cc
>> Welcome, today's DART Talk
will be given by Jon Odorico
from the School of Medicine.
He'll be discussing "Cell
Therapies and Transplantation."
>> Good evening.
Thank you Adam,
for that introduction.
Judging by the turnout today,
which is pretty good,
the weather didn't keep
too many people away,
but thank you for inviting me
to the DART Symposium.
I'm happy to address you tonight
and the viewers online
about our research in the field
of stem cell therapies
for treatment of diabetes
and transplantation.
So you are all the stalwarts
and the diehards,
so I think you'll be rewarded
for coming out on a nasty night
like tonight.
I hope you will be anyway.
What I'll try to cover tonight,
in the series of about
50 minutes, is where we are
in the field, current status
basically, of differentiating
embryonic stem cells
to pancreatic beta cells,
or insulin producing cells,
for a replacement therapy
for patients where they've lost
insulin production
or need additional insulin
production to treat
or reverse their diabetes.
I'll talk about some
of our research, some other
research in the literature.
And we'll also talk about
somatic cell nuclear transfer
derived human ES cells
as a means of generating
genetically matched human
ES cells, so that one might
obviate the process of rejection
and not need immunosuppressive
medications
after transplantation
of such a therapy.
We'll talk about a few
of the innovative ways that
scientists have thought about
to get around the ethical issues
with regard to somatic
cell nuclear transfer
derived human ES cells,
or therapeutic cloning.
And then we'll finish
with some lessons from pancreas
transplantation,
and whether the aspects
of autoimmune may play a role.
So by way of introduction,
I think it's fairly well-known
by many that there's a huge
public health impact
of diabetes.
It's extremely costly.
It's extremely morbid for
patients that develop diabetes.
There's a high mortality.
There are many complications
that develop.
In fact, it's the leading cause
of blindness and kidney failure
in the United States.
It's one of the most prevalent
diseases in westernized
countries.
It costs, and you can see
some of the numbers here,
enormous amounts to take care
of these patients,
for our government
and for our healthcare system.
And there's an epidemic
of diabetes.
And it's estimated that
the incidence or prevalence
of diabetes may increase 40%
in the next 50 years.
So it's a huge problem.
And it affects both
the young and the old.
There are several different
types of diabetes.
Diabetes can be loosely defined
as an absolute or relative
insufficiency or deficiency
of insulin producing beta cells
that are usually present
in the pancreas.
The main two types
are Type 1 and Type 2.
Type 1 is usually
juvenile onset.
It's usually associated with an
absolute deficiency of insulin
and a destruction of beta cells
by autoimmunity.
And most of those patients
require insulin,
exogenous insulin
or insulin injections
from the very beginning
of their disease onset.
In contrast, Type 2 patients,
initially, it's more associated
with an insulin resistance.
And that is that the end organs,
muscles, heart, brain, etc.,
do not see or perceive
the insulin that the pancreas
or islet beta cells are
producing and therefore,
the pancreas increases
its output of insulin.
But that is insufficient,
and results in hyperglycemia
or elevated blood sugars,
which then over time
is thought to cause burnout
or apaptosis
or death of those beta cells.
So ultimately those patients
may also require insulin therapy
and be relatively
insulin deficient.
But it takes some time, usually,
for that process to develop
in any particular patient.
In contrast to the autoimmune
ideology of diabetes in Type 1,
Type 2 is a polygenic
and familial component.
Also, many patients that may not
require insulin are often
treated with diet and exercise
and hopefully weight loss,
as ability to control
their diabetes.
So the two types are very
different, but there are
other types that might represent
a mixed phenotype.
There is also gestational
diabetes.
And there's also something
called maturity onset diabetes
of the young, which is
associated with genetic
deficiencies of some
of the pancreatic developmental
transcription factors.
So diabetes is a
multipresentation type disease.
For the purposes of
the therapies that we'll talk
about for the rest of the talk,
we'll be mostly focusing on
clinical application
of Type 1 diabetes.
And that is because there's
an absolute deficiency,
and one wants to replace
those lost beta cells.
So primarily, we're talking
about therapies for Type 1.
So what are the current
therapies for Type 1 diabetes?
These two images represent the
two accepted clinically accepted
therapies right now in 2008.
On the left, you see
an insulin pump.
This is a multigenerational
insulin pump.
It has a censor that senses
blood sugars.
That's implantable.
It has a delivery system
that's the size of a pager.
It has a reservoir for insulin.
It delivers it through
that small catheter directly
into that subcutaneous tissue.
And this is an advanced
technique that has shown
a lot of promise, but has become
accepted in some centers
as the state-of-the-art.
However, it's still associated
with some complications.
And many patients still have
imperfect blood sugar control.
And in some patients,
it's even more dangerous
than just standard injections.
So this is a therapy
that has some advantages
and some down sides.
Pancreas transplantation,
on the other hand is a therapy
that's also effective;
90% of patients, after a
pancreas transplant do not
require insulin, and basically
have a vacation or cure
from their diabetes by one year
after transplantation.
And many of those pancreases
continue to function
for many years.
Patients can remain off insulin
from one or two days after the
transplant, for 10 or 15 years
in some cases.
However, this too is associated
with some side effects
and potential complications.
Whereas insulin pump therapy
may not require major surgery,
pancreas transplantation
requires major surgery, and
is, therefore somewhat risky,
associated with bleeding
complications,
technical complications,
return to the operating room for
various corrective procedures,
as well as the issues
with immunosuppression.
Patients, after transplant,
require immunosuppression
to prevent rejection.
And that's associated
with short-term side effects,
long-term complications,
including increased risk
of infections and malignancy.
So those are two therapies
that are clinically available.
Neither of them
are perfectly effective.
Although pancreas
transplantation can absolutely
normalize blood sugars.
However, it's more risky and
in summary, insulin pump therapy
may be less risky,
but it does not absolutely
prevent metabolic complications,
such as retinopathy, neuropathy,
ischemic changes in the limbs,
nor does it absolutely
correct blood sugars.
In many patients, despite their
best efforts of a physician and
the patient, they can still have
erratic blood sugar control.
So these are
imperfect therapies,
but it's what we have today.
Patients and physicians are
looking for something better.
And as is often the case
in medicine, we're looking for
less invasive ways to do things.
So a less invasive way
to correct blood sugars
with automatic minute-to-minute
control, with the production
and release of insulin,
is to, in addition to
a pancreas transplant
is an islet cell transplant.
Islets can be isolated from
the pancreas, according to the
diagram shown here.
And this has now been done
in many hundreds of patients
and humans.
You have a donor here.
And the pancreas is removed
and then through an enzymatic
and mechanical digestion,
and then a purification process,
the islets can be purified
and then put in an IV bag
and then infused by gravity
into one particular site
that's used clinically,
in general, is the liver.
There they are carried
by the portal blood flow
or the liver blood flow.
And they lodge or embolize
in the liver
and they take up function.
Now this is a therapy
that is used clinically,
although it's still considered
experimental.
It's also been used in patients
that have been transplanted
at the University of Wisconsin.
In fact, we did our last
islet cell transplant
just yesterday.
The islet cell transplantation
is proof of principle
that a cell therapy
can normalize blood sugars.
And here we have some curves
of hemoglobin A1C, which is
glycosylated hemoglobin
and is a measure of long-term
blood sugar control.
High is bad, in the 8-12 range.
Six is normal.
And you can see that after
islet cell transplantation,
most patients can return
to normal glycemia.
This is a glucose curve over
approximately a week and a half
in one of our
transplantation patients.
And you can see that after
their islet cell transplant,
they've had very stable
blood sugars.
So this has been done in
many centers across the world.
It is still experimental,
however.
There are some limitations
of the therapy.
There's a shortage of donors.
And this is the same
for pancreas transplantation.
There's also islet damage
that occurs during the process
and the engraftment period,
such that if you transplant
a million islets for instance,
probably 300,000 of them
survive.
There are scientists and
investigators working on ways to
improve and prevent that damage
and improve engraftment.
However, the current
state-of-the-art is that
there is a lot of wastage
in the system.
Islets, also, just like
a pancreas transplant
are highly immunogenic,
meaning they stimulate
the immune system to attack it,
i.e., the rejection process.
The shortage of donors and
the islet damage that occurs
during and after transplantation
accounts for the fact that the
success rates may be reasonably
good in the short term;
80% of patients may
come off insulin at some time
after islet transplantation.
However, in the long-term,
by three to five years,
most patients have returned to
insulin therapy, unfortunately.
So it's good in the short-term.
It may not be durable,
but science is studying that,
and hoping to advance that
and improve its success rate.
But the fact that you have
islet damage, and these come
from donors, means that
none of the islets that
are produced are of absolute,
off-the-shelf quality
in all cases.
So it's not quite reliable,
the product that's produced
during the isolation process,
although it's still
a state-of-the-art process.
Or it's still an art,
I should say.
So with this proof of concept,
human islet transplantation
as a cell therapy, one can ask
is an embryonic stem cell,
if it can produce beta cells,
or as an adult stem cell,
if they can produce beta cells,
can they be used as a
cell source for transplantation.
And in many ways Type 1 diabetes
is a stereotypical disease
amenable to a cell based
therapy.
As I told you before
diabetes is due to loss of
a specific functional cell type,
the beta cell is damaged
by autoimmunity.
It's a simple protein factory,
in a sense, in a very simplistic
sense, so there's low anatomical
complexity of the system.
We don't need integration of
the cells in a host environment,
like a neuron would have to
integrate with other neurons
to take up functions
after it's transplanted.
Likewise, a heart cell
needs to function in beating,
if it's transplanted.
That needs to be in synchrony
with the other heart cells
of the host.
So there's low anatomical
complexity and an absence
of a need for integration
in the host tissue.
In addition, we have the islet
transplantation as a cell based
treatment proof of principle
that's successful in some cases,
but not in all cases.
There's a shortage
of donor cells.
And as I mentioned,
there's a variable quality
of islets recovered or isolated
from the cadaver donors.
So Type 1 diabetes
is a stereotypical disease.
Other diseases have been thought
of, of course, for stem cell
therapies are Parkinson's,
ALS, Alzheimer's, heart diseases
and vascular diseases as well.
Many others, you can pick an
organ system, and it's possible
that stem cell based therapies
might be useful to treat
diseases in that system.
All right, if we are thinking
about embryonic stem cells
and differentiating them
toward beta cells for
a diabetes therapy,
you have to think about
development a little bit.
And this is a schematic
of development.
And you can see here
the blastocyst embryo producing,
going through gastrolation
and developing of the three
germ layers, which the
differentiated cell types
present in our body are derived.
And embryonic stem cells
are a culture derivative
of these inner cell mass cells
of the blastocyst.
When one wants to think about
differentiating them,
one wants to think about forcing
them through this pathway,
through developmental signals
and other mechanisms.
So we asked the question
a number of years ago,
simply, initially not to drive
differentiation towards
beta cells, but can ES cells
enter this pathway
of development, again,
once they're in culture.
So the answer to that question
is yes.
ES cells can become endoderm,
from which the pancreas
is derived, and they can become
beta cells, which are one of
the endocrine cell types
of the pancreas.
These are a number of reports
now, that have appeared in the
literature and data,
including our own,
that says that yes,
human and mouse ES cells
can become pancreatic beta cells
and produce insulin.
To tell you a little bit
about our data, I wanted to
just give you a brief background
of pancreas development,
a very brief background and tell
you about one molecule called
Pancreatic Duodenal Homeobox 1.
PDX-1 is a homeobox
transcription factor
that is essential
for pancreas development.
In the early stages of
development, when the gut tube,
or uncommitted endoderm
is becoming or thinking about
becoming pancreas, or liver,
or thyroid, or lung, or
the other gut derived tissues,
which are derived
from the endoderm.
It has to express
some transcription factors
that are specific
to the pancreatic lineage.
This is one of them.
And PDX, as I said, is essential
for pancreas development,
in that mice
who are born without it
fail to develop a pancreas, and
are born with neonatal diabetes.
There is an analogous situation
in humans that's been
discovered,
mutations in the PDX gene.
So this is an important gene
in human development as well.
And we also know from mice
experiments that PDX-1
expressing progenitor cells
can give rise to all
of the functional cell types
of the pancreas,
which I'll show you in a minute.
So in green here, you can see
the PDX expressing cells
in the pancreatic buds.
There are two buds in fact,
which bud off of the endoderm
tube, which is shown here
across the middle,
and the dorsal bud here
which forms first
and the ventral bud here.
So PDX are these green cells
highly expressed
in the pancreatic buds,
fated to become pancreas.
Now, PDX is also important
for insulin production
in the adult islet.
Shown here on the right
is a human islet isolated
from a cadaver donor.
And it shows in red
the PDX expressing cells.
And so PDX transactivates
the insulin locus and therefore
is very important
in the expression
and production of insulin.
Now the adult pancreas in fact,
has two functions
other than blood sugar control
for which the endocrine cells
are primarily responsible,
but it also has functions
to secrete enzymes
into the GI tract
to help us digest food
and to produce bicarbonate
to buffer stomach acid,
the function of which
is taken up by the acinar cells
and the exocrine cells,
enductal cells.
So the islets only make up
one percent of the pancreas.
Therefore, it's a fairly rare
cell type in our body.
The pancreas is not a
particularly large organ either.
An interesting fact
that may come into play later
is that endocrine function
of the pancreas is not necessary
during development
to control the embryo
or fetus' blood sugars.
In fact, the mother does that
in utero for the fetus
to promote growth.
But the blood sugar control
in the embryo does not require
the presence of insulin cells
during development.
So we wanted to look at,
can embryonic stem cells
differentiate into beta cells.
And a number of years ago,
some scientists in my lab
set out to test that hypothesis
question and spontaneously
differentiated embryonic stem
cells in culture, first through
an embryoid body phase
in suspension culture,
where they make these
embryo-like structures, and then
with subsequent differentiation
in the presence of serum,
but without any added
growth factors.
And what we found was that,
in fact, progenitor cells
do appear in these cultures
marked by PDX expression
in red here.
And within those clusters,
emerge a subset of cells that
express some of the endocrine
hormones, insulin and glucagon.
At a later stage, some of those
PDX expressing cells
express insulin in the cytoplasm
in granules and they also
continue to express PDX.
And, in fact, many of them
co-express the marker C peptide,
which is a marker of endogenous
production of insulin,
and is denovo production,
and is a critical marker,
and demonstrates bona fide
production of insulin.
We found that in mouse ES cells.
In human ES cells as well,
we could get PDX insulin
co-expressing cells.
That also expressed C peptide.
And these three markers together
are highly reminiscent
of an adult beta cell.
Now, we've done this many times
and others have reproduced
this work.
However, the observation
was made that it seems that
there were a large number
of PDX expressing cells that
could emerge in these cultures,
but very few
endocrine producing cells.
This image on the left
is a large patch
of PDX expressing cells.
But however, as you can see,
only two cells
within that cluster
are also expressing insulin.
We counted cells
in many cultures
of different cell types,
insulin, glucagon, etc.
We saw that there were very few
endocrine expressing cells
within the cultures.
In the range of 10-100 cells
expressing some of these
hormones
out of several million cells.
So there were quite few
endocrine cells.
And, in fact, there were
too few endocrine cells
to really test their function,
because the assays used
could be inhibited
by the other cell types there
that might take up insulin
or affect the insulin output.
Because there are few numbers,
it's been difficult to study
the function of the cells.
However, again, in summary
we do know that mouse
and human ES cells are capable
of differentiating into
pancreatic progenitors, and
that islet hormone expressing
cells can be produced,
and that the phenotype of
these cells is very reminiscent
of adult beta cells.
However, we also learned, and
I don't have time to show you,
that serum inhibits the
production of the cells.
And if you withdraw serum
at a later stage,
and reduce the serum component
in the cultures,
you can enhance
the endocrine differentiation.
So how do we then, if we have
a few endocrine cells
and we want to make more
endocrine cells,
if we're going to think about
generating millions of cells
for millions of patients.
How do we direct
the differentiation
of embryonic stem cells
into beta cells
and only beta cells?
How do we get rid of those
other cell types?
Well, in these cultures,
I didn't show you, but there
are probably other cell types
within the cultures.
We're not interested
in getting those.
We're interested in enriching
for a cell type
of the pancreatic lineages,
and that could be
either a differentiated
cell type represented
in green here by the terminally
differentiated beta cells
that are fully functional.
Or it could represent
a precursor cell
at a variety of stages
shown there in the other colors,
which might then differentiate
then along that lineage, but
they'd be relatively committed
and therefore generate
primarily endocrine cells, or
at least pancreatic cell types.
So how do we get to this
type of approach?
How can we direct the
differentiation in culture?
Well I think it's important
to think about, and what
the field has come around to
is that we need to take clues
from development.
And we need to take clues then,
from developmental studies
in lower organisms,
such as mice, and zebra fish,
and other lower organisms.
So we need to learn from those
mouse and zebra fish studies
and apply what we learn
there to human ES cells
and culture.
However, we know that human
ES cells don't necessarily
behave like mouse ES cells
and don't respond to the same
growth factors.
The mechanisms controlling
pancreas development in mice
might not be the same as
pancreas development in humans,
although there are certainly
some similarities.
So the same growth factor
that works for mice
that's been discovered
in a series of studies
in lower organisms,
might not function the same way
in human ES cells and culture.
It's certainly worth testing,
but we have to be wary
of the fact that we might
come up empty or be misled.
So some degree of empiricism
is probably necessary
in a series of studies
to balance rational approach,
take clues from development
and empiricism,
just a shotgun approach
in figuring out
what are the growth factors
or combinations that
might promote differentiation
towards a pancreatic cell type.
Keeping that in mind,
if we want to investigate
a large number
of developmentally relevant
growth factors,
or unknown growth factors,
looking at multiple different
combinations and concentrations,
we're talking about
a large array of experiments.
So we and others are working
along those lines, combining
both a rational approach
and an empirical approach.
But we're also
doing some other things.
So in addition to looking at
what are the intercellular
signaling molecules
that are elaborated by cells
and tissues during development
to drive and induce
differentiation
to various cell types,
we're also looking at
transcription factor
directed differentiation.
That is, can the over expression
of a particular
key transcription factor,
like PDX in embryonic stem cells
or subsequent transcription
factors are necessary
for later stages,
can those be used
to drive differentiation.
We're also looking at ways of
purifying cells in the cultures
by pulling out the cell type
of interest
using magnetic bead sorting
or flow cytometry.
What I'm going to tell you
about today is mostly focused
on developmental signals.
So there's been a series
of studies in the literature
that address this.
There are some very elegant
studies and represent
the state-of-the-art.
So this is one study where
a series of investigators
in Sweden said,
we don't know the signals.
Let's take the embryonic
pancreas and mix that
with ES cells; and maybe there
will be something elaborated
in the embryonic pancreas,
an unknown signal
that's produced that will
direct differentiation.
So this group took human
embryonic stem cell cultures
that were partly differentiated.
In a nonspecific fashion,
they collected certain areas
of those cultures
that were relatively enriched
in or pancreatic endoderm
and expressed PDX,
but were not able
to express insulin in culture
for those investigators.
And they took those derivatives
and co-transplanted them
into mice
with a mouse embryonic pancreas.
So what did the pancreatic
signals do?
The pancreatic signals
appeared to stimulate
the differentiation
of the human ES cells
into insulin producing cells
and other cell types.
So we have here a series
of immunostains for markers
of insulin,
a human nuclear factor.
And we can see that
there are insulin cells
that are detected in this tissue
underneath the kidney capsule
that are also expressing
a marker for a human nucleus,
so they are human derived
rather than mouse derived.
Similarly for glucagon
and similarly for amylase.
You can see that
there are other cell types
within this tissue that also
have a human nuclear marker,
and are derived from human
ES cells that are not expressing
these markers,
so it's possible that there are
other cell types here as well.
But nonetheless, they
demonstrated that there's
some signal within the mouse
embryonic pancreas that
seems to drive differentiation
towards an endocrine cell type.
And as their control, they used
a human -- and that did not
have the same effect.
So this was effect specific
for the embryonic pancreatic
tissue.
So that suggests that there's
some differentiation promoting
factor, some signal in this
early stage pancreas that is
promoting differentiation.
Other scientists have gone
a bit further and have looked
at specific growth factors and
a cocktail of growth factors.
These growth factors were tested
also based on a large volume
of developmental literature
and a specific factor
that has been shown now
and reproduced in several labs
to promote differentiation
towards endoderm.
And then with subsequent
additional growth factors
toward a pancreatic lineage,
they showed that activin
was an important growth factor
in this.
Activin is a factor
that stimulates nodal signaling.
By promoting nodal signaling,
they can promote definitive
endoderm differentiation.
So their culture protocol
is shown here in yellow
with activin and some of these
other factors shown here,
including FGF10
fibroblast growth factors,
cyclopamine, which inhibits
sonic hedgehog signaling,
retonoic acid, and a number of
other factors
in specific sequence
at specific time points
appear to optimally promote
endoderm, then pancreas,
then endocrine differentiation.
With such a protocol,
they were able to show that
insulin producing cells
were enhanced
and were dependent
on a high dose of activin.
I should say that although it's
clear that activin is important,
the absolute relevance
and importance of some of these
other growth factors that they
used in this culture protocol
has not yet been determined.
So with activin, they showed
that high doses of activin
and induction of nodal signaling
can promote endocrine
differentiation,
and specifically beta cells.
And here is a culture
in the center treated
with high doses of activin.
It shows in the green cells that
there are more endocrine cells
in the cytology panel below
that shows approximately 7%
of cells are producing insulin.
So this shows that activin
and nodal signaling
are important
for endoderm differentiation.
You can modestly enhance
the prevalence of endocrine
or insulin producing cells
in the cultures
derived from human ES cells.
However, this is still not
a purified culture.
And other functional studies
they did showed that the cells
do not behave necessarily
like mature beta cells
in response to glucose.
In response to high glucose,
they do not release
a large amount of insulin,
although they do express it.
That's really the current state
of the field at the moment.
And where do we have to
go to next to think about
a cell therapy?
What do patients want?
They want a relatively
purified population.
It doesn't have to be absolutely
purified, but it has to be
at least highly enriched.
We need to think about, can we
enhance that differentiation
with matrices and
tissue engineering constructs,
additional growth factors,
a degree of empiricism
and rational approaches.
And then we need to really show
that these cells function well
in vitro and in vivo.
For that, there are a number
of animal models of diabetes
that are available.
There is chemically induced
diabetes, that is established
model systems in mice, and rats,
and primates.
In addition, there are
autoimmune diabetes models
in mice and rats.
So those would be model systems
to test this cell therapy.
And not until we develop
a cell therapy that has
a robust ability
to restore normal glycemia
or good blood sugar control
in animals with diabetes,
can it then move forward
into the clinic.
So as one thinks about
delivering this to the clinic,
again, we have to think about
balancing efficacy and safety.
And before the FDA or any
regulatory agency would approve
an initial phase one
clinical trial, there would
have to be a series of tests
to look at efficacy and safety.
From the safety point of view,
we want to make sure that
these cells don't develop
tumors, or malignancies,
or are transformed,
or are unstable genetically
after transplantation.
That's an important
consideration.
We also want to make sure
that there's an absence
of transmission
of unusual infectious agents
that might be carried along
in the cell line,
unrecognized in culture.
So we'll have to go through
a series of tests.
Most of the ES cells to date
do not have any infectious
agents or any unusual viruses.
They have been tested
extensively, but they'll need
to be tested again
immediately prior
to going into cell therapies.
We want to avoid some
cell based adverse events
or adverse effects.
In islet transplantation, when
you put islets in the liver,
they cause focal fat deposition
in the liver, it turns out.
So that might not be
the best site.
And we don't know what
that focal fat deposition
would have in terms of
the long-term effect, whether
that would be a problem.
Unlikely to be a problem,
in fact, but there are
other cell therapies
like delivering cells
into the heart
that might cause some problems,
namely, arrhythmias.
Neural cells put into certain
areas might create problems
with integration and synapses
with the wrong cell types,
which might be problematic.
So all those things will need
to be tested and optimized
before advancing
to later stage clinical trials.
So those are some of the hurdles
and the challenges
moving to cell therapies.
There's a lot more to be done
in the diabetes field.
The earliest trials
of human ES cells
are phase one trials that are
just getting underway now
for spinal injury
with some investigators
in California.
And we're anxiously awaiting
the early results of those.
So, moving on to the next
segment of the talk,
we've covered current therapies
for diabetes
and what's going on in my lab,
as well as what's going on
in the literature
and the current status
of the field.
I'd like to move into
the next part of the talk,
and talk about generating
genetically matched
human ES cell lines.
Of what is the benefit of that?
Why are we interested in that?
This will deal with a little bit
the ethical aspects of
somatic cell nuclear transfer.
So one's interested in
generating genetically matched
human ES cell lines, because
one might get around the issue
of histo-incompatibility
and rejection.
And therefore a patient
who gets this therapy
might not reject the cells
without immunosuppression and
immunosuppressive medications,
or anti-rejection medicines
might not be required.
Is a patient that gets cell
therapy from a human ES cell
possibly in the future, would it
require a genetically matched
cell line to advance
to clinical therapy?
I would say probably not,
although there are
some advantages
and some applications.
But I'll mention a little later,
there may not be as much
of an advantage
in the application of diabetes.
I'll talk a little bit
about that in a little while.
But first, what are the
strategies that people have
come up with as ways to generate
histocompatible cells
for transplantation?
Well, histocompatibility
is largely determined
by the HLA
or human leukocyte antigens
that are present
in all somatic cells.
Those are the transplantation
antigens, so one could think
about generating cell banks,
or HLA specific cell banks
of ES cells,
much like a bone marrow bank.
However, that hasn't
really solved the problem
of histo-incompatibility with
bone marrow transplantation,
so you'd need millions,
and possibly billions
of different combinations
of different ES cells
to really establish a bank
that was useful in all patients.
One could think about deleting
or inserting these HLA genes
that are specific for the donor,
or are specific
for the prospective recipient
and deleting the ones
from the donor
or inserting new ones
that are specific
for the prospective recipient.
That hasn't been accomplished
yet, but I'm confident
that it might be in the future.
And I'm sure some investigators
are working on that.
Another potential way to do
this is to think about
generating hematopoietic
stem cells from ES cells,
transplanting those into
a patient, let's say,
with diabetes, and using
the human ES cell line
to make those
hematopoietic stem cells.
Creating a state of chimerism,
such that the recipient
no longer is going to react
or reject the cells
from that particular
human ES cell line.
And you could make a beta cell
perhaps from that same line,
and therefore they wouldn't
reject it and come back
with a therapy for a beta cell.
So that's a possibility, but
I think there's a lot of work
that needs to be done there.
Nuclear reprogramming has been
from very earliest days
within a year or so
after Jamie Thomson discovered
or isolated human ES cells,
nuclear reprogramming
was thought as a way to generate
genetically identical
human ES cells.
And that's the idea
of therapeutic cloning.
So therapeutic cloning, though,
is not reproductive cloning.
It is not likely to have
the disastrous consequences
that some believe, yet there are
ethical challenges and issues
with it that many have had.
So what are we talking about
with therapeutic cloning?
Therapeutic cloning can be
described by this diagram here.
And what potentially
would be involved,
is you have a patient that's
a prospective recipient,
or potential recipient
of a cell therapy.
You take a skin cell
or a somatic cell
from that individual, and
then go through the methodology
used to clone Dolly,
you would recover the nucleus
from that cell
and genetic material,
get an oocyte from a third party
individual, usually a volunteer,
a woman volunteer, who has
allowed recovery of oocytes.
Then one removes the nucleus,
inserts the donor nucleus,
go through a period
of development in vitro,
generate an embryo.
And then, from that blastocyst,
generate or isolate
the inner cell mass and generate
the human embryonic
stem cell line for the purposes
of differentiating
those cells in vitro,
to generate a cell type
that would be useful
for that patient.
And then transplant those cells
back into the patient.
So as I said, there are
a number of ethical issues
with this.
However, first I just want
to tell you that somatic cell
nuclear transfer derived
ES cells do show some evidence
of histocompatibility.
Although some people feel that,
or there is evidence
that there may be some
histo-incompatible component
to somatic cell nuclear transfer
derived human ES cells.
That would still require
anti-rejection medicines
if transplanted.
And the reason that's suspected
is that the clones might inherit
mitochondrial DNA
from the oocyte, and therefore
might present antigens,
either directly or indirectly
to the host immune system
that are foreign
to that nuclear donor
or prospective recipient.
And therefore, they may not be
purely incompatible.
But studies that have been done
so far, although not a series
of perfect studies, but
the studies that have been done
so far in mice, pigs and cows,
suggest that nuclear transfer
derived tissues
are genetically compatible,
or histocompatible,
with the nuclear donor.
So it's likely that they would
require very little
immunosuppression,
or none at all, if such a thing
could be applied to humans.
Another thing that I wanted
to tell you is that when
one thinks about histocompatible
tissues derived from somatic
cell nuclear transfer
derived human ES cells,
they're useful, potentially
for cell therapies.
But they're also potentially
useful for other types
of therapies, such as
is outlined here
in a very elegant experiment
done by a group of scientists
in Boston, which worked
toward correcting
a genetic defect in mice.
So, such a therapy could be
applied, not only to a patient
that needs a new cells because
of deficiency or organ failure,
but they could also be applied
to patients that have genetic
defects in the following way.
So what these scientists did
is they had
an immunodeficient mouse,
up here in the upper part
of this diagram.
They took some skin cells or
fibroblasts from that animal,
cultured them.
And then used somatic cell
nuclear transfer to take the
nucleus from those skin cells
that are syngeneic
to that animal, and then
generate embryonic stem cell,
in this case,
a mouse embryonic stem cell
from those skin cells.
And those skin cells, of course,
were genetically deficient.
They had this genetic defect
of the RAG2 deficiency, which
made them immuno deficient.
And because they have this RAG2
deficiency, they are unable
to make T and B cells.
And the animals
don't have an immune system.
So they set about repairing
this deficiency
by putting a wildtype gene
into these ES cells and
correcting the genetic defect
with -- recombination
using a wildtype locus.
Then they took those
genetically corrected ES cells,
differentiated them
into embryoid bodies, then
over expressed another gene,
which drove hematopoietic
cell development
or blood cell development.
Then they took those blood cells
and put them back into
the immunodeficient animal.
And new syngeneic blood cells
were formed in that animal,
partially correcting
the immune deficiency.
So this is an example
of how a genetic defect,
an isolated genetic defect
could be corrected
by a combination of gene therapy
and stem cell therapy, and
somatic cell nuclear transfer.
I think a very elegant
experiment.
Why do people have ethical
problems with somatic cell
nuclear transfer?
Well, it all hinges
around these two stages.
The ethical challenges are,
the main probable is that one
is generating a embryo
for the sole purpose of research
or destruction.
And that, in essence, is
one of the major problems with
somatic cell nuclear therapy.
Another major issue,
another ethical issue is that
in order to get a large number,
one needs to get
a large number of oocytes
to make this happen.
The process, as we currently
know it, is extremely
inefficient.
There's a lot of wastage
such that a large number
of oocytes are necessary
to do one experiment, to
generate one embryonic
stem cell line that might be
genetically matched
by somatic cell nuclear therapy.
So where does one get
a lot of oocytes?
One gets them from
women volunteers who have been
hyperovulated or hyperstimulated
with hormones, and then doing
an ovary recovery procedure,
which is although
not extremely risky, and is done
routinely for in vitro
fertilization procedures,
does require a procedure,
invasion of the patient,
and has some potential risks.
It's certainly not without risk.
It's associated with some pain
and discomfort, as well.
So if one thinks about doing
that, one has to put many women
simultaneously
through that process.
And that's what was done
in Korea a number of years ago.
That was considered
ethically very questionable
in many western countries.
Nonetheless, we need to improve
somatic cell nuclear transfer
so that perhaps one can do this
without an oocytes
or the volume of oocytes
that are necessary, perhaps
the ooplasm might be used.
However, one still has issues
with generating
a human blastocyst or embryo
for the sole purposes
of destruction.
Most cases, ethical and
political debates about science
come after a major discovery.
It's fairly unique that
the ethical and political debate
surrounding somatic cell nuclear
transfer have generated
a whole new industry of science,
and that is to develop
innovative methods
for creating histocompatible
matched human ES cell lines
without destroying an embryo,
or without needing
a large number of oocytes.
So since a few years ago,
a number of approaches
have been put forth.
Really, a whole cottage industry
has arisen around this.
These are some of the methods
that have been published
for generating histocompatible
human ES cell lines,
without the process
of producing a human embryo
and without
some of the ethical issues.
I won't go through
all of these in a lot of detail,
but one of them is an altered
cell nuclear transfer.
The idea here is to generate
an embryo that cannot implant.
The way to do that,
the way the scientists did that
is to knock out the CDX2 gene,
which is required
for trophectoderm
or placental development.
And, therefore, although you've
got a functional intercell mass
from which one could derive
ES cells, you would not get
implantation of that embryo.
However, one still produces
an embryo earmarked for
destruction, albeit an abnormal
embryo in this method.
In addition, it still requires
a large number of oocytes.
So in my mind, it does not get
around the major ethical issues.
Another way to think about
doing this is to not destroy
the embryo.
And what one can do,
as is commonly done in prenatal
genetic diagnosis, associated
with in vitro fertilization,
when you generate the in vitro
fertilized embryo in culture,
you can then sample it and
take out one cell of that embryo
or one blastomere,
and then test that
for the genetic defect
that might run in the family
of that family that's having
the in vitro fertilization.
So you can keep embryos that
do not have that genetic defect
and then implant those.
So some scientists
went ahead and did that, and
showed that you could generate
a human ES cell
at least transiently,
that has some phenotypes
of a human ES cell, from
a single cell or a blastomere,
and that does not destroy
the embryo necessarily.
Although in this publication,
the embryos were not implanted
and did not go on
to have normal development.
So that's a little bit
of an unknown, yet it does show
the principle that possibly
one could develop human ES cells
of a single cell of the embryo
without destroying the embryo.
Still, a large number of oocytes
at the current rate of
producing those embryos in vitro
would be necessary to do that
on a wide or large scale.
Another potential method
is parthenogenetically derived
embryonic stem cells.
Those were recently described
for human ES cells,
and previously in mouse
and primate ES cells.
Parthenogenesis is the process
of activating development
without a sperm,
that is, auto-activation
in culture of an oocyte.
One of the down sides here
is that the oocyte,
or parthenogenetically derived
human ES cells would be
genetically identical
only to a woman.
So it would be a therapy
one could develop cells, then,
only for females
and not for males.
The last method,
just recently described here
by Jamie Thomson and Dr. Yu
at the University of Wisconsin
is induced
pluripotent stem cells.
I'll go into that methodology,
or technology,
in a little bit more detail.
Again, as a potentially
useful way for generating
genetically matched
human ES cell lines.
So what was the concept here
is that one can take
a somatic cell,
a skin cell for instance, as
they did in their publication,
from an adult.
In their publication, they used
some neonatal skin cells
and some foreskin cell lines
rather than adult cell type.
We'll get into
the differences there.
But taking that somatic cell
or skin cell from an adult,
and then modifying it by
over-expressing four
key pluripotency genes
in that somatic cell,
at least transiently,
showing transient expression
and then that would promote
dedifferentiation
or reprogramming towards
a pluripotent stem cell
like phenotype.
The genes they used were Sox-2,
Oct-4, Nanog and Lin-28.
Others in mice and humans
have shown some other genes
may play a role.
The minimal set of genes has not
actually been determined yet,
But the IPS or
induced pluripotent stem cells
that were generated
by this group
are really strikingly similar
to human embryonic stem cells.
They have high telomerase
activity.
They express some of
the phenotypic markers
of human ES cells.
They can differentiate
into all the germ layers
and teratomas in mice.
When compared to human ES
cell lines by a study looking at
hundreds and thousands of genes,
they were virtually identical.
So their study is shown here.
This was published in Science
this past year,
approximately six months ago.
They used these four genes
and over expressed them
in these foreskin cell line,
IMR90 shown there in panel B,
in this image
taken from their publication.
And you can see that
in the cells that had
all four genes expressed,
they had the largest number
of IPS colonies
that were produced.
And they sequentially subtracted
one of each individual genes
and showed that the two
or three key factors were really
Oct-4, Sox-2 and Nanog, which
are common pluripotency genes
that have been described
to be expressed
in human ES cells
and in mouse ES cells.
So you see that we can induce
a pluripotent state
by over-expressing these genes
and somatic cells.
So the question next is,
will these IPS cells be useful
clinically in the future?
I think the question remains--
the answer to that question
still remains as we don't know.
But many are excited by this
development, though it still
does not obviate the possible
utility of wildtype human
ES cells and improvements
in somatic cell nuclear transfer
or therapeutic cloning,
primarily because these are
genetically re-modified cells.
So one of the issues with
IPS cells is they did this
with a neonatal
and foreskin fibroblast cells,
and a foreskin
fibroblast cell line.
When one wants to think about
therapy, we need somatic cells
from an adult patient,
an aged patient, perhaps.
Someone who has cells
that are undergoing senescence.
So what the right cells are,
and whether it would be possible
for the skin cells from an adult
is not exactly clear
at the moment.
But it will be an important
thing to test.
These cells are also highly
genetically manipulated,
and have lentivirus
inserted into their genome.
So because of that,
there's potential
for transmission of virus,
and some of the problems
with gene therapy, and some
of the risks of gene therapy
associated potentially with
transplanting these cells.
So it will be very important
to find other ways to insert
and express these genes.
And it will also be important
to test these cells for safety
prior to clinical therapy.
The other aspect
is a more mundane question,
but a simple question.
Because they have the insertion
of these genes in a certain area
of the genome,
have that insertion
or that process disrupted
differentiation potential
for differentiation
into differentiated cell types?
That is, will it prevent
differentiation or lead to
incomplete differentiation?
And we don't know that.
But that is potentially
surmountable by generating
multiple cell types
from a somatic cell for that
particular patient
and then testing them.
But nonetheless, that will be
important to test
whether they have the same
differentiation potential
as wild type human ES cells.
So in the last couple minutes,
I just want to talk about
whether genetically matched
human ES cells
will be useful clinically,
for specifically patients
with diabetes.
And the reason why
that might be a question
hinges around
the autoimmunity issue.
Patients with Type 1 diabetes,
who might be recipients
of this therapy
have autoimmunity.
They have lifelong autoimmunity,
meaning that they still retain
memory T cells that are poised
to attack self-cells,
if new self-cells
could or would be transplanted.
That is, many years
after transplantation,
they still have
active autoimmunity as part of
their immune make-up.
The reason why we know that
is from mouse studies,
and from some human experiments
in the early days
of pancreas transplantation.
In the early days
of pancreas transplantation,
before immunosuppression, some
segmental pancreas transplants,
or partial pancreas transplants,
were done between
genetically identical twins
that were discordant
for diabetes incidence.
One had diabetes, one did not.
But they were genetically
identical twins.
The transplants
were technically successful,
again this was before the era
of immunosuppression,
so the patients
were not immunosuppressed.
The patients remained
off insulin for several months.
But within three months,
or four months, the patients
returned to insulin therapy
and lost insulin secretion.
And when those grafts
were looked at, they had
the pathological findings
of recurrent autoimmunity.
There was immunological attack
on those beta cells.
So it's clear that autoimmunity
may destroy self-beta cells
if allowed to develop
from an embryonic stem cell
or transplanted
from an embryonic stem cell
in a particular patient.
However, we also know
that in a small group,
a series of patients done at
the University of Chicago,
that twin transplants
with low amounts
of immunosuppression,
less than is needed
for normal organ transplantation
can be successful.
That is, only a small amount
of immunosuppression may prevent
that autoimmunity attack
from attacking a new pancreas,
but not as much as is necessary
for a typical organ transplant.
So I think I'll stop there.
We've covered a lot of things.
I hope you got something
out of that.
I just acknowledged some of
the scientists in my lab
who have done some of the work
I showed you,
showed there in the upper right,
and some of the people that have
helped us with the research,
some of the studies
I have not showed you.
And our funding agencies
for which we are very grateful
for their continued support.
So I'll end there.
I'd be happy
to take any questions.
>> For the islet
transplantation, what does
that accomplish that pancreas
transplantation doesn't?
>> It accomplishes all the
same things, hopefully,
a pancreas transplantation does.
However, it's a less invasive
procedure.
The patient isn't
under the knife for six hours.
There's no big scar.
There's less risk of bleeding.
So it's a much less invasive
procedure, and perceived to be
a little bit more safe
in that regard.
However, on the down side,
at the current state of the
therapy, it's less durable
than pancreas transplantation.
Whereas islet cell transplant
recipients may achieve
insulin independence
for a number of years,
most return to requiring insulin
within five years, whereas
that's not the case with
the pancreas transplantation.
Those patients generally have
preserved function,
the majority of patients
have preserved function
for more than five years.
>> You talked about
Type 1 and Type 2 diabetes.
For Type 2, it is obviously
much more prevalent.
But were you saying that
that's basically,
Type 2 diabetes is basically
that you become resistant and
your receptors don't really
work to bind insulin anymore?
>> That's generally felt to be
the main mechanism.
Although it's not really known,
and it's clearly multifactorial
and takes on different forms
in different patients.
So scientists are still studying
what exactly are the mechanisms
of that insulin resistance, or
the causes of Type 2 diabetes.
However, most patients,
or many patients do end up
having a relative deficiency
of insulin as time goes on.
And many patients, although
right after their diagnosis
of Type 2 diabetes,
can be treated with diet
or exercise or pills,
eventually go on
to require insulin therapy.
So there are some similarities
in terms of the end result.
So when people think, however,
about stem cell therapy
for diabetes, for the most part,
they're focusing on
Type 1 diabetes.
And if a therapy of any sort
might be found to be effective
for Type 1 diabetes,
then I would envision
people starting to apply it
to a subset of patients
with Type 2 diabetes.
But one would require a lot more
production of insulin,
a more robust production
of insulin,
because that potential
for insulin resistance.
>> Focus on maybe, finding a way
to re-express the receptors
or you know, find out exactly
what's wrong with them, because
I feel like since it's
so much more prevalent,
like everything I hear
seems to do with Type 1.
>> Well there's a lot
of research going on
for Type 2 diabetes.
Much of the focus of the
American Diabetes Association,
in fact, is on Type 2 diabetes.
Basic science, clinical aspects,
a variety of things.
Whereas the Juvenile Diabetes
Research Foundation primarily
focuses on Type 1 diabetes,
but not exclusively.
So there's a kind of
separation there, if you will.
But there's a lot of work
going on with looking at
the nature of the disease
of Type 2.