deliver them right.

So these are harvested collected cells
from which we've gotten rid of and

then we have a delivery device
which is much more like you would

have been using few cells I.V.
and clinically approved systems for

being able to do that and
then how can you store.

Can you still in a bag in
which you can talk them out so

that then you can ship it back to
the site and you can deliver them so

we try to look to see whether we could
move harvested M.S.E. population and

take them through this entire system and
what that work and we'll work for

clinical trials and I don't know if you
can see this apologize for this but

it's a group at the university in Germany
in which we've been looking at this and

looking at all the data in terms of being
able to use a close system to do it so

that this next step will be to
take these pieces and try and

see if we can put all of this together.

The next piece then can
we do it on a really

large scale and again many things have
been worked out on large scale and

I'm going to just highlight a couple
of strategies that we figured out here.

One thing that was really important to us
can be divisive to ship bulk media for

any large scale manufacture It's a lot
of fluid and it's a lot of liters

that you need to really do
the manufacture meet in one place.

If you really have to do
a commercial scale production.

We have to figure a way to do this
don't doubt that this had been sold for

all the bio production needs that
are being done by a pharmaceuticals and

that thing that we chose is this thing
called the Advanced granulation technology

it's it's not life because it's quite
difficult to granulation getting the.

Substances into solution and
stability seem to be quite good.

So you know how to do this we just
checked whether we can take our media and

do the same thing and the answer was yes.

Then the next question was
Can we get that I sized bags.

And can we make a close system in which we
can take this media take that I feel so

that you can really now go in bulk culture
and I just wanted to give you examples

of two of the sort of off the shelf
commercial systems that are available.

One of them is from G.M.
which is this sort of a bag technology

the other sort of a modified intermediate
scale technology that you can do and

we just check to see whether
you can do prefer systems and

whether you can actually just fill
this with granulated media so

that you can add water and sets and
get a fresh back that you can use for

all of these storage requirements and for
manufacturing equipment and about scale.

And that's an example of
doing that with a G.T..

So you design your bag in the right way so

that you've got the media
loaded in one side.

This is your culture system which you
break and then you matter to this so

that you sounded like the media and
then you break the scene here and

you have a culture device that goes into
a bag or a shaker wherever you want to

properly and you have appropriate
connectors to be able to do this.

That's a single use by
the actors between tested and

they've been taking advantage
of the existing development and

adapting or tweaking that they're then
trying to solve a problem from scratch or

looking at a completely unique
solution terms of being able to do it.

And importantly get testing that there's
a close system that we can deliver in time

to have success.

I want to try and emphasize all of that
in terms of being able to do this.

So the sort of universe of
back solutions actually exist.

The companies we sell them.

You know so it's just a matter of adapting
existing things to be able to do the.

The last piece I want to tell you about in
terms of the technology which I think is

going to be important.

Is how do you select says this is sort
of a fundamental problem that's been

facing us not just going on
differentiated M.S.E. but

certainly a problem with other stem cells
as well as when you want to differentiate.

Because one bigness with our technology
right now for differentiating sets is that

there's nothing that we can do which gives
us one hundred percent or one kind of set.

So what we have to do is
get some efficiency and

then purify if you need to purify
population of differentiated cells so

we've looked at a whole variety of
different technologies in terms of being

able to do that and again our girl is
a successful complete system which

works with all the media agents bags that
sector that we put together and the close

systems that I just described which will
allow you to do it on a clinical scale

relatively quickly because people are
moving towards therapy relatively quickly.

And so we looked to see what kinds of
technologies are available which was

safe and then later pleaded liable and
which could be used at a gym be scaled and

which could be G.M.P. able.

Which unfortunately for
us right now facts this time.

OK.

There were to be technologies that seem
to fulfill the criteria one of them.

Luckily for me came from the company
I work for which is done and beads.

There's another company which manufactures
another magnetic based technology which is

minute twenty The difference
is the Dyna beads a much

larger They're not that much larger
than the size of the cell the don't get

ingested minute any beads are much
smaller than the size of a cell.

So that's an advantage of working with
them and the disadvantage is that they get

taken up by the self so there's a person
cons to that but both are politically

approved sets of beats which have been
used for purifying isolating cells.

For political trials.

OK So clearly a clinical
great product existed and

what we needed to test was whether
we could use it in the up close

sort of system to be able to do
any kind of selection should we.

Needed in the future.

And yes we have a prototype.

But we haven't yet tested it and

shown successful large scale
isolation using the system.

However we've been successful in using the
system not to do positive selection but

to do negative selection and negative
selection becomes quite important.

And I'm not going to
show you this data again.

So I just thought this might be a good
time to show this is that we can take

Dinah beads as an antigen which is present

on differentiated embryonic stem cells and
we can get rid of done differentiated sets

during the process of different station so
that you did use the risk of us.

OK So depletion works we can use
clinical great bits to be able to do it.

Positive selection we haven't yet
shown on a large scale but

we think that we have a process and
a technology to be able to do that.

So I'll just

spend the next ten or
fifteen minutes on looking

at new stem cells just to highlight
the sort of conceptual differences.

So we can take a closer can isolate the we
can look at the properties of the cells

and we can do the same sort of like this
analysis that I just pointed out in being

able to look at whole gene expression
morphology ability to differentiate their

body to make different tissue and we think
that all of those things are now possible.

Skip all of that and

go straight in the interest of time
to looking at neural stem cell.

It's an embryonic stem cells.

So the only things I'm going to
highlight here are the differences

is the additional scope of the problem
as far as we're concerned so

hopefully I can grow much faster so but
one important difference here between

adult stem cells and mesenchymal stem
cells and embryonic stem cells is

that there's really no therapy
even just on differentiated.

Yes I just use them as a bank
to get whatever kind of

intermediate progenitor because if
only differentiated cells that I need.

So that's one big problem that
I need to keep in mind it means

I always need some selection process but
gunplay available technology and

my processes are going to take time
because I have to differentiate them.

The second time.

And involved with multiple
culture conditions.

OK Because what is optimal for
growing cells is not optimal for

differentiating them.

So it means that my processes for
growing embryonic stem cells or

having a complete system are going to
be multiple components or stages and

it will require that I solve multiple
media problems along the way.

And my scale up issues are going to be
different because when I make a seed bank

it's going to be relatively small
event I make a master bank it's

still going to be relatively small
unlike what I had to do with.

Mess and common sense which are being
used at an undifferentiated state but

I have to expand downstream
in a different way.

So my scales will be different
at each of these stages.

So keeping all of those in mind I just
quickly go through a lot of slides and

again we just highlight some
things on that front and

I'll skip this except to point out that
just like with M.S.E. we needed to make

sure that whatever we did on the expansion
side also read on the dedication side.

OK so.

So we use the same strategy we
look at which party base and

which growth factors might be important in
terms of the self renewal of the sales.

We figured out which were the key
signaling pathways and how we could

use small molecules our growth factors to
make a defined media be used the same sort

of slow steps strategy in terms of being
able to go with this be started with

the see them replacement event to a see
them free media and in fact part of

the Syrian Free Media Development was done
in collaboration riches in Athens Georgia.

Under the guidance of AT and Robbins.

Who in fact you guys interact with.

And we were able to share with
the same rigorous criteria that

you can get sets which can be maintained
in an undifferentiated state.

You don't need feeders you can grow them
without feeders in a growth factor copter

we did the same sort of experiment here we
needed to worry about whether we needed

them in a large scale for
certain conditions and

we showed that this can be
grown in suspension culture and

this was done in collaboration with Alan
and in fact this slide is from Adam.

So just using this mapping our process me
went through the same sort of strategy

that we did before and I'll just use
this to summarize a lot of the data and

to show you some of the controls
in the next few slides.

So we'd look to see that we
could get a piece and the I.V.S.

clinical or the I.P.S..

And that the derivation could be done in
the same media that we were going to use

to make all of these steps and
the answer was yes.

Did we know we had to make sure that
because good I'm in a small scale

when you're making seed banks or we were
making a master bank in the same media

substrate enzyme passage in conditions
lastic way that we were using and

the answer was that but that was true.

Then we had to try and
go to the next step.

So here we decided that we wanted to
introduce important step in our normal

manufacturing because this was going to be
too long to see whether we could freeze

and thaw the cells as part of our regular
process with reasonable viability and

the answer was yes as well and
we had a media and

just like we said that all of
this needs in a free media.

We had to make sure that all
of that process would be true.

So just going to try and
focus on this steps and

say that this seemed to be like
a generalized step that we need.

We need to initiate different station we
need to optimize that process we needed to

have some kind of selection protocols and

then we needed to have
something on shipping.

I won't tell you anything about shipping
and I won't tell you anything about

selection of Depletion Protocol
since I mentioned them already and

I'll just use one example and
that is how to make the payment.

It's using a large scale process like this

because that to differentiate
itself population.

It's difficult it's got multiple
processes and hopefully

the problems that you have to solve
are similar to that in any other center.

So let me quickly now with
the next few slides then.

So we looked at many different.

It turned out that there were maybe one
standard protocol that we could use and

that was to make some stem cells from

stem cells make it look like me to
finally get dopaminergic neurons.

So we took that process and
we said make it scalable and

the answer was yes we could take
conditions bodies from them.

Initially we did it by six.

But then we took off
clinical grade nine and

did I did that way in the same media and
then he showed that he could grow it.

Went through a step that you
make from the same dead bodies

we harvested and the reason I've
highlighted this is that this is a process

we haven't been able to automate and
so this is a manual process.

It's OK at this scale that we're doing it
now but this is going to be an issue and

it's a technical challenge that we
haven't sorted out here we take it and

we harvest suspensions we grow them on and

here in substrate we can also grow them in
suspension I want to show that data and

you get a pretty homogeneous people
population of stem cells and

we know that after freezing and telling
them that we can differentiate them.

OK.

This is just to show that we can do
this all with clinical grade lines

which have been good.

I haven't an idea clinic
conditions skipped that slide.

We then had to figure
out how to make them.

And again we took advantage of
the literature in a standard strategy

of being able to look at all of this and
it again.

I won't go through all of this it turned
out that there were two useful growth

factors that we could use eight and
sunny catch up and be expressed for

the period of ten days
we would get the new.

Stem cells to convert to make brain
phenotype that made brain phenotype

included neurons and separate sets and
undifferentiated sets but it was a critic

of the crime and before you got that
I'd kind of dopaminergic neurons.

At Schiphol of this this is all very thick
ation musing mouse models to show that

the growth factors would identified with
that eye trunks because they also work in

mouse in vivo transgenic Spivey looked
at knockout so what expression studies.

So then the result was
that we had a process

by which we could start with a clinic
a great line in clinical grade media

grow them up freeze them at the muster
bank stage get them to the neural stem

cell stage freeze at the new stem
cells stage if we needed to and

go through two additional steps where we
could get about ten to thirty percent

of the cells being dopaminergic neurons
of that eye phenotype we can't get

more than that unless we take the cells
and we use a purification protocol and

as I pointed out we can deplete unwanted
sets but in a positive selection.

We can do it on a large scale we can
do it on a small scale and it works.

For animal studies etc but

it doesn't work on a lot scale yet
in our hands but sufficient.

And skip all of this this is just the last
shipping part can be fleeced or says and

can we talk to them and with the surviving
will be have reasonable viability

the F.D.A. has some specific cutoffs that
we need for that and the answer was yes.

So at this stage that we want to
transplant today to clinical environment.

This would be the stage we
want to ship to the hospital.

This would be the stage that
you taught them out and

this is the stage your transplant.

So that's a quick summary that this is
what we can do with these cells and

then we can get them in
the appropriate stage and

this just highlights the stages
that what we need to do this thing.

This is just to show what maybe
are with positive selection.

Remember I said that we can't
really do it at a large scale but

we can do it on a small scale.

So we've been doing some testing and
it turns out that we can you.

Use two antibodies to be able to get
positive selection one of them is what you

see here that's B.S. and Cam cam it's
not just expressed in dopaminergic

neurons it's actually expressed on
my daughter ignorance many kinds but

it's certainly a niche is away
from another support cells and

stem cells which
are present in the culture.

So you can get a significant
enrichment of your dopaminergic

population along with other neurons.

If you use that marker.

If you combine it with one additional mark
and that's low affinity and Jeff receptor

you can get a purity of about eighty
percent in terms of being able to do it.

We can combine it by using two
antibodies at the same time but

we can combine it by doing a sequential
purification process the viability for

that sequential process is not such
that we can use it at a large scale.

It's fine for
using a small scale to do animal studies.

Skip all of the depletion and

this is just a pretty picture to show
we can get relatively pure isolation.

Be done here.

So like M A C.

There are issues with manufacturing but
with embryonic stem cells.

We have one other important issue and
which I just want to highlight in one

slide since I have a couple of minutes and
then stop which is right out the test.

She'll do and
negative results are reasonable and

I want to emphasize this even though it's
not truly a bio engineering problem but

it's a problem which I think lends
itself to a bio engineering solution.

I think and
this is how do I do to my studies and

you need a little bit of background on
this front is that it is a benign tumor.

That's formed because embryonic stem cells
don't have a signal to differentiate.

So if you transplant them in
a particular site they grow and

they only signals they
have it in the to mass.

So they differentiated.

In advantage.

And since embryonic stem cells
can differentiate into multiple

phenotypes what you will
see within a is made.

The ectoderm ended a meter them
said maybe born with maybe.

Well you know you might see a nervous
tissue along with you know so

it's a complex Juma in that sense but it's
a benign tumour because it's self limiting

because all the cells once they've
differentiated then you don't

know you don't get that too much so and
this happens because down signals for

the cells from outside regulating
the process of difference.

Normally we test for if you do
embryonic stem cells by transplanting

in a particular site in any compromised.

So that you don't reject the cells from a
human in terms of being able to do it and

you do about two to five million sets.

OK In terms of getting that and that
success rate generally between eighty to

ninety percent so about ten percent
of the time even though you've done

the transplant just because of the way
the surgical process is it doesn't work.

OK So the problem for me is the following

it looks like success that I have depleted
all my cells because my treatment for

Parkinson's really cries I
use less than a million sets.

So if I take my own product and
I transplant implantation experiment.

I always get a negative result right in
the time period that I look at it but

it doesn't mean that I won't in the future
get it that I don't have as it will

embryonic stem cells and I don't know
how to sort that and I don't know how

to regulate the process by which I can do
that and I can't really do a large enough

which would be the sort of knee jerk
solution that when you've got a low

frequency of any occurrence to a large
enough N. and you pick it up but

I can't do two thousand animals I can do
that you know just great too expensive.

Both in terms of animal lives and
in cost rights in terms of doing it.

So that's a big issue.

So if we could figure out a three D. model
if we could figure out a way to be able to

test this we can look at equivalence.

And that would be really a huge
solution to this thing is this.

This is the last slide here this
can be a different station and

function relatively rapidly and
the answer is yes we can use.

Sorts of Monaco about a technique that
I just talked about and luckily for

us when we look at dopaminergic different
station to look at any of the lineage

in terms of different station my
developmental biology colleagues have

worked out things in a very nice detailed
way so I just have to go to the literature

and I can really pretty much identify
markers that each of these stages in this

process or any other difference in process
that we've looked at so I have very clean.

Marcus to be able to
define stages of cells.

Keep all of this in the last slide here
I want to show is this and this is

important for manufacturers scalability an
issue about generalizing things remember I

started out by saying famous C. ideally
wanted to make sure that myself that

I develop with a package or that I def
leppard work for all M.S.E. like cells.

So for the field.

We had this new finding right.

Just three years ago we had telling us
that there is another way to get pretty

potent sets and then that was replicated
by more than a hundred fifty labs within

less than a year and now everybody can
make induced pluripotent stem cells right.

So everybody's now wanting to use their
personalised stem cells right for

some kind of therapy.

So I can develop a separate media for
everything right.

So what do I do so for me my big hope
was the same media processes and

protocols that are developed I'm burning
steppes should work with I.P.S. cells.

So my first test was are the protocols
the same I had this whole protocol for

defensive dopaminergic neurons.

I should now be able to take multiple
stem cell lines and I should be able to

run them through the same system and
I should get the same result.

If that was true then it might be tweaking
but that would be like with any other line

that I was using but it's not like I have
to develop holes in your systems and

so the quick take on answer is that
the protocol to a very similar.

I took three different lines to which had
been denied by a group at Hopkins and

one which had been derived from
the Scripps Institute and I tested them.

And basically the same Xeno
preconditions and the.

Package that would use could be used
to drive I.P.S.E. stem cells and

then I could take those and differentiate
into dopaminergic neurons which is what

you're seeing at low and high powered
they're staying B.T.H. and I apologize.

It's much better on the screen and
hopefully you'll have a copy of that and

you can show that you can
purify the same day and

at the same markers in
the same growth factors work.

It was something we expected but
it was important that we show it right and

of course we want to show on every three
lines you have to really do it with a much

larger N. and the take that if it's
truly become N.T.S.C. like seven using

the you know reprogramming chains then
almost everything that we've described for

and yes the protocol has worked for
us so far right.

And we've now done it with more than these
three but that's the most complete data.

I have right now so.

So we feel relatively confident that
this might actually be lucky for

us right that it's not going to be
something unique epigenetic changes

a micron and it changes which have
been shown to be different going to be

sufficiently significant in terms of
the culture and manufacture process.

So we have a process and
that process seems to work for

yourself as an I.P.S. he said.

So this is just some acknowledgment.

So a lot of the M.S.E. work was done by
this large group of people with a knife

technologies as you heard because
Abbott did with this group in Germany.

We also collaborated with groups
in Singapore particularly for

figuring out the E.S.C. did M.S.E.
culture conditions and protocols and

identifying the key growth factor partly
from the Bioinformatics Institute.

You saw some of the in the work with
the University of Tokyo and of course

we've worked with Case Western Reserve
University to look at growth factor and

sent essence of stem cell an embryonic
stem cell work that we did here.

Apart from the stem cell team and

the clinical great dedication was done
by stem lifeline which is a company

based out of California and a lot of
the work on dopaminergic differences.

In fact ninety percent of the work was
done in collaboration with doctors and

a little bit of the work which I did show
you here on transplant was done with

Judy Anderson snap and
I have a small group of us.

I didn't show you this data but we can
take the same neural stem cells that

we harvest from this clinical guidelines
and make pick a source from them and

we can use those for

different kinds of therapy and
that work has been done with John Hopkins.

Dr and Dr Jeff what's to be happy to take.

Thank you

so so it's a guess.

Again we don't have insight into
all the details even there I think

fifty percent off it is just
biological variability.

It's hard to select that
I kind of patience and

you made a bet that you're going to
go with a single type of line and

it's not a poor population as we have
selected something so that's a big issue

as far as we're concerned if you look at
the data YOU KNOW YOU CAN GO Pers talk

analysis as far as F.D.A. regulates
the approval but you can do it for

your own interest and if you do that
you can parse out the patients.

You know and
you get a subclass responding and

there's a subclass which
don't respond as well.

And if you.

Could describe this patients who have very
good very dramatically improved his out

and unfortunately for them.

That's a big problem.

Problem is we can't proactively identify
which ones are going to fit on but

it's useful to remember but this is
true for breast cancer therapy as well.

Right.
Sept and

works on being a subclass of patients so
in fact now it looks like you know one

of the major drugs that we use for
anticoagulation right.

Works only in ten percent of the patients
they've identified the gene.

That's important.

So one function for this.

We don't know what the cause is but
I also think there's a process issue.

They can make it through certain
kinds of manufacturing processes and

the passage number based on
what was known at that time and

that's the best quality that we had at
that time but they didn't optimize for

the function that they wanted to keep
the first clinical indication right.

And it's important to do that are

less.

Absolutely.

So the reason for

the two reasons why this did what it
did in terms of the new S.F. right.

If you have the guidance is from the F.T.

the following right that if
it's minimally manipulated and

it's less than seventy two hours and
culture that's that off guideline.

And it's been used in and
out all of this way whether

it's an analogous used to
it's in Bebo use is yours.

But then what that means
is that there's no F.D.A.

regulation and then you can even market
in the Senate but it's a process right.

And so that's one reason
why they're doing it and so

they they have no incentive to purify it
there's no incentive to do anything in

fact it was better not to do it right in
terms of being able to do that the data

that that product works better than appear
to fight product is actually not there and

the few studies which half suggested
purification might be useful

as actually suggested
beautification would be important.

However that enforces a whole
set of regulations and

a whole set of sort of different criteria
in terms of what you can approve and

what you can label and
what you can sell it.

Have that that didn't seem to
be a good strategy for them.

Yes So

that right.

I can say that families see I
would absolutely say that's true

in the nervous system.

Because that we have very clear data that
appear if I population doesn't work but

if we actually had support cells in this
case the Astra sites then the survival

rates a significantly higher right.

So

we don't know but it's important
to find out because the way F.D.A.

or anybody that gets the process is.

The product is not just the cell
you think is a benefit but

it's the composition of everything
that goes into that tube and so

that would be a normal question that
will be asked is everything else

in that you do whether it's the residue
in media of it as it will damage so

that is it a good fact that the antibodies
used for selection or the support says

that may be carried over and you have to
identify them and in fact what the F.D.A.

says is for a consistent product you're
not just going to say I have for

example if I was looking at
the payment edge of neurons that you

can see one hundred fifty
thousand DR magic neurons.

But I also have to say what
the other cells are and

that that energy ratio remains
the same from a lot to a lot.

So you're absolutely right.

We need to know and
that becomes an important requirement.

So so far.

Every cell type we've looked at it's been
possible to adapt existing technology.

To doing that.

Did that patient sometimes has been
difficult but it's been possible to do.

So it's not that I've had to identify
any fundamentally new methodology

to be able to sort or to be able to
select or to be able to go in suspension.

You know for
any of the things that I pointed out so

beads don't work as well I actually had to
figure out that meant I made media define

them that inhibitors of defense station
rather important component of the media

right before I could use it for
a stem cell grow.

When I grew things in suspension.

It turned out that there was a Mass Effect

would differentiate simply because
of the micro environment changes but

I could correct for
that if I could do a size selection.

So that I never let things go beyond a
certain size and those could be automated.

And those could be stated up you know
in terms of that I'd hate to be able to

do these things but it took time
to figure those things out but so

far it's not like I had to say well
I can start with beads at all and

it's never going to work and I need
ultrasound I need you know heat methods of

selecting what I need normal methods
of suicide gene control simply because

there's no other strategy or
choice available so far.

OK

thank

you very much.