- Good afternoon.
I'm Tomiko Brown-Nagin,
the Dean of the Radcliffe
Institute for Advanced Study.
Welcome to this year's
Kim and Judy Davis
Lecture in the
Sciences featuring
renowned neuroscientist
Dr. Mu-ming Poo.
Mu-ming is the Founding
Director of the Chinese Academy
of Sciences Institute of
Neuroscience in Shanghai
and Senior Investigator in
the institute's Laboratory
of Neuroplasticity.
He also leads the academy's
Center for Excellence
in Brain Science and
Intelligent Technology.
Before relocating
to Shanghai, Mu-ming
worked as a researcher and
professor in the United States
for close to 40 years.
And he is the Paul Licht
Distinguished Professor
in Biology Emeritus at the
University of California,
Berkeley.
Mu-ming is well known for the
breadth and innovativeness
of his scholarship.
His research has drawn insights
from both physics and biology,
and he's made
critical contributions
to several subfields
of neuroscience.
Mu-ming has shaped
our understanding
of the neurobiology
of learning and memory
and of the brain's
ability to adapt
over time, known as plasticity.
For his work in
this area, Mu-ming
received the 2016
Neuroscience Prize
from the Gruber Foundation
at Yale University.
More recently,
Mu-ming has focused
on developing non-human primate
models of brain diseases
such as Alzheimer's and
Parkinson's to advance
scientific understanding
and, eventually,
to treat these diseases
more effectively.
The development of
non-human primate models
is potentially more fraught
and certainly more complex
than it might sound.
This work involves both gene
editing and cloning primates.
And the resultant
ethical questions
have certainly captured
public attention.
A common starting point in
conversations on this topic
is the observation
that animal models
have been and are powerful
tools in biomedical research.
Non-human primate models
represent the next phase,
advocates argue, promising
critical insights into human
neurobiology that
other avenues cannot.
Some ask whether the ethics of
gene-edited and cloned animals
should vary across species.
For example, is the
cloning of primates
for research ethically different
from the cloning of rodents?
Another common and
important question
is whether the cloning
of non-human primates
might open the door
to human cloning
and, therefore, a whole host of
other ethical considerations.
Now I won't attempt to
answer these questions.
Trust me.
Yet, what's clear is that
there's a complex interplay
between scientific advancement
and the web of standards--
international,
national, institutional,
and indeed moral--
that aim to ensure ethical
research practices.
This isn't a theoretical
discussion alone.
Last year, the Institute of
Neuroscience that Mu-ming
directs announced it had
produced the first ever primate
clones via other technique of
somatic cell nuclear transfer--
the result, a pair of
genetically identical macaque
monkeys.
Earlier this year,
Mu-ming and his colleagues
shared that they had
successfully disabled a gene
in macaque monkeys related
to the sleep-wake cycle
and then created the
world's first clones
of a gene-edited primate.
These are watershed moments
for science and technology,
forcing factors
for ethical debates
and opening to
potentially game-changing
biomedical research.
I hope that today's lecture will
be an opportunity for us all
to engage with the
scientific possibilities
and the ethical
questions at hand.
Before I turn things
over to Mu-ming,
I want to extend my
thanks to Kim and Judy
Davis for their generous support
of the Radcliffe Institute
and of the dean's
lecture series.
I'm also grateful to the members
of the Radcliffe Institute
Leadership Society and to
all our annual donors who
make the work of the
institute possible.
Thank you very much
for your support.
And now please
join me and warmly
welcoming Dr. Mu-ming Poo.
[APPLAUSE]
- Thank you very much,
Dean Brown-Nagin,
for this very nice introduction
and very insightful comment
at the end.
And I hope I would
have time to discuss
this about the ethical issues.
Many worthwhile debates
and consideration
should be given to
the future research
in the non-human primates.
But, begin with my
talk, I would show you
what we think the usefulness
of this model system would be.
Let's begin with the brain, the
human brain, probably the most
mysterious objects on Earth.
Now I heard that
last week you were
having lectures on
the outer universe,
on the astrophysical,
astronomy aspects.
Now this is the inner
universe, equally mysterious.
And we hope to understand
how the brain function.
Now the outer
cover of the brain,
the so-called cerebral
cortex, is most developed
in the primate species.
The inner part, more
complex, ancient,
evolutionarily ancient part, are
very similar to other species,
mammalian species as rodents.
But this outer cerebral cortex
is the seat of humanness.
It produce many
complex functions
of the higher cognitive
function of the brain.
And that's what we
need to understand.
Now, the brain is very
complicated networks
of nerve connections.
The human brain is
composed of about 10
to the 11th nerve
cells or neurons.
And each cell is
making connections
with at least a
thousand other cells,
making 10 to the 14th
[INAUDIBLE] number
of synapse in the brain.
These form a very complex
interconnected network.
Within this network,
there's neural circuits,
specific pathways
of signaling, that
are responsible for
specific brain functions.
Now, in the cortex, here is
a drawing by Ramon y Cajal
more than 100 years ago, only
a few cells in the cortex.
In fact, this cortex is
packed with this network.
They are not only
complex connections.
The cell types are complicated.
They are at least
hundreds of different cell
types in the brain.
The extent of diversity
is yet to be figured out,
and many people are
still working on it.
Josh Sanes here is one of the
pioneers in this area trying
to figure out how many different
types are there in the brain.
And they are different
in morphology,
and they are different
in physiology,
in their firing patterns.
Now all these differences
gives the complexity
of the signal processing.
Just the example, here is the
52 cells from a mouse cortex,
50 cortical neurons.
We only plotted projections,
the long range connections
of these neurons.
Of 52 cells across
the entire brain,
you can see the complexity.
We labeled different neurons
with different colors.
Now, when you think
about billions
of cells in the human brain,
all with all the connections
around and carrying out
different functions,
to figure this out is really--
it's the daunting task.
Now, eventually,
we'd like to know--
to understand at least three
levels of cognitive process.
I would say these
three levels can
be distinguished as a
cognition of the outside world.
This is nearly all
the animal does it.
They need to recognize the
external world in order
to survive, in order to
respond appropriately.
This includes the
sensory perception signal
from an environment, integration
of multiple sensory signals,
transform the sensory
signal into a response,
a sensory motor
transformation, attention
to a particular stimulus
from the environment,
memorize the experience,
learning and memory,
categorize different objects
in the environment or making
decisions.
All these are many animals
have these functions.
So you can use many
animal models from worm
to flies to rodents
to monkeys and humans.
And so this is
[INAUDIBLE] our cognition.
I say the first
level of cognition.
But there is another
level of cognition
that is called the cognition
of self and non-self.
This is more complex.
And we need to perform
complex executive function
in dealing with a lot
of peoples in the world.
We need to have self-awareness.
We know self from a non-self.
We have empathy of
feelings of others.
We have complex social behavior.
We have theory of mind, knowing
what other people are thinking.
All of these are cognition
based on very important aspects
of knowing yourself from others.
And that's the basis
of all social behavior.
Now this is not
all animal has it.
I mean, we can give an example
in just a minute about empathy.
But it's probably best studied
in non-human primates, closer
to human, with all
this complex cognition.
Now, finally, there's
another level of cognition.
I would say this is
the jewel of the crown.
That is the language.
And this is the ability to
have vocal communications that
are very complex with
syntax and grammar,
open-ended construction
of sentences.
Many animals can have
vocal communication.
Even chimps can learn
hundreds of symbols
corresponding different
things, but chimps were never
able to learn to form sentences
with syntax and grammar.
This is well proven over the
last many decades of work
with teaching chimps
the human language.
So this is human-specific.
And we can study this by looking
at humans with injured language
dysfunction, but
that's very limited.
For the mechanism studies,
you need perturbation that
are experimentally controlled.
So how do you study language
circuits in the brain?
Perhaps, monkey
would be-- you might
be able to study the evolution
of language, early prototype
of language in monkeys.
For example, we are
now teaching monkeys
to learn sequences,
reverse sequences,
the recursive
structure of symbols.
The monkey can learn to do this.
And what happened
to their brain when
they learned to do
this primitive language
construction?
What happened to the brain?
Is there any way of
constructing these circuits that
are more capable than
simple vocal communication?
These are the
problem that one can
study with non-human primates.
Empathy-- empathy
is a very big word,
very important-- the power to
understand and imaginatively
enter into another
person's feelings
and identify with
their feelings.
And this is very human.
And there are many
level of empathy
if we look at the evolution.
Now we have very
primitive form of empathy
we call emotion contagion,
mimicry, or sympathy
and compassion,
pro-social behavior.
These can be found in
many animal systems.
I mean, you can
have examples that
very much show this behavior.
Perspective-taking, that's
a little bit difficult,
to know what other
people are thinking.
One of the things is
the gaze-following.
If you look at each other's eye,
and then you shift and shift
your gaze to a
different direction,
that person would
turn their head
and see what are you looking.
The person would know-- if they
know what you are thinking,
this type of gaze-following is
use of this perspective-taking.
All these, the higher you
go, it's more difficult.
Now, in the monkey, there
is a very interesting set
of systems developed in
the primate system, first
discovered in a monkey,
called mirror neurons.
A monkey would have neurons in
their motor cortex, premotor
cortex.
These neurons will fire
if they grab something,
but they also fire when
somebody else grabs something,
show the same motion.
The monkey-- the
same neuron would
fire when the monkey
is doing it itself
or seeing other monkeys doing it
or other human beings doing it.
These are called mirror neurons.
They were found
in monkeys first.
And in human, if you do
functional MRI imaging, brain
imaging, you can find that,
when people show empathy,
there are specific areas
in the brain active.
That can be shown.
So they are very specific.
Now, lower species--
let's say rodents.
There's no convincing
evidence that rodents
show this empathy-like
neuro-structure.
So one can look at empathy.
There's a gradient of empathy
from the affective, just
emotional empathy, feeling
other parties' injury,
for example, the pain, or
gradually to the higher empathy
we call cognitive empathy,
the perspective-taking, theory
of mind, the understanding of
what other people are thinking.
These are higher
level of empathy.
They all depend--
the higher you go
to the right, the more
dependent on your self
and non-self distinction,
to know other people
different from you.
That's the self and
non-self distinction.
This is a gradient probably
of evolution of this capacity.
So it would be nice to
understand the circuit basis,
knowing how these develop.
Now self-awareness,
there's a way to test it.
One [INAUDIBLE] in the last 40
years, the primate biologists
or animal behavior
studies have shown
you can actually use mirror
self-recognition as a way
to show self-awareness.
If you put a dye or a marker on
the face of a two-year-old kid
and give her the
mirror, she should--
she will scratch that mark.
That's called face
mark test, knowing
that what she is seeing
in the mirror is herself.
And this ability of
mirror self-recognition
will appear in 85% of the
kids by two years old.
But, if you test by one year
old or one and a half year old,
the majority of
them cannot do it.
In fact, in old-year-old
kids, very few
can do this mirror
self-recognition.
So what happened?
What happened to
the baby's brain
when they were able to
do self-recognition?
Is it learned?
Or is it inborn?
Is it, once they develop
to a certain stage,
they will have this ability?
Now this will be useful.
So we thought that, if we can
make a monkey develop this
ability, then we might be able
to figure out how the monkey
developed this self-awareness
because the monkey can never do
this--
can never pass this mirror
self-recognition test.
Put a mark on a monkey's face.
Even with a mirror in the
cage from the early period
after birth throughout life,
the monkey will never learn.
So this was an
experiment demonstrated.
For 40 years, this
hasn't been successful.
So we thought that
this would be useful
because there are some
psychiatric patients
or autistic patients.
They appear to be
impaired in this test
if you do this face mark test.
It's not clear
whether they are just
not interested in the
face, their own face,
or they fail to
recognize themselves.
This is unclear.
So it would be nice to know
what self-awareness as reflected
by mirror self-recognition is.
So this is the experiment.
So two colleagues of
mine decided to put--
to train a monkey sitting
in front of a mirror.
Here is the monkey sitting
in front with head fixed.
And Neng Gong and
the student Cheng
put a laser pointer from
the side on his face.
This is a higher power laser.
It gives you a little
bit of warm feelings.
So the monkey would feel--
see a spot in the
mirror and a feeling
of warmness on the face.
So they make connections,
associations between a mirror
image and their own body.
So this is how the experiment
starts if we can see this.
So they were rewarded.
In beginning, they don't
know the connection.
So it takes training.
A few weeks, they start
to know the association.
And they would be
rewarded if they
touched the spot correctly.
So now, once they are
trained after a few weeks,
you can do face mark test.
They pass it.
Actually, 5 out of 7 monkeys
passed the face mark test
once they can do this
in front of mirror.
That shows that they can--
now the face mark test is
they scratch their face
with a mark in front of face.
But the primatologists,
such as Frans de Waal,
people studying primate biology,
they don't agree with this.
They thought-- they think that
this is a conditioned response.
The monkeys are just
conditioned to do
this when they see a spot,
to do this on their face.
So training to touch the
face is a conditioned monkey.
So the monkey may not
recognize themselves.
It's just a response.
So we have to do another
experiment later.
This experiment, we
do not touch the face.
Our laser spot is
on the back of the--
on the backboard.
The monkey have to touch--
looking at a mirror image,
touch that spot precisely
and quickly.
Once they touch right,
they get a reward.
Now, in the beginning,
they cannot do it.
They don't know where
their hands are.
By chance their hand is in the
right spot, they get a reward.
So, after many, many weeks,
very slow, many weeks,
they are establishing--
they begin
to know that the image of
the hands in the mirror
is their own hand.
So you have a--
they move their hands
with their joints.
So they have a self
body awareness.
That self body
awareness is linked
with the image in the mirror.
So then they're suddenly aware
that that image is themselves.
Now we do mirror face test.
Now we never train
the face, right?
Never trained to touch the face.
The face mark test
is this training.
Now we secretly put some dye
on the face of the monkey.
And, in the cage,
they will see this.
They will touch that spot.
This is the standard.
People think this means
they recognize themselves.
There's spontaneous activity
of pulling their hairs.
We never trained it.
They just sit in
front of mirror.
They will pull their hair.
And they become
interested in places
they have never seen before.
[LAUGHTER]
So they're--
[LAUGHTER]
Right, so the one
with yellow collar,
that was untrained monkey.
It was sitting in the same cage
for half a year, never learned.
He just was just watching
the other monkey do all this.
He would never show.
We do a face mark test.
They never acquire the ability.
So, without establishing
this association
between the face, the mirror
image, and themselves,
they would never learn that is--
to recognize themselves.
So the implication
of this study is
that monkey can learn
mirror self-recognition.
Now this mirror
self-recognition was
used by many animal behaviorists
to be a sign of self-awareness.
If they can show this, then
the animal has self-awareness.
But our study seems to say that
the mirror self-recognition is
only an experimental tool
to reveal self-awareness.
The monkey is learning
to associate themselves
with the mirror image.
Once they learn that, they
can use the mirror as a way
to review themself,
review the self-awareness.
So this self-awareness maybe
can be in many animals.
It's just we have to find
a way to reveal that.
The mirror self-recognition
is just an experimental way
of revealing that.
So this is actually twisting the
original idea of using mirror
self-recognition as a tool for--
as a sign for self-awareness.
So now impaired mirror
self-recognition
in patients, in autistic
or schizophrenia patients,
is that indicating defective
visual-proprioceptive
association or rather than
a lack of self-awareness?
That's an interesting question.
Now, again, we need to show.
Maybe we can train them--
train the association
or find a way
to establish the association
if that's a defective aspect.
So this could be--
mirror self-recognition may
be a way of therapeutic tools,
I say, to help these patients.
Now, finally, the babies--
now so the baby, two years
old, why two years old?
It turns out that,
during that period,
the babies start to
be aware of this.
So, after our experiment
was published,
a colleague of [INAUDIBLE],,
Sid Kouider in Paris,
he started to think that
maybe the baby can be trained.
So he took one-year-old
baby, put a wristwatch,
which gives some
touching sensation,
but with a light on the watch.
So the baby would see a light
in the mirror and some feeling
on the hand.
And the baby learns,
one-year-old baby.
Many of them learned quickly,
much faster than the monkey.
So this learning or the ability
of mirror self-recognition
is probably a
learned experience.
I mean, they start to
understand what it means.
The parents telling, oh, look
at that baby in the mirror, baby
in the mirror.
After a while, they learn
that that means themselves.
So maybe that's how they learn.
So this is the still interesting
for the cognitive development.
So now another twist
of my presentation,
I want to talk about social
burden of various brain
diseases.
According to the WHO,
the brain disease
is now counting number
one in just overall.
All the brain diseases together
have counting about 28%
of all the social burden,
higher than cancer
and cardiovascular disease.
Now this is important
for an aging society,
the neurodegenerative
disease, and also
for the modern
society, depression
and addition problem.
Altogether, it's a
problem we have to face.
Now, to understand all the
bases, scientific bases,
and the pathogenic
mechanism of these disease,
it may take many, many decades.
We still don't know exactly
how the Alzheimer's disease
develop.
What is really the
causal factors?
So we need to have
therapies urgently
within next 20, 30 years.
Otherwise, the medical care
system will be bankrupt.
And so this is the
problems we are facing.
The problem facing brain
disease treatments,
pathogenic mechanism is unclear,
including many neurological
and psychiatric diseases, but it
takes decades to figure it out.
It is also difficult to
identify specific drug targets
because, many of
the dysfunctions,
specific circuits dysfunction.
Now all these other
brain circuits
are formed by similar neurons--
even though there are
many diverse types--
and similar synapses.
So it's hard to find
specific targets that address
certain dysfunction.
So this is a difficulty.
It is also very slow
to develop the drugs.
I mean up to 10 years for
development costs billions.
And the failure rate for brain
disease drugs is more than 90%.
Some people say more
than 95% or even 99%.
So one of the main reasons
is inappropriate animal model
for preclinical drug testing.
Now, before you go
into clinical trial,
you have to do three
types of tests.
You test the safety of the
drug or the metabolism,
like the metabolism of
the drug in the animal.
And, also, you
test drug efficacy.
Large animal like
macaque monkeys
are used for safety
tests and drug metabolism
because they are
close to humans,
but the drug efficacy tests
are all tested on rodents
because only rodents have
disease models, brain disease
models.
And that appears to
be inappropriate now
because their physiology
and their anatomy
are so different from human.
That's why many of the drugs
that passed the drug efficacy
tests in preclinical
animal studies
failed in the clinical test.
So this is one of the reasons
I think having animal models is
very important.
So, since 2001, the
transgenic gene-edited monkeys
has appeared.
And you have many
different type.
You can have overexpression
of human genes
in monkeys, disease gene monkeys
producing disease phenotypes.
You can do now editing, genetic
editing, CRISPR-Cas9 type gene
editing in monkeys.
You produce a specific gene
knockout or even knock-in.
All these allowed
the possibility.
Now we can edit the
embryos of the monkeys
and produce progenies that
have disease phenotype.
So I will show you some
examples today of these studies.
Now the first example
I want to show you
is a MECP2 transgenic monkey.
And this is a protein.
MeCP2 is a protein
identified by Huda Zoghbi
to be the origin or the
causal gene for Rett syndrome.
The mutation of this gene
causes Rett syndrome, primarily
a subcategory of autism.
Now overexpression and
duplication of this gene
are also found to have
some autistic phenotype.
So we decided a few
years ago to over express
a human MECP2 in
monkey hopefully
to get some autistic
phenotype in the monkey
for studying the
development of autism
and perhaps even
therapy of autism.
So this we express using a
neuron-specific promoter,
AAV viral transfection in the--
the expression in the
oocyte, the monkey oocyte,
and then in vitro
fertilize the oocyte
and then take the
developed embryo
and implant it in a surrogate
mother to get offspring.
So we obtain a number of these
transgenic monkeys, actually
seven of them.
These seven monkeys
all have transgenes,
the extra human genes inserted
in different chromosomes.
Here you'll see that
they are randomly
inserted with different variable
copies in different chromosomes
in the monkey.
Now these monkeys show a
phenotype, show behavior
phenotype.
You can show that there's
specific expression
of this transgene in these,
specifically in neurons,
in nerve cells.
One of the phenotype
is that they
seem to show abnormal
locomotive phenotype.
They tend to make circular
motions in their cage,
much more than the
usual wild-type monkeys.
These are the
transgenic monkeys,
clearly move a lot
doing circularly.
We stereotype them, you
can see, stereotype motions
in the cage and constantly.
And we consider this
as a similar thing
the stereotype motions
of autistic patients.
Now they also show high anxiety.
We're using this threat-related
anxiety and defense test.
Facing the monkey,
eye to eye the monkey
will show anxiety by their
vocalization of grunts.
The grunts productions
are highly elevated
in these transgenic monkeys,
showing the higher anxiety.
This is standard
anxiety test in monkeys.
And, most importantly, the
social behavior-- and this
is one of the key for
autistic phenotype.
When these monkeys
are in a group,
these transgenic monkeys
shown here on the--
showing here interact much
less than the wild-type monkey.
They interact in pairs.
If you take two monkeys
and isolate in new cage,
they interact very
little with each other,
much lower than
their interaction
with wild-type or interaction
between wild-type.
So there's clear social
dysfunction in this monkey.
You can also test the cognitive
function via the so-called WGTA
cognition test, but
mostly memory tests.
You can see that, in
all these memory tests,
they are doing now on
average OK, similar
to the wild-type monkey, but
they are highly variable.
All these transgenic,
seven transgenic,
varies a lot, while,
in the wild-type,
they are very much
a clear cluster.
They're similar.
They seem to be
slower in learning,
but they eventually can learn.
So there's not big difference
in their cognitive function
except high variability.
So this is a problem.
You have a transgenic with
the insertion of a human gene.
There are different places
with different variable copies
in different chromosome.
The phenotype is diverse.
So that's not a good model.
We cannot do experiments where
you compare experimental with
a control.
We can also generate a second
generation of the monkey,
obtain one of the monkey that
germline transmission yielded
five monkeys, which also
show similar chromosome
insertion persisting
in a second generation.
They are social-- social
interaction also reduced
in the second generation.
So this is a phenotype that is
transmitted through germline.
Now that's a first example.
The second example is a little
bit more interesting to me.
It's the circadian
rhythm disorder.
As you know, we
have a daily cycle
with a metabolism, very distinct
difference in metabolism
during the day and during
the evening, controlled
by a group of circadian
rhythm gene transcription
factor that cycles.
They show cyclical expression
in the normal tissues.
So these defective
circadian regulatory genes,
such as PER2, BMAL1,
CLOCK gene, these
are the key transcription
factor resulting
in many different
diseases including
premature aging and also
neurodegenerative disease.
For example, here the
circadian disorder
is found in the AD patients and
Huntington's disease patients
that they don't sleep well.
This is well known.
The control daylight--
clear daylight rhythm
disappeared in these patients.
Their cycling hormones
in their blood,
such as melatonin, also reduced,
dampened in their oscillation.
And the BMAL1 expression, one
of these transcription factor,
are clearly reduced in
Parkinson's diseased patients.
So we decided these might
be very key proteins that
affect many function
that may be of interest,
including brain disease.
So we knockout these
functions by editing the--
using CRISPR-Cas9, editing the--
inject it in the fertilized
egg, fertilized embryo,
monkey embryo, and then
have embryo develop in vitro
and then transplant it
in surrogate mother,
produce the offspring.
Now the offspring produce--
some of them show complete
knockout of these protein
with basically no
expression of BMAL1,
some with partial knockout
or the mosaic knockout
because CRISPR-Cas9
editing sometimes
doesn't work in all
cells and only part
of the embryonic cells.
So you have an embryo that
have partial knockout.
So this we call mosaic.
This is one of the standard
findings in this technology.
So we found that this knockout
monkey with a BMAL1 [INAUDIBLE]
show defective daylight
locomotive activity.
Usually, in wild-type
monkey, it's
very clear day activity,
no activity at night.
But, in the knockout monkey,
it's more spread out,
and you can see more
activity during the night.
Their period of activities
also show abnormality.
Many different
periodicity of locomotion
appeared in this
knockout monkey.
More clearly is
the sleep patterns.
Now, in the wake,
the knockout monkey
wake more during the day--
during the night.
And the amount of non-REM
sleep, non-eye-movement sleep,
or the eye-movement
sleep are all
reduced in the knockout monkey.
And it's also more
exaggerated if you
do a three-night complete
daylight for three days.
And this distinction
between the sleep pattern
is even more exaggerated.
You can see that the
very much reduced REM
sleep and non-REM sleep,
so sleep disorder.
Now a more interesting
study is circadian rhythm
that we found in the normal
animals are much more dampened.
Melatonin, testosterone,
DHEA, and then, interestingly,
stress hormone, the
cortisol, in the blood
are flat in the knockout monkey.
Now this is very interesting,
the high level of cortisol
maintained in this monkey.
It turns out that
this is directly
the cause for another
phenotype we're seeing,
the stress and depression.
We can see that these monkeys
are all hiding in a corner.
They tend to be off the ground
more time, leaving the ground,
while the wild-type monkey
moves around, all move around.
But these monkey all stay at
one region, move very little.
So this is the stress.
In particular, when the human
being, the animal care person,
comes into feed,
the normal monkey
comes in and move and
look for the food.
And the monkey with knockout
is hiding or always goes
to the corner and
hide themselves.
They will show this very
depressed, anxiety phenotype.
And you can also use EEG to
look at some other phenotype.
In the schizophrenia, there's
a event-related potential
you can detect in
the human patients.
The schizophrenia
show a much reduced--
the MMN, Mismatch Negative wave.
And this is associated
with the odd-ball effect.
You have a constant sound.
Then you have deviant
sound comes in.
That deviant song
is the odd ball.
That odd ball creates this event
that can be detected in EEG.
And the animal--
knockout animals
show very similar
reduction or disappearance
of these MMN peak, so similar.
I'd say we're in EEG pattern
with the schizophrenia
phenotype.
And, if you look at the genetic
changes in these knockout
monkeys, a large number
of cellular processes
are altered, including immune
system, response to stress,
and many also neurodegenerative
pathways all activated.
This is using genetic
analysis, looking
at the genes, a large group
of circadian-related genes.
So we have a knockout
showing a lot of phenotypes.
The point is that we can--
we maybe use them
for disease studies,
looking at the perhaps
screening for drugs.
Now the gene editing
that I showed
you, these two types, viral gene
transgenics or the CRISPR-Cas9
editing have problems.
The viral expression
gives random insertion
of copies that's
not controllable.
The CRISPR-Cas9 editing has low
efficiency, off-target effects,
and mosaic expression.
These cannot be solved easily.
And then long generation time,
you want to produce monkeys,
five years is each generation.
I mean, we can speed up this
into about two years per cycle
with some tricks
in endocrinology,
but it's still too slow.
So now also, most
importantly, you
see that all these
monkey we generated
are coming from
different oocytes
with a different
genetic background.
So you need a large
number to be a model.
Unlike a mouse model, you
have a cell-- or mouse lines,
which are uniform, inbred
many, many generations.
They are unified or homogenized
in their genetic background.
So they are a good model.
20 mice control, 20
mice experiments--
you get a good enough
statistical significant
difference for your
treatment parameter.
So, to have a uniform
genetic background,
really is a key for a model.
So that's why we decided
to clone five years ago,
to clone the monkeys by
somatic cell nuclear transfer.
And you've heard
about this method.
This is another old method.
It's been used to close the
Dolly the sheep 20 years ago.
And 20 different other--
20 other mammalian
species have been
cloned by this method
except non-human primates.
Now many efforts have been
made, particularly in the Oregon
National Center for
Primate Center where
they have extensive studies
there for a 10-years period,
but finally stopped because
there's no progeny produced.
So we've spent
five years on this
and finally succeeded doing this
using cultures of fibroblasts,
fetal fibroblasts from
aborted female tissues,
and fused the fibroblasts
with an oocytes
from a donor monkey in which
the nucleus has been removed.
So the fusion of the fibroblast
introduced the new nucleus
in replacement of the
original oocyte nucleus.
And that reconstructed oocytes
was put into a surrogate mother
to produce the clone.
Now the key to this is the
use of a epigenetic modulator,
a demethylase, KDM4D.
This is an enzyme that,
shown by Yi Zhang--
I think he is in the audience--
here in Harvard to be a very
much helpful in producing--
to reopen the developmentally
relevant gene that
are suppressed in somatic cell.
That injection of the
demethylase is very important.
So here is an example
of the procedure.
What you do is you use a
pipette to focus quickly
to that white spot.
That's where the
oocyte nucleus--
removing quickly
without taking too much
of the oocyte cytoplasm.
And you have to do
that efficiently
to have an intact or relatively
intact oocyte to develop.
After that, you fused
the fibroblasts.
The fibroblasts are introduced
into the vitelline membrane
between the oocyte cell and
the vitelline membrane outside.
So the fibroblast is
treated with a virus
that promotes fusion.
So the fibroblast is actually
fused with the oocyte
to produce the-- to
introduce the nucleus,
the fibroblast nucleus.
Now the key for this--
this optimization
of this procedure
took a couple of years
by a postdoc Zhen Liu.
He practiced to a stage that
he produced the minimum damage
to the oocyte and very quick
fusion to get, finally,
the oocyte is healthy
enough to survive.
And together with
the treatment--
critical with the treatment
of the demethylase, the KMD4D,
together with a histone
deacetylase inhibitor,
all these are genetic modify--
epigenetic modifications that
re-open a developmental gene.
Now two key papers by Yi
Zhang's lab show that the--
and [INAUDIBLE] this
is a human embryo--
that there are a lot of
genes, these suppressed genes,
persistently suppressed
genes, can be reopened
by using this demethylase.
And they identify
this particular KDM4D
as the most effective.
So we show that,
indeed, the genes that
are suppressed in the
fibroblasts, in somatic cell,
can be reopened by using
this demethylase, histone
demethylase.
And that helped, helped
to have a higher pregnancy
rate and a higher fetus--
as well as a good fetus form,
as shown by the ultrasound.
So two babies were produced
at the end of 2017.
We called Zhong
Zhong and Hua Hua.
And it's very clear that
they are clones because you
look at their nuclear DNA.
You can do an ear tissue
and check their nuclear DNA.
They are all identical
to the fibroblast cell
that we fused, but
different from the donor,
oocyte donor, or the surrogate
mother, completely different
using the--
looking at the short
tandem repeats, that way
of looking at DNA gene.
But mitochondria
gene, which are mostly
coming from oocyte--
that's dominant,
lots of mitochondria
in the oocyte.
So, if you look at
the clone, there
are mitochondrial genes
identical to the donor oocyte,
but are different
from the donor--
the surrogate or
the fibroblasts.
So this is clear,
black and white.
These are clones.
So this was published.
And these are two
heroes who did it.
Zhen is a postdoc
fellow, and Qiang Sun
is our platform director.
Now, having this work--
so you remember we
have produced a
circadian rhythm disorder
monkey with clear phenotype.
So we took-- we decided
to take that monkey that's
already one-year-old.
We take the fibroblasts
from that monkey
and do the same way of cloning.
So we generated
earlier this year
five monkeys, BMAL1 knockouts.
And their gene clearly
belong to the fibroblasts
of a donor monkey, which show
the most strong phenotype,
disorder phenotype.
And this is the statistics.
We take the fibroblasts,
second passage.
Third passage is just to
transfer the fibroblasts.
Actually, these are statistics--
100 embryo used for
fusion, for transfer.
In the first pass--
the second passage, we produced
seven pregnancies out of 23
surrogate, about one third.
And we produced one live baby.
But, after two passage,
we have only 55 embryo
transferred, only 12 surrogate.
We produced five pregnancies
and two healthy babies.
So we don't know.
This is still a small
number, but, overall,
the total efficiency is low.
We'd like to have it higher.
Now, normally, the pregnant
monkeys, the successful birth
rate is about 70%, 60% to 70%.
Right now our pregnancy,
5 out of 16, is still low.
It's one third.
So we want to improve
the technology
so that we can at least reach
the natural birth efficiency.
Then we can generate
more monkeys.
So this is showing these
monkeys are really clones.
And this is the first clones.
So the clones show the
same depressed phenotype
as the donor monkey.
So there's no good depression
phenotype yet at this time.
This phenotype is
very important to have
a depression phenotype
for studying depression
because we really--
this is a good--
the drug for depression,
if you know about it,
are not very effective.
And, also, it's a drug that's
been developed 40 years ago,
no new drugs.
We need to really deal
with these problems.
Now these are two
papers published.
One, the first paper,
is the knockout monkey
for BMAL1 knockout monkey.
The second paper is the cloning
of that same monkey-- the one
with the higher, best phenotype.
We clones this.
This was published
earlier this year
in a journal, which you
have never heard of.
It's called the
National Science Review.
It's the PNAS of China,
Chinese equivalent of PNAS.
Obviously, this creates
excitement and debate, which
was mentioned by the dean.
The debate is is this a
approach we should take.
Ethical issues are
we haven't proved
that the monkey model is
useful unless we develop
a drug with these models.
And, until then,
this would always
be a question, whether you
should just use a rodent model.
So why non-human model
is useful animal model,
I've said higher
cognitive function.
It's also a very good model
for study evolutionary origin
of human intelligence.
I know this is a subject I
don't have time to talk about.
Now we want to know
how the human origin--
how human evolution gives
us our intelligence.
How do we study this?
What is the origin of
human intelligence?
This is one of the 25 questions
of science posed 10 years ago.
Everybody agrees it's
an important question.
How do we address this?
Non-human primates
are the question.
There's a recent paper on
transgenic monkeys done
by a different
group in China who
put a human gene
into a monkey embryo
and created a neoteny of the
brain and higher cognitive
function.
So now that gene,
MCPH1, human gene,
might be important for the
origin of the neoteny, one
of the genes at least.
So this is the way we will be
able to ultimately understand
what are the gene
response during mutation,
during human evolution, that
give us the human intelligence,
important, basic questions.
Of course, for the
medical purpose,
disease models, and the key is
to have an ethical standard,
which the dean mentioned.
We have to guarantee
to the society
that we are treating an animal
in the best possible way.
In fact, our cages are
now bigger and bigger--
the European
standards are actually
bigger than the American
standard, the US standard--
housed in pairs, for
example, better conditions.
And that's what we are doing.
The treatment of animals
is clearly a problem,
but that's not the only issue.
The only issue is the monkey
should be an appropriate animal
to use for experiments.
That's another different
totally at all.
Some people even
think mice should not
be used for animal
experiments as well.
It depends on the
extreme of point of view.
OK, so if you are
interested in this question,
the National Academy
of Sciences held
a very good, very important
workshop last October
in which I participated--
it's called transgenic
neuroscience research--
to figure out-- people from the
US and China, Japan, Europe all
come together and talk
about the ethical issues
and the potential
benefit of this approach.
And this was just
published by the National
Academy of Sciences if you're
interested in looking at this.
OK, I think I passed my time.
I should stop with
people involved
contributing to this work.
This is a group of people.
And thank you for
your attention.
[APPLAUSE]
- I was interested in the data
early in your presentation
about how you observe gradations
of empathy in these non-human
primates.
I wonder if you accept the
premise that humans are capable
of self-transcendence
of unselfish action--
that's going a little
beyond empathy--
and whether you observe
that in non-human primates.
- Right, we haven't
followed enough.
So we have to,
first of all, create
an experimental
manipulation, let's
say, that create the empathy
that is similar to humans,
right?
So people thought that empathy
is very clearly demonstrated
by chimps, especially
if you read
the books written by Frans de
Waal on the empathy in chimps.
But, in monkeys, how
much the chimps behave--
a chimp is very close to human.
I mean, human is the
third chimp, right?
But monkeys are somewhat
distant from that.
The phenomenon that
are seen in the chimps
are not always seen in monkey.
So I guess one could think about
experimental studies trying
to train a monkey to show
empathy and then watch
how that evolve.
You cannot do
experiments on chimps.
They are no longer
experimental animals.
So the monkey is
still experimental.
So it's an interesting question.
- Thanks you.
- Now we tested, actually, our
monkey to see whether they can
show gaze-following.
That they fail.
Monkeys fail.
Even after training with
the self-recognition,
the gaze-following,
they couldn't do.
Yes, please.
- Thank you, Professor Poo.
I am very stressed
by the depression
that was in the
monkeys, especially
since they are so close to us.
And I just wanted
to state that I
think we should find
more human-based research
and human-based exploration
for these questions.
And we should not be
imposing on other primates
and maybe even other
animals, but certainly not
other primates, for
the very same reason
that they are like us in
these emotional issues
and dimensions.
Thank you.
- Right, I appreciate
your comment.
- Then you should [INAUDIBLE].
- Yes, right.
- Did you find any surprises?
- Pardon me?
- Did you have any surprises?
- Surprises.
- Were you surprised by
anything of your findings?
- Yes, in fact, I think
the very clear phenotypes
of BMAL1 knockouts--
there are BMAL1 knockout
mice already done.
They show very clear
circadian disorder.
It's been done here actually
in MIT a few years ago.
But to have an anxiety--
a very clear anxiety
and even schizophrenia
phenotype, if we-- now we don't
have all the schizophrenia
assay for the monkey yet.
But, based on EEG, I'm surprised
to see that these monkeys show
a psychotic phenotype.
That was the surprise.
Now I agree that the--
unless we show that these
phenotypes are useful
for let's say developing drugs
for schizophrenia or depression
or the anxiety, that better
drugs appear because of this,
we cannot convince people
that this is useful approach.
But, based on
principle, you might
think that a model, a human
disease model, especially
for these complex brain disease,
a model that's closer to human
is in principle a better model.
But, unless we prove that we
can use this model to generate
drugs that are now failed for
all the other drug development
because of the use of rodents,
we cannot convince the society.
So this is the single biggest
problems we are facing now.
I think we can demonstrate
this in three years.
In fact, we have already
new drugs being tested.
I think within three years
we can show the society,
with a limited amount of
monkey we are producing,
unlike the ones used by
pharmaceutical companies--
do you know how many monkeys are
used by big pharma these days?
70,000 in US monkey used just
for safety and drug metabolism.
And how many mice
used for testing?
Innumerable.
But the drugs all failed.
The latest drug in Biogen you
heard, an AD drug, also failed.
So I think that the only
window of opportunity is there.
Within three years, if we can--
I think you will see
that this is demonstrated
that we can use the depression
model or other model
or circadian disorder model--
or even we have other model
ongoing, not AD model yet.
AD models develop very slowly.
I mean, it's a
neurodegenerative disease,
but, other acute
monogenic disease,
we develop drugs that's really
effective in human trials.
That's what we need to do.
Basically, I'm sorry.
I said too much.
Questions.
- Not at all.
That's very interesting.
So, particularly for
the affective disorders,
which are so common,
we really think of them
as a mix of genetic
vulnerability,
but a very important
environmental component
of early trauma.
And cross-fostering studies show
us that parenting is important.
So the model you're
working on here
seems to really stress
the genetic component.
Do you think about
bringing in environmental?
- Right, yeah, actually, we have
a large population of monkeys.
We actually now find the OCD
model and depressed monkey,
naturally depressed,
naturally a monkey
that show a phenotype like OCD
phenotype among the population.
There's also a PD,
naturally occurring
PD monkeys found in a
large population, colonies.
So one can look for it.
Those are not just
genetics, maybe combined
genetic and
environmental factors.
- So, yes, I noticed that
you had a-- this is a general
question.
You had a number of
specific techniques
for putting specific genetic
sequences into the DNA.
- The DNA, yeah.
- How far are we, or
is it even possible, to
where you can essentially
generally edit the DNA?
If you have the sequencing
of it, which is not too hard,
and you want to make it
a different sequence,
how far are we
from being able to?
- Oh, we can do it now.
I mean that's called knock-in.
You can-- you design
a sequence and put it
into a specific site in the DNA.
You can-- the CRISPR-Cas9
technology is very powerful.
You can do just
cutting and pasting.
You can do that in
all species now.
- You can almost produce
any sequence you want to?
- Yeah, you can create
any sequence you want
and put it into the DNA.
- OK, thank you.
- But now, to control exactly
where you put it in, there are
still off-target effects.
Off-target effect means
you are not putting exactly
the right place editing.
You are editing
some other places
that are not intended
place called off-target.
- Like over-- put in more
than you wanted in your--
- Yeah, or cutting more than
you want or put in a site
not precisely what you--
besides the editing
you want to do,
you accidentally
edit in other places.
- OK, so that's why they
had this study where--
- That's called-- that's
so-called off-target effects
of the gene editing.
- That kind of
probabilistically--
- Yeah, so it's probabilistic.
- --if you do it 100 times--
- And, also, it's being solved.
The problem is now being solved.
For example, the
most popular thing
is related to Cas9 technology
called base editing.
You just cut-- change
bases, nuclear bases.
That produced
off-target effects,
but that off-target
can be corrected.
There are ways to correct it.
So the technology is
improving with time,
very rapid development.
- And it doesn't make
any difference really
whether you're talk about
bacteria or eukaryotes.
We're able to--
- It doesn't-- yeah, it's
all the same technology.
- It moves a lot.
- Right.
- I'm wondering about issues
on sacrification of monkeys
because--
- Separation?
- Sacrifice.
- Sacrifice.
- Because the
psychiatric disorder
has much to do with connection.
So the result from
imaging is quite
limiting in human
research, but I
feel anxious whether
the killing of monkeys
for such a motivation is right.
- Well, you don't kill the
monkey unless it's necessary.
For example, the drug
company use toxicity tests.
If the drugs they
put are too high,
and they produce toxic effect,
the monkey is in distress.
They'll kill the monkey.
Otherwise, the monkey--
after experiments,
you put it into a monkey
farm to have nice treatment
for the rest of their life.
In general, unless they
are really distressed,
you don't kill the monkey.
[MUSIC PLAYING]
That's the procedure, OK?
- That'll be the last question.
- Thank you very much.
- And please join me
in thanking Dr. Poo.
[APPLAUSE]
