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.