Ethanol Effects on Murine
Muscle-Derived Stem Cells
Experiment and Analysis by Donald Kline, Jr
Pittsburgh Central Catholic High School
Brief History of Stem Cells
Word stem cell first coined by Alexander
Maksimov in 1908
 1992- stem cells first cultured successfully
in vitro, a major step for future
treatments of many disorders.
 2006- Led by Yamanaka, IPS or Induced
Pluripotent Stem cells are discovered

Stem Cell Overview
Basic Definition- cell that can produce
lineages more specialized than themselves
and can renew itself
 Spectrum of Stem Cell Behavior:

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totipotent embryonic cells
pluripotent cells
multipotent cells
unipotent cells.
osteoblast
adipocyte
hematopoetic/endothelial
cells
muscle cell
chondrocyte
Adult MuscleDerived Stem Cells
(MDSCs)
hepatocyte
nerve
cells/neurons/glial
cells
Stem Cell Applications
Duchene's Muscular Dystrophy, limb
regeneration, cancer research, and many
others
 Results of this experiment could be
applied to the effect of alcohol on general
muscle regeneration or on the embryos
of pregnant mothers

Mice in Experiments

Most Common Laboratory animal
◦ Share a relatively large amount of genes with
humans
◦ mammals
◦ Genome completely sequenced
◦ Breed rapidly, easy to handle, small, inexpensive

First inbred by Clarence Little in 1909, who
promoted the use of mice in the laboratory
Muscle-Derived Stem Cells
(MDSCs)
Adult Stem Cells taken from muscle
tissue
 Isolated using a variety of techniques to
remove the non-stem cell populations
 Confluency needed for stem cells to
differentiate in vitro

◦ Confluency allows the cells to fuse and begin
forming multinucleated myotubes
Blood Vessels
Nerves…
Cross-sectional View of Muscle
Sarcomere
Mouse-Derived Stem Cells
(Experimental Focus)

Mouse cells used were Muscle Derived
Stem Cells (strain pp6 ft 15 mouse)
◦ Pp6 is preplate six; the last step before
‘purified’ stem cell culture
◦ f in the ft15 stands for female, while the t 15,
also called c, is the specific strain of mouse
cells used
The Preplate Technique
Enzymatic
digestion
Muscle Derived
Stem Cells
Muscle
biopsy (mice)
Pre-plating
technique
After 1
hour
Pre-plate 6
After 24 h
(repeated 4
times)
Qu-Petersen et al., Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. The
Journal of Cell Biology, 2002. 157(5): p. 851-64.
Cell Differentiation


Adult Stem Cells found in most animals are said
to be pluripotent: able to differentiate into many
cell types
The Murine pluripotent stem cell differentiation
into myotubes was analyzed
◦ Several ways to track this process
 Desmin stain (myosin precursor and intermediate filament)
 Myosin stain (essential to muscle motility, appears in myotubes
near completion of differentiation)
 Hoechst stain (nuclear stain)
Ethanol
“Pure Alcohol” EtOH
 Fermentation reaction
 One of earliest reactions applied by
humanity
 Depressant
 Health Issues- Fetal alcohol syndrome,
alcohol addiction, impairment of nervous
system and motor system

Purpose

Explore effects of Ethanol on myoblastic
stem cell differentiation
Hypothesis

Null Hypothesis: Increasing EtOH
concentration would not significantly affect:
◦ The Differentiation of the cell, measured by %
differentiation
◦ The average number of nuclei in each fusion
Materials
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Centrifuge
Macro Pipets
Micro Pipets
FTC 15 mMDSCs (pp6)
2% DMEM media
Mouse Cell Culture Hood
37 degrees Celsius Incubator
70% Ethanol Solution (sterilization)
Sterile Gloves (ethanol)
Live Cell Imaging Robot
Suction Filtrater
Trypsin (remove cells from
adhering to flask)
Vortex (EtOH concentrating)
Absolute Ethyl-Alcohol (EtOH)
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C Flask
Hemocytometer
Culture Flasks
Conical Tubes
Microscope
Wells
ScienceCell
Media
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10% DMEM media Hemocytometer
Materials (Special for
Differentiation)
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100% Methyl Alcohol
10% Goat Serum
Mouse Anti-Human Myosin Heavy-Chain Stain (primary)
Goat anti-mouse Fitz (Myosin secondary)
Hoechst’s stain
Monoclonal Anti-Desmin Pro Mouse (Desmin primary)
Anti-Mouse IgG (Desmin secondary)
Fluorescent Inverted View Microscope with Northern
Eclipse Imaging Software
Procedure
1. pp6 mMDSCs were acquired
2. Expand mMDSCs through repeated culturing
1. Pass cells (aspiration, trypsinization, replating)
3. Transfer cell suspension into 15mL conical tube
4. Rinse Flask with 5mL of PBS twice
5. Add the rinse solution to the 15mL conical tube
6. Spin at 150g for 5 min.
7. Supernatant was removed by suction filtration
8. The cell pellet was resuspended in 1mL of DMEM media
9. Density of cell suspension was determined by use of a
hemocytometer
10. Cells were re-plated at a density of 5,000 cells/well
Procedure (Differentiation)
1.
2.
3.
4.
5.
6.
7.
The cells were grown until they were in sufficient quantity, and then were pipetted into wells at a
density 5,000 cells/well.
The cells were allowed to continue growing until they began to differentiate.
The variable concentrations of Ethanol were then added.
After several days, the media was removed and the cells were fixed in 100% Methanol.
The following differentiation assays were performed:
1.
After performing the methanol fix, the cells were rinsed three times with PBS.
2.
The cells were blocked in 10% Goat Serum for one hour, only enough to cover surface of wells.
3.
The Block was removed and the cells rinsed 3Xs w/ PBS.
4.
For Myosin, the primary (mouse anti-human myosin stain) was added at a ratio of 1 uL primary
to 300 uL PBS. It was allowed to sit for one hour.
5.
The primary was removed and the cells were washed 3 times with PBS.
6.
Myosin secondary was added (Goat anti-mouse Fitz) and allowed to sit for one hour at a ratio
of 1:300uL. It was then covered in foil for an hour and every kept in the foil until analysis was
completed.
7.
Fifteen minutes before the end of the previous step, a Hoechst stain was added at a ratio of
1:500uL (Hoechst stain is the nuclear stain)
8.
After sitting in foil for one day, the desmin stain was performed. This process has the same first
three steps, so it began at step four
9.
For Desmin , the primary (Monoclonal Anti-Desmin antibody produced in mouse) was used at
a ratio of 1:300uL for one hour.
10. The primary was removed and the cells were rinse three times with PBS.
11. The secondary (Anti-Mouse IgG fragment of sheep antibody) was added at a ratio of 1:500uL
and allowed to sit for one hour.
The cells were then analyzed under an inverted fluorescent microscope with Northern Eclipse
Imaging Software.
Pictures of the fluorescent myotubes were taken and the data was analyzed.
Differentiation Procedure: Flowchart
Confluence
Rinse
Several
days after Fix in 100%
Add
Rinse
Variables
Methynol
Myosin OR
1 Hour Add
1 hour
Desmin
Primary
Rinse
Block
1 Hour
Add
Secondary
Hoechst
Stain
Cover
in foil
Analysis Using Inverted
Microscope and Northern
Eclipse Image Software
Examples of Fluorescent Myotubes
1% EtOH Desmin merged
with nuclear stain
1% EtOH Myosin Merged
with nuclear stain
1% EtOH Nuclear
Stain
Desmin Visualization: Percent
Differentiation
Desmin % Differentiation
50
45
% Differentiation
40
35
30
25
20
P<.05
15
10
5
0
Control
.5% EtOH
1% EtOH
Variables
3 % EtOH
5% EtOH
Desmin %
Differentiation
Desmin Visualization: Nuclei/Myotube
4
Nuclei/ Myotube (Desmin)
3.5
3
Nuclei
2.5
Nuclei/Myotube
2
1.5
P<.05
1
0.5
0
Control
.5% EtOH
1% EtOH
Variables
3 % EtOH
5% EtOH
Dunnett’s Test Results: Desmin
T Critical Values: p<.05:3.26
p<.01:4.71
ANOVA
0.5%
EtoH
1% EtOH
3% EtOH
5% EtOH
p>.05
p>.05
p>.05
p<.05
T values
.306
1.56
2.42
3.53
Nuclei/
Myotube
p>.05
p>.05
p>.05
p<.05
.983
.608
.988
3.42
Percent
Differentiation
0.008677
0.001314
T values
Conclusions: Desmin
% Differentiation: Statistically significant at
5% Ethanol (p<.05)
 Nuclei/Myotube: Statistically significant at 5%
Ethanol (p<.05)
 At the concentration of 5% , the Ethanol
does appear to significantly reduce
differentiation and average nuclei per
myotube. The null hypothesis can be rejected
at this concentration.

Myosin Visualization: Percent
Differentiation
Myosin % Differentiation (Avg.)
45
% of Nuclei Differentiated
40
35
30
25
Differentiation
20
P<.05
15
10
P<.01
5
0
Control
.5% EtOH
1% EtOH
Variables
3 % EtOH
5% EtOH
Myosin Visualization: Nuclei/Myotube
Myosin Nuclei/ Myotube
4.5
Nuclei Per Myotube
4
3.5
3
2.5
Nuclei/Myotube
2
1.5
1
P<.05
0.5
0
Control
.5% EtOH
1% EtOH
Variables
3 % EtOH
5% EtOH
Dunnett’s Test Results: Myosin
T Critical Values: p<.05:3.26
p<.01:4.71
ANOVA
0.5% EtOH
1% EtOH
3% EtOH
5% EtOH
p>.05
p>.05
p<.05
p<.01
T Values
2.79
2.84
4.12
5.34
Nuclei/
Myotube
p>.05
p>.05
p>.05
p<.05
0.068
0.587
2.27
4.49
Percent
Differentiation
0.000294
0.001017
T Values
Conclusions: Myosin
Percent Differentiation was significant at 3%
(p<.05) and 5% (p<.01) ethanol concentrations
 Nuclei Per Myotube: statistically significant at
5% ethanol Concentration
 The Differentiation of the cells measured using
myosin was significantly affected at 3% and
above, while 5% significantly affected the
amount of nuclei/myotube. The null hypothesis
can be rejected at these levels.

Overall Conclusions
Under tests of both desmin and myosin, the
effects of Ethanol were significant at 5% for
nuclei per myotube.
 With myosin, % differentiation became
significant at 3% while for desmin it was at
5%.
 The quantity of 5% ethanol rejects the null
hypothesis for all tests

Limitations/Extensions
Cells can vary in viability
 Cells may not represent single defined
population (mMDSC’s lineage not defined)
 Increase amount of replicates
 Test effects of other health hazards on stem
cells (such as smoking, radiation, etc)
 Use a fluorometer to quantify data for
greater accuracy

Sources/Acknowledgements

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NIH
Science
Cummins, James H. An Overview of Current
Scientific Research in Muscle Regeneration.
Children’s Hospital of Pittsburgh.
Special thanks to Dr. Deasy for assisting me with
the project and use of the Deasy Lab
Special thanks to Pitt undergraduate Jordan
Nance for teaching me the majority of
procedures
Special thanks to John Huard and Dr. Garibeh for
use of the Huard lab