Dr Valerie New York University Medical
Center,
Human Muscle in tissue culture,
neuromuscular diseases are considered to
be disorders of the motor
unit.
It means the disease can
originate either in
spinal cord or in peripheral nerve
or in the muscle fibers.
With all techniques you for examination
of muscle pathology like histochemistry,
electron microscopy and electro biography.
It is possible to identify involved in
disease process system.
However,
pathogenesis of majority of neuromuscular
diseases still remains to be known.
Tissue culture of human muscle provides
unique opportunity for examination
of muscle which was developing in
the environment where all other factors
which possible are causing disease in
humans are eliminated
in,
in the even in the muscle grown in such
environment where neural humoral
vascular and other factors are absolutely
eliminated.
We will be able to reproduce the changes
present in the patient.
Muscle biopsy.
Primary character of muscle disorder
could be anticipated.
Multinucleate cross created
muscle fiber in myogenesis
in Vitra and in viva are the
result of fusion of
mononucleated spindle shaped myogenic
cells.
During myogenesis.
Those cells attached to each other,
they form multinucleate myotube
and finally multi nucleated
cross created muscle fiber
growth of adult human muscle in
Vitra is initiated by so called
satellite cell described for the first
time in 1961 by Alexander Mora from
Rockefeller University.
Satellite cells originate from
embryonic myoblast during myogenesis.
What happened is that all myoblast
probably are originated from one pre
myoblast.
During myogenesis,
they divide,
they connect with each other,
they fuse and they may cross trid muscle
fiber.
However,
sometimes for any known reason,
one single myoblast doesn't fuse and
this cell gets incorporated under
basement membrane of a dark muscle fiber
and is forming so called satellite cell.
Satellite cell is embryonic cell in the
origin and is placed between
basement and plasma membrane of dark
muscle.
Satellite cells account for about
2% of all muscle nuclei
even thought it was possible to
grow embryonic chick muscle for many years.
Only recently,
it has become possible to grow adult
human muscle.
And two approaches for growing adult human
muscle could be established.
One is that whole muscle biopsy
is sin and tissue culture is
established from single cells.
This technique appears to be very suitable
for examining all steps of muscle
development.
Like here,
we can see my house of single cells
here they elongate and get attached to the
bottom of the Petri dish.
Here they divide again.
And finally,
this ammonia created spindle shaped
cells are getting close together.
In order to fuse,
we can see here on higher magnification
how two myoblast get together.
There is a third myoblast,
which was joining them.
Finally,
they make multinucleate myotube and
cross created muscle fiber.
Another approach which we prefer in
our laboratory is to establish
muscle culture from adult
human muscle biopsy.
We are establishing this making
small one millimeter cubic ex
plants which sometimes are cut
longitudinally and sometimes are cut cross
and we try to put about six or
even at the present moment,
more explant on the bottom of the
gelatin and human plasma coated Petri
dish.
After a few days,
about three or four days,
we can observe that there are some single
cells which are merging out from this original
muscle X plan on higher power.
One can observe that majority of the cells
are Fibroplastic cells and
very few are spindle long
shaped,
probably MYOB blastic cells.
However,
after about 10 days in in
vitro,
the cells fuse,
they form multinucleate
tubes,
they mature become cross created.
But unfortunately,
after about three weeks,
all these fibers are dying and they are become
overgrown by connective tissue.
This problem was bothering us a lot
because our goal is to
obtain very rich and mature
muscle culture.
In order to be able to apply all
techniques used for examination of muscle
biopsy,
like histochemistry,
electronic copy biochemistry and so on.
For this reason,
we were very pleased when we developed
technique which we call plat
explant re explantation technique,
which enabled us to diminish
amount of connective tissue in the cultural
muscle.
What we are doing is that we are picking up
this original muscle explant to the
entirely new Petri dish.
And we are initiating like a new culture
but from previously already present
ex plants.
And after about one or two days,
single spindle shaped cells are
coming out from this plat which
fuses and after a few days they form multi
nucleated mao tubes.
And here we can see like amount of
connective tissue was greatly reduced in the sculpture.
All this staining is the P N H.
The heterogenous staining are presenting
multi nucleated muscle fibers.
And this picture was taking about after eight
weeks in Vitra
using explant explantation technique.
We are achieving quite advanced maturation.
We can see that muscle fibers are
becoming cross created and majority of the new
climate migrate to the periphery of the fiber.
Like I said before,
we are attempting to
apply all techniques using for examination
of muscle biopsy to the culture human
muscle.
And uh I would like to show a few
examples how we are doing this.
One approach is to air dry,
entire culture including original
explant and apply histo chemical
reaction.
On this example,
the P N H we can see on
lower low power,
many many fibers coming out from the egg plant.
And here longitudinal view on higher power,
very strong staining of cultural human muscle,
only nuclear area remains in stain.
Another approach is to pick up
entire outgrowth and
leaving original X men alone.
And put this outgrowths cross on the top
of the adult muscle fiber,
we freeze this and we perform serial cross
sections.
This light illustrates like cult
human muscle are very strongly positively
stained with A T P S reaction.
Perform at P H 9.4 A
T P A reaction perform after acid
preincubation.
Suing the heroes give somehow
weaker staining and phosphor staining
appears very characteristic in farmers of
spots or legs which we like to call them.
Those legs are positive stains or phosphor.
As we all know that
human and animal muscle is differentiated for
different histo chemical fiber types.
We can see on this example when A T P S reaction
at P H 9.4 is performed that this
muscle majority on this muscle are staying dark
indicating that there are type two fibers
reverse being through with A P A reaction
performed after acid preincubation.
However,
those two light fibers here are staying
dark here indicating that they belong to
different uh group of fiber types.
And here with D P N H,
the heterogenous,
we are able to identify types
of uh those two groups.
However,
when car master were put on the
top of the sedar master and serial cross
sections were performed,
we were not able to observe
reciprocal reaction between
different histo chemical staining.
And this fact indicates that
cultural muscle grown in
Vitra without neural influence,
are not able to differentiate for his
different histo chemical fiber
types.
For electro microscopical examination,
we like to fix entire
dish with muscle cultures inside
with grower aldehyde and post in.
And then we embedded we are embedding in.
We found extremely necessary not
to perform blind studies on
the the culture,
but to be able to perform selective
examination of culture for electron microscopical
examination.
What we are like to do is after we
embed the dish,
we stain this with metal and blow,
We observe fibers of our
interest.
We mark them under the bottom of the dish.
And then using by developed by us
technique which we call drilling technique,
we select against this area and we pass them
out.
And this small disk comes from this big
disk here and shows the same fibers which were pre
previously selected and shown here.
We put this whole small disk on the track,
we trim this,
we cut the thin section and e examine under electronics.
It is extremely important
perform to perform
selective examination of fibers for electronics
because as we all know in the disease
process,
very often,
not entire fiber is affected by the
disease process and very often in one
entire muscle biopsy,
only very few fibers are affected.
And the same is true in the tissue culture
fibers.
This is why we found this extremely important to
be able to selectively examine cultural muscle.
For electron microscopy.
Using this drilling technique,
we are able to pick nuclei area.
If we are specifically interested in this or we are
able to decide that we would like to look at
the periphery of the fiber.
And we are really,
but after small in section are cut,
we really know where we are and what structures we
examine.
And this picture here shows very young multi
nucleated area of very young my tubes
with some beginning of myofibril formation
here on the periphery from two sides.
However,
this fiber is much more mature.
We can see much better organization of myo
fibras here and we can
see beginning of zen formation here is not
even nice zen
present the same on this side and
mitochondria are present and rough in the plasm cri.
However,
we can see that even this fiber is very young
because center of this fiber is loaded by
ribosome rough in the plasm cri.
And all this um signs
indicate that it is very young,
actively developing muscle fiber.
However,
this picture shows us much
more mature about six weeks in Vitra
mass fiber which is able to contract and
maintain in this form for a longer period of time.
You can see nicely organized my filaments and Zs
formation is present.
As we all know Aceon
receptors in normal muscle
are localized to post synaptic
membrane.
In endplate area of the muscle,
we can never see any aceto colon receptor
extra junction.
They are all located in the end plate
area.
Visualization of alaia
receptors became much easier
after immunoperoxidase staining
of alpha Banga toxin was
performed first by Daniels and Vogel
and it became later used.
What this staining um
enables us to see is
immunology reaction after
alpha Bangar toxin obtained from the venom
of snake multi
binds to a receptors
and then antibodies against alpha Bangar
toxin are applied.
They combine and immuno peroxidase
staining is applied to the end to visualize this
reaction.
And we can see here and plate of the
normal master showing that
postsynaptic membrane is darkly stain.
And there is present staining
of presynaptic area as well
as a normal muscle.
Like I mentioned previously,
only end plate area gives
positive staining.
However,
when muscle is denervated,
like we can see here there is the few
staining extra junction.
So we can see that there are a receptors
not only present in the end plate area,
but they are present all over the membrane
is a difference to normal master here where
extra junctional area remains in
stain.
This work was done uh with cooperation of
doctor um bender and the
ringer,
we were interested to see
if a neurally grown
culture muscle will have
similar appearance of
receptors to
biopsy muscle or it will have
similar appearance of receptors to
denervated muscle or it will be entirely
different story.
And what we have done we applied
immuno perox staining to a
neurally grown culture.
And this picture represent rat
culture muscle.
And you can see here,
diffuse staining
Oxin indicating a receptor
binding site on this culture,
muscle fiber.
Those two slides shows us
show us staining of cultural
human muscle here and
here this immuno peroxidase
staining of ace receptors,
the insides.
And this is electron microscopical picture
which shows diffuse
staining of the plasma lemma.
Those are two adjacent fibers
of human culture.
Whole preparation is and only I
is performed.
And this slide shows comparison
between immuno perox of
alpha toxin in the human culture,
muscle,
in the rat culture,
muscle and in the chicken culture muscle.
So as we can see when
muscles are growing without nerve
influence,
they do not differentiate for different histo chemical
fiber types.
And as with respect to a
receptors,
they behave like denervated muscle
in viva having Aceon receptor
spread in the entire area of
tic membrane.
We are interested to see if
muscle in culture will be innervated if
there will be localized area of Aceon
receptors.
And what we do now this preparation shows
is this is human muscle in tissue culture,
which we are trying to innervate
by embryonic rat spinal
cord here are nerve
coming to this human muscle.
These studies are in progress now and
we are going to search
for endplate formation in this
culture.
Muscle.
In general,
one can see following
goals in culturing human muscle
fibers.
We one would like to see in abnormal
muscle if it would be possible
to reincarnate a characteristic
morphological defect which is present in
muscle biopsy to the culture muscle.
Another thing it would be possible to
reincarnate a characteristic biochemical
defect which is present in the muscle biopsy into the
cultural muscle.
And third point,
very important is if it would be possible to
prevent or to reverse the defect by
in Vitra treatment in normal
muscle.
I started kind of like from the end before and
I analyzed the normal appearance of muscle
fiber and then we can discuss later
inducing of characteristic defect in normal muscle
fibers.
I would like to start with some
of our positive data when we were able
to reproduce some
morphologic defect present in muscle
biopsy into the culture,
muscle.
We started to grow muscle from eight
patients with ophthalmaplegia
syndrome.
Ophthalmoplegic syndrome is characterized by all
this uh data
which are put here on this slide and
morphologically they are characterized by
ragged red fibers present in the muscle
biopsy.
Ragged red fibers are the best
visualized by trichrome modified Trichrome
staining which shows red ring around the
fiber and by succeeding the heterogenous
staining which is showing very dark rink
around the fiber on the electro
microscopical examination.
One can see that they are big
collection of abnormal
mitochondria.
They are very peculiar pattern.
They are enlarged,
they are in cluster,
some have crested,
twisted in fingerprint pattern,
some are filled with amorphous material here
and here and here.
Some have para crystal included in it
in general.
They are enlarged in a mouth and in size
and they are put in clusters.
Cyto oxidase is
very useful in examination of
muscle mitochondria because we don't
have to pay attention to the entire tissue.
And only mitochondria are stain cytochrome
oxidase stain out the membrane of
mitochondria and cry.
This is normal human muscle
and this light demonstrate muscle
biopsy from patients with ragged fiber and cyto
oxidase staining form.
We can see abnormal mitochondria
here,
crystal crystal not being stained with cyto
oxidase but outer membrane
and reminder structures are
stain
when tissue cultures were established from
muscle biopsy.
From patients with ragged red fibers.
We haven't noticed any difference in the
ratio of growth time of forming of
myotube or maturation of muscle fiber.
We can see here very rich outgrowth from the
original muscle biopsy,
we can see very nice developed cross
trid mature master fibers.
However,
in some of the fibers,
there were some collection of gran
material present which we were
not able of course to identify on
the light microscopical level.
However,
when cytochrome oxidase reaction was
performed on the sculptural muscle,
we kind of noticed that collection
of this granu materials stay
very richly with cyto oxidase
indicating that they might be mitochondrial
in the original
fact that only very few
fibers are stained are
abnormal in this muscle ground
from patients with rare fibers is not really
surprising because when we look in
this three example,
from different patients with rare fibers,
we can see that the
fibers are really very few
and the entire muscle looks
quite normal.
So when we expect pathological changes
coming only from this very few fibers,
we really wouldn't expect entire dish of
culture to be affected
when using this breeding technique,
which I explained before we selected
area of this rich cyto oxidase staining.
And the we examine this
area under electron microscopy,
we indeed observed that this area
corresponds to abnormal looking mitochondria.
And here we can see from cultural master giant
mitochondrial filled with amorphous
material and very peculiar shape,
peculiarly shaped.
And there are some crystal stain positively
with cyto oxidase on the edge of this
fiber of this mitochondrial here and here
too and kind of like amorphous
material and stain
is being in the center as
compared to normal cultural mitochondria
which are small and very rich in Christian.
This next four slides
will show similarities which we often
observe between mitochondria
present in the given patient muscle biopsy
and in the culture grown from this
patient muscle.
And uh if you could just look at this
mitochondrial here,
we can see very peculiar shape
part is filled with amorphous material.
There are some crest that twisted in peculiar
pattern and there is like another small mitochondrial in
the center of this big one.
This is patient muscle biopsy.
This is this patient muscle
grown in culture.
And we can see very similar mitochondria
here to this one which we observed
before and in the neighborhood of this
abnormal looking mitochondria.
There are some mitochondria which are quite normal in
appearance.
And here is another example beside
stain mitochondria.
Here from patient master biopsy,
we can see cry being packed here in the
center and here being packed in the
periphery in very peculiar fashion.
And from this patient culture master,
we can see that mitochondria appear in
very similar pattern,
cry packed,
strongly stained and center
being and stain and filled with amorphous material.
However,
we will never be able reproduced
as far para
crystal inclusions which are
always almost present in the abnormal
mitochondria and red fibers.
Here is like exaggerated example of big
paracin inclusion present in patient
master biopsy.
We we were able to reproduce
abnormal mitochondria as far to the
size and shape is concerned,
but we we're not able to reproduce parasol
and inclusion probably
much longer time is necessary for parasol
inclusion to develop than we are dealing
in tissue culture condition.
Another step forward which I would
like to discuss now is
our ability to in one
case to reincarnate in culture
characteristic morphological defect
and partially biochemical defect
which was present in patient muscle biopsy.
This man was very
healthy until age 22
he was in the Marine Corps.
However,
at age 22 he started to feel
weak and now he has from 12
years,
slowly progressive proximal muscle
weakness,
but some distal muscles are involved as well.
Otherwise he doesn't have any complaints
in muscle biopsy of this patient.
We could observe vacation present
in some fibers,
electron microscopically,
his muscle was badly damaged and
we can see it was very peculiar fashion.
It was in.
There were inclusion of
multi laminated
bodies here and here.
Some lightly stain,
some darkly stain but on cross
section remained and look
like cross section of the cabbage.
This master was hardly destroyed,
very destroyed and we couldn't observe
an accumulation of glycogen there.
When tissue cultures were established from this
patient muscle biopsy,
we could notice that even very
single and young cells already were highly
vacated and this vacation were
carry on till muscle was quite
mature.
And we can see here,
multinucleate mature myotube,
highly evacuated stain preparation and
end stain.
All these vacuums present
electron microscopically.
The sculpture fiber similarly to
his muscle biopsy was very
damaged.
And we could see all this field is
loaded by these vacuums which under electron
microscopy appear like multi laminated
bodies,
bitter mind cross section of the cabbage
as well.
This muscle culture was so damaged that
I couldn't really find any muscle which
was normal looking.
Every muscle fiber which I choose for electronics
examination was loaded by this
abnormalities.
Biochemically.
Doctor Salvador De from
Columbia University Medical Center,
examine muscle biopsy of this patient
and his cultural muscle.
And he found in both cases highly
elevated acid phosphatase.
However,
specific biochemical defect probably
lies which is responsible for these
changes in both this patient cultural muscle and in his
muscle biopsy still remains unknown.
As the next example,
I would like to show
it two
different forms of glycogen
storage diseases.
In one form when we were able to
reincarnate in culture,
characteristic morphologic and biochemical
defect present in the patient's muscle biopsy.
And one example of um glycogen
storage disease,
which we were not able to reincarnate um
defect
as we know
breaks the glucose through two
pathway.
Major pathways is through phosphorylase.
And minor pathway is through alpha
glucosidase called acid as
well.
So alpha glucosidase works here and
breaks glycogen to glucose.
And phosphorylase works as a major
pathway in macarthur
disease.
Myoor is absent in the
patient muscle and this slide shows
negative staining of phosphorylase
from muscle biopsy,
from patient with me disease,
so called myoor
deficiency.
Because phosphorylase is absent from this
muscle glycogen cannot break to
glucose in this muscle and is accumulating
hardly as a cytoplasmic
glycogen.
In this patient,
muscle biopsy,
what could be visualized under electron microscopy.
However,
interesting enough when we were growing
muscle from patient with mac
disease,
what was previously reported by uh
doctor doctor and as
well,
we were able to detect phosphorylase
staining histo chemically here is
rich rich outgrowth.
All these fibers are cultural fiber from patient
with um phosphor deficiency.
And we can see here,
typical characteristic of a standing
for master culture.
We made like further
examination in this area and
we have done electro microscopical
studies of culture muscle from patients with mac
disease.
And there we couldn't find any
normal is either,
we haven't seen any accumulation of glycogen
mas was becoming cross rated and very
nicely developed.
And again,
biochemically amount of phosphor a present in
this cultural fibers.
From patient with me,
phosphorylase deficiency was
normal.
Didn't differ from a phosphor a
present in normal culture muscle.
So this was an example of glycogen storage
disease,
which we were not able to reproduce
defect.
We were surprised because my phosphorylase
deficiency was always considered genetically
determined inability of muscle to
produce phosphorylase.
However,
tissue culture studies indicated
that this muscle
growing in Vitra is able to produce
phosphorylase.
And there are probably some other factors
operating in patient which
enable for phosphorylase to be
maintained.
Another example of
glycogen storage disease is
lack of gly of alpha glucosidase,
which is lysosomal enzyme in
another way called acid maltese.
And when acid maltese is absent,
glycogen cannot be break
down to glucose.
And the characteristic feature of this
disease is formation of glycogen
accumulation of glycogen in lysosomal
bound,
we call the sugar.
And uh our first patient
with this disease which ma whose
muscle we have been grown in culture was
32 years old men
who had four years history of
progressive muscle weakness.
He was very healthy till this time.
He was a football player in high
school and he participated in all kinds
of variety of and he was very good in this.
However,
now he developed for the last four years,
proximal muscle weakness
on his biopsy muscle.
There were highly vacated muscle and
both two types.
Type one and type two of fibers were
involved in this cul process.
And then when acid phosphate staining was
performed,
it indicated that these cules
stain positively with acid phosphatase.
What indicated Liz character
of these vales
on the electron microscopy?
We could see this characteristic
appearance of glycogen in
this lysosomal bound
sax membrane bound sacks
here here and here.
And this is characteristic appearance of a
form of acid maltese deficiency
master looks quite normal.
There is no really increase of cytoplasmic glycogen
only accumulation of glycogen in
membrane bound lysosome which we like
to call sugar balls.
When muscle of this patient
were grown in tissue culture,
we were able to see
vacuolization of culture muscle.
After about 10 days in Vitra is
living culture and this is embedded culture
preparation.
We can see vacation when
this vacated area was
selected for electro microscopical
examination.
We could notice even in quite
young fibers,
already some accumulation of
glycogen in this membrane bound lysosome
here and here.
However,
this whole fiber is not very affected yet.
We can see this area is developing has
beginning of zen formation.
There is a lot rougher
the ri.
However,
in ribosomes nice
mitochondria.
However,
already the accumulation of glycogen could be noticed.
However,
when we waited a little bit longer,
we could see that this muscle is becoming
very destroyed.
And all this area is packed
by this glycogen located in
lysosomes here and here here and here which
resemble very much appearance of the
same abnormalities in the spa master biopsy.
And this light illustrates
similarities between patient muscle
biopsy and his culture muscle.
And we can see here there is this
dark membranous membranous material present
in this patient muscle biopsy which could be
detected as well already maybe not
so in pronounced degree in his culture
muscle.
Here and here
again,
doctor from uh Colombia
University Medical Center uh
perform biochemical
examination of this muscle
biopsy in our culture.
And he found that in both this
patient muscle biopsy and in this patient
culture,
muscle acid maltese was missing was
zero as compared to high
amount of acid in the
control muscle biopsy.
And in the culture normal
culture,
this was with natural subs
subtract Marty hydrolysis.
So this case was really our
first example when we were able
to reproduce in Vitra
entirely both morphological and
biochemical defect present in the patient muscle
biopsy.
And we were able in this way to indicate
primary myopathic character of this
disorder.
Later on at this moment,
we are able to grow five
mass muscle from five
patients with acid maltese deficiency and
three heterozygote.
And the this um
picture shows electron
microscopy of
juvenile form of acid maltese
deficiency.
And we can see that there are much more pronounced
abnormalities and even so still this
character of abnormalities,
glycogen bound is
characteristic and present.
There is a lot of glycogen
cytoplasmic as well.
And when this muscle was put to culture to grow,
we have noticed that
vacuolization in these fibers happened
much earlier than in adult form of acid maltese
deficiency.
We could see even very young fibers already are
being vacated.
Even single fibers are being
vacated and on the
electron microscopy as well,
we notice much more
pronounced abnormality than
we have seen in.
From patient with adult form of acid maltese
deficiency biochemical defect in
this juvenile form was reproduced as well.
Last month,
we received muscle biopsy from um
Amsterdam from Doctor Belem from Wilhelm in Augustus
University.
And uh after 20
hours in the plane,
this muscle arrived finally to our laboratory
and we were able to grow this and this
muscle even contracted.
And this was from
patients with biochemically proven,
again,
proven again,
acid maltese deficiency.
And we can see here abandoned nice
growth of culture muscle from this
53 years old plain.
However,
even very young muscle fibers here are already
evacuated and they have glycogen
present and bound in this glycogen
accumulated in lysosomes.
Higher magnification shows this much
better glycogen present in the
membrane bound lysosomes here and
here here in a relatively
young and developing
fiber.
Moreover,
another interesting characteristic which we have never
observed before in cultural muscle was present of
this small sarcoma here
which have never been described in skeletal muscle and we
have never seen this in the skeletal muscle
before.
And here is another fiber from the same
culture,
having this small circo
present there as well.
And the all this that shows
that tissue culture provides
very good system for examination
of muscle pathology.
And we hope that with further
improvement and very exact
morphological electron microscopical and
biochemical examination of cultural human
muscle.
We will be able to learn more and more
about pathogenesis and eventually treatment of
human neuromuscular diseases.
Thank you.