Ethanol Effects on Murine Muscle-Derived Stem Cells

Experiment and Analysis by Donald Kline, JrPittsburgh Central Catholic High School

Brief History of Stem CellsWord 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 OverviewBasic Definition- cell that can

produce lineages more specialized than themselves and can renew itself

Spectrum of Stem Cell Behavior:◦totipotent embryonic cells◦pluripotent cells◦multipotent cells◦unipotent cells.

Stem Cell ApplicationsDuchene'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 ExperimentsMost Common Laboratory animal

◦Share a relatively large amount of genes with humans

◦mammals◦Genome completely sequenced◦Breed rapidly, easy to handle, small,

inexpensiveFirst 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 tissueIsolated 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

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.

Cell DifferentiationAdult 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” EtOHFermentation reactionOne of earliest reactions applied by

humanityDepressant Health Issues- Fetal alcohol

syndrome, alcohol addiction, impairment of nervous system and motor system

PurposeExplore effects of Ethanol on

myoblastic stem cell differentiation

HypothesisNull 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• 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)

• C Flask• Hemocytometer• Culture Flasks• Conical Tubes• Microscope• Wells• ScienceCell Media

• 10% DMEM media

Hemocytometer

Materials (Special for Differentiation)

• 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

Procedure1. pp6 mMDSCs were acquired2. Expand mMDSCs through repeated culturing

1. Pass cells (aspiration, trypsinization, replating) 3. Transfer cell suspension into 15mL conical tube4. Rinse Flask with 5mL of PBS twice5. Add the rinse solution to the 15mL conical tube6. Spin at 150g for 5 min.7. Supernatant was removed by suction filtration8. The cell pellet was resuspended in 1mL of DMEM media9. 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. The cells were grown until they were in sufficient quantity, and then were pipetted into wells at a density

5,000 cells/well.2. The cells were allowed to continue growing until they began to differentiate. 3. The variable concentrations of Ethanol were then added.4. After several days, the media was removed and the cells were fixed in 100% Methanol 5. 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 wells3. The Block was removed and the cells rinsed 3Xs w/ PBS4. 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 hour5. The primary was removed and the cells were washed 3 times with PBS6. 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 four9. For Desmin , the primary (Monoclonal Anti-Desmin antibody produced in mouse) was used at a ratio of

1:300uL for one hour10. The primary was removed and the cells were rinse three times with PBS11. The secondary (Anti-Mouse IgG fragment of sheep antibody) was added at a ratio of 1:500uL and

allowed to sit for one hour 6. The cells were then analyzed under an inverted fluorescent microscope with Northern Eclipse Imaging

Software7. Pictures of the fluorescent myotubes were taken and the data was analyzed

Differentiation Procedure: Flowchart

Confluence Add Variables

Several days after Fix in 100%

Methynol Rinse

Block1 hour

Rinse

Myosin OR Desmin

Add Primary

1 HourRinse

Add Secondary

1 HourHoechst 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 Nuclear Stain

1% EtOH Myosin Merged with nuclear stain

Desmin Visualization: Percent Differentiation

Control .5% EtOH 1% EtOH 3 % EtOH 5% EtOH0

5

10

15

20

25

30

35

40

45

50

Desmin % Differentiation

Desmin % Dif-ferentiation

Variables

% D

iffer

entia

tion

P<.05

Desmin Visualization: Nuclei/Myotube

Control .5% EtOH 1% EtOH 3 % EtOH 5% EtOH0

0.5

1

1.5

2

2.5

3

3.5

4

Nuclei/ Myotube (Desmin)

Nuclei/Myotube

Variables

Nuc

lei

P<.05

Dunnett’s Test Results: Desmin

ANOVA 0.5% EtoH

1% EtOH 3% EtOH 5% EtOH

Percent Differentiation

0.008677

p>.05 p>.05 p>.05 p<.05

T values .306 1.56 2.42 3.53

Nuclei/ Myotube

0.001314

p>.05 p>.05 p>.05 p<.05

T values .983 .608 .988 3.42

T Critical Values: p<.05: p<.01:

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

Control .5% EtOH 1% EtOH 3 % EtOH 5% EtOH0

5

10

15

20

25

30

35

40

45

Myosin % Differentiation (Avg.)

Differentiation

Variables

% o

f Nuc

lei D

iffer

entia

ted

P<.01

P<.05

Myosin Visualization: Nuclei/Myotube

Control .5% EtOH 1% EtOH 3 % EtOH 5% EtOH0

0.5

1

1.5

2

2.5

3

3.5

4

4.5Myosin Nuclei/ Myotube

Nuclei/Myotube

Variables

Nuc

lei P

er M

yotu

be

P<.05

Dunnett’s Test Results: Myosin

ANOVA 0.5% EtOH 1% EtOH 3% EtOH 5% EtOH

Percent Differentiation

0.000294

p>.05 p>.05 p<.05 p<.01

T Values 2.79 2.84 4.12 5.34

Nuclei/ Myotube

0.001017

p>.05 p>.05 p>.05 p<.05

T Values 0.068 0.587 2.27 4.49

T Critical Values: p<.05: p<.01:

Conclusions: MyosinPercent 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/ExtensionsCells can vary in viabilityCells may not represent single

defined population (mMDSC’s lineage not defined)

Increase amount of replicatesTest effects of other health hazards

on stem cells (such as smoking, radiation, etc)

Use a fluorometer to quantify data for greater accuracy

Sources/Acknowledgements

NIHScienceSpecial 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