Transcript Creatine Effects on Oxidatively Stressed Stem Cells

Creatine Effects on OxidativeStressed Stem Cells
Anthony DeRenzo
Pittsburgh Central Catholic HS
Grade 11
February 6, 2010
What is Tissue Engineering?
• Broadly Defined: Tissue Engineering is the
development and manipulation of artificial
implants, laboratory-grown tissues,
genetically engineered cells and/or
molecules to replace or support the
function of defective or injured parts of the
body.
Principles of Tissue Engineering
Cells
ECM
Defect
Hormones
Phil Campbell, Carnegie Mellon
Regeneration
Blood
Supply
Overview of Skeletal Muscle Structure
*Muscle
*Muscle Fiber Bundles (fascicles) and the ECM
*Muscle Fibers (multinucleated structures)
*Basal Lamina Membrane
*Satellite Cells (mixed population)
*Sarcolemma Membrane
*Sarcoglycan Complex
*Dystrophin
*Actin-Myosin Complex
C2C12 Cells
• Subclone of the mus musculus (mouse) myoblast cell line.
• Differentiates rapidly, forming contractile myotubes and
produces characteristic muscle proteins.
• Mouse stem cell line is used as a model in many tissue
engineering experiments.
• Useful model to study the differentiation of non-muscle
cells (stem cells) to skeletal muscle cells.
• Expresses muscle proteins and the androgen receptor (AR).
• AR- DNA binding transcription factor which regulates
gene expression.
Creatine
• An organic acid naturally
synthesized from amino
acids (methionine,
glycine, arginine)
primarily in the kidney
and liver, then
transported in the blood
for use by muscles.
• Approximately 95%
located in the skeletal
muscle.
• Direct relation to ATP
production and storage.
Supplemental Creatine
• Most popular bodybuilding
supplement on the market.
• Has such an impact because it
super-hydrates muscle cells
with water.
• Enhances muscle growth, and
strengthens fibers.
• Increased energy levels,
strength, and recovery rates.
Accelerates weight loss and
builds lean body mass.
Oxidative Stress and Hydrogen Peroxide
• Caused by an imbalance between the reproduction of
reactive oxygen and the ability to repair toxic damage.
• All life forms maintain reducing environment within
their cells.
• If disturbed, peroxides and free radicals are
produced.
• These cause harm to cell components (protein, lipids,
DNA).
• Apoptosis
• Hydrogen peroxide stress
• Two electron reduction state
• Formed by dismutation of O2- or direct reduction of
O2
• Lipid soluble
• Can be caused through overproduction of oxygen
during physical exercise
Purpose
• To examine the effects of Creatine
Monohydrate on the proliferation,
differentiation, and survivorship of normal
and H2O2 stressed C2C12 cells.
Hypotheses
Nulls: 1. Creatine Monohydrate will not have an effect on
the proliferation, differentiation, and survivorship of
normal and H2O2 stressed C2C12 cells.
2. There will be no evidence of synergy between
Creatine Monohydrate and peroxide stress.
Alternates: 1. Creatine Monohydrate will have an effect
on the proliferation, differentiation, and survivorship of
normal and H2O2 stressed C2C12 cells.
2. There will be significant evidence of
synergy between Creatine Monohydrate and peroxide
stress.
• Cryotank
• Three 75mm2 tissue
culture treated flasks
• Twenty-four 25 mm2
tissue culture treated
flasks
• 10% fetal bovine serum
• C2C12 Myoblastic Stem
Cell Line
• Trypsin-EDTA
• Pen/strep
• Macropipette + sterile
macropipette Tips (1 mL, 5
mL, 10, mL, 20 mL)
• Micropipettes + sterile
tips
• DMEM media -1% and
Complete Media (4 mM
L-glutamine, 4500 mg/L
glucose, 1 mM sodium
pyruvate, and 1500 mg/L
sodium bicarbonate + [
10% fetal bovine serum
for complete])
Materials
• 75 mL culture flask
• Incubator
• Zeis Inverted Compound Optical
Scope
• Aspirating Vacuum Line
• Laminar Flow Hood
• Laminar Flow Hood UV Sterilizing
Lamp
• Labeling Tape
• Creatine Monohydrate
• Hydrogen Peroxide
• Hemocytometer
• Sterile PBS
• Ethanol (70% and 100%)
• Distilled water
Procedure (Stem Cell Line Preparation)
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•
•
A 1 mL aliquot of C2C12 cells from a
Cryotank was used to inoculate 30 mL of 10%
serum DMEM media in a 75mm2 culture flask
yielding a cell density of approximately 106 to
2x106 cells.
The media was replaced with 15 mL of fresh
media to remove cryo-freezing fluid and
incubated (37° C, 5% CO2) for 2 days until a
cell density of approximately 4x106 to 5x106
cells/mL was reached.
The culture was passed into 3 flasks in
preparation for experiment and incubated for
2 days at 37° C, 5% CO2.
Procedure (Proliferation)
•
•
•
•
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After trypsinization, cells from all of the flasks
were pooled into 1 common 75mm2 flask
(cell density of approximately 1 million
cells/mL).
5 ml of the cell suspension was added to 24
25 mm2 tissue culture treated flasks,
creating a cell density of approximately 10 5
cells per flask.
1 mM stock concentration of hydrogen
peroxide created by adding 0.1 ml of
peroxide to 8.7 ml of sterile water.
The following concentrations of variable (next
page) were added to the flasks. 4 flasks for
each group (2 for proliferation, 2 for
differentiation)
The cells were incubated (37°C, 5% CO2) for
the remainder of the study.
Experimental Groups
0% Peroxide
10 uM Peroxide
0%
0.6 ml sterile water
Creatine 4.4 ml cell culture and medium
0.05 ml peroxide
0.55 ml sterile water
4.4 ml cell culture and medium
.01%
0.05 ml Creatine
Creatine 0.55 ml sterile water
4.4 ml cell culture and medium
0.05 ml peroxide
0.05 ml Creatine
0.5 ml sterile water
4.4 ml cell culture and medium
1%
0.5 ml Creatine
Creatine 0.1 ml sterile water
4.4 ml cell culture and medium
0.05 ml peroxide
0.5 ml Creatine
0.05 ml sterile water
4.4 ml cell culture and medium
Procedure (Differentiation)
• The differentiation experiment was identical to the
proliferation experiment with the following exceptions:
• On Day 3 of experimentation, the original media was
removed and replaced with 1% DMEM media (serum
starvation) to induce myotube differentiation.
Imaging
• On Days 1, 3, and 6 pictures of two areas of each
flask were taken with a Nikon Inverted Microscope.
• These pictures are a visual representation of the
proliferation and differentiation of the cells.
Proliferation
35
30
Cell Count (104 cells/flask)
25
20
stressed
15
unstressed
Baseline
10
5
0
0.00%
Day 1
0.01%
Day 3
Day 1
0.10%
Day 3
Day 1
Day 3
P Values (Single /Double Factor)
Stressed vs. Non
Stressed
Stressed vs. Non
Stressed
0.01% Creatine
Stressed vs. Non
Stressed
0.1% Creatine
2.23 E-07 (SF)
Significant
0.013721 (SF)
Significant
0.00275 (S)
Significant
0.06457 (DF)
Insignificant
0.058231 (DF)
Insignificant
Conclusions:
1. The first null hypothesis can be rejected in
every case.
2. The second null hypothesis can be accepted
in both cases.
Proliferation Unstressed vs. Stressed
0% Creatine
Unstressed
Stressed
Day 1
Day 3
Proliferation Unstressed vs. Stressed
0.01% Creatine
Unstressed
Stressed
Day 1
Day 3
Proliferation Unstressed vs. Stressed
0.1% Creatine
Unstressed
Stressed
Day 1
Day 3
Differentiation Unstressed vs. Stressed
0% Creatine Day 6
Unstressed
Stressed
Differentiation Unstressed vs. Stressed
0.01% Creatine Day 6
Unstressed
Stressed
Differentiation Unstressed vs. Stressed
0.1% Creatine Day 6
Unstressed
Stressed
Qualitative Analysis (Differentiation)
• Unstressed vs. Stressed 0% Creatine
• Appearance - Significant
• Evidence of myotube formation in unstressed test group.
• Unstressed vs. Stressed 0.01% Creatine
• Appearance - Significant
• Evidence of myotube formation in unstressed test group.
• Unstressed vs. Stressed 0.1% Creatine
• Appearance - Significant
• Evidence of myotube formation in unstressed test group.
• In all examples, creatine was not able to remediate the
oxidative stress. Therefore, there were no synergistic effects.
Limitations and Extensions
• Only used qualitative assay of differentiation /
Utilize quantitative assay (MyoD expression)
• Test more variations of concentrations
• Use other types of stress (UV, heat, infection,
various chemicals)
• CyQUANT™ Cell Proliferation Assay
• More quantitative than counting cells on a
Hemocytometer
• Fluorescent dye binds to nucleic acid in the cell
References
• John Wilson, Biostatistician for the University of
Pittsburgh
• Conrad M. Zapanta, Ph.D BiomedicalEngineering
Laboratory, Carnegie Mellon University
• Mark Krotec, PTEI
• http://www.netdoctor.co.uk/focus/nutrition/facts/oxidat
ive_stress/oxidativestress.htm
• http://cropsoil.psu.edu/courses/AGRO518/Oxygen.htm
• http://www.creatinejournal.com/
• http://www.bodybuilding.com/store/creatine.html