Creatine Effects on Oxidative-Stressed Stem Cells

Anthony DeRenzoPittsburgh Central Catholic HSGrade 11February 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.

Cells

Principles of Tissue Engineering

ECM

Hormones BloodSupply

Defect Regeneration

Phil Campbell, Carnegie Mellon

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.

Materials• 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])

• 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)

• 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)• 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 Groups0% Peroxide 10 uM Peroxide

0% Creatine

0.6 ml sterile water4.4 ml cell culture and medium

0.05 ml peroxide0.55 ml sterile water4.4 ml cell culture and medium

.01% Creatine

0.05 ml Creatine0.55 ml sterile water4.4 ml cell culture and medium

0.05 ml peroxide0.05 ml Creatine0.5 ml sterile water4.4 ml cell culture and medium

1% Creatine

0.5 ml Creatine0.1 ml sterile water4.4 ml cell culture and medium

0.05 ml peroxide0.5 ml Creatine0.05 ml sterile water4.4 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.

• 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.

Imaging

0.00% 0.01% 0.10%Day 1 Day 3 Day 1 Day 3 Day 1 Day 3

0

5

10

15

20

25

30

35

stressed

unstressed

Baseline

ProliferationCe

ll Co

unt (

104 c

ells

/flas

k)

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

0.06457 (DF)Insignificant

0.00275 (S)Significant

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.

Unstressed Stressed

Proliferation Unstressed vs. Stressed0% Creatine

Day 1

Day 3

Proliferation Unstressed vs. Stressed0.01% Creatine

Unstressed Stressed

Day 1

Day 3

Proliferation Unstressed vs. Stressed0.1% Creatine

Unstressed Stressed

Day 1

Day 3

Differentiation Unstressed vs. Stressed0% Creatine Day 6Unstressed Stressed

Differentiation Unstressed vs. Stressed0.01% Creatine Day 6Unstressed Stressed

Differentiation Unstressed vs. Stressed0.1% 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/

oxidative_stress/oxidativestress.htm• http://cropsoil.psu.edu/courses/AGRO518/Oxygen.htm• http://www.creatinejournal.com/• http://www.bodybuilding.com/store/creatine.html