Antioxidant Remediation of

Oxidative Stressed Stem Cell LineIshan Chatterjee

Grade 10 – Fox Chapel Area High School

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Introduction: Stem Cells• Stem Cell -- unspecialized cell characterized by the

capacity to give rise to various differentiated cell types

• To model human stem cells, C2C12 mus musculus (mouse) myoblast cell line used

• C2C12 cells used to model cell differentiation from stem cell state to skeletal muscle state through myotube structure formation

• Stem cells have uses in research and treatment– Cancer, Type 1 Diabetes, Parkinson’s Disease,

Huntington’s Disease, Celiac Disease, cardiac failure, muscle damage, neurological disorders 2

Introduction: Stem Cells• Can be used to heal damaged tissue• Current solution inadequate: organ and tissue

donation supply fall far short of demand and risk of rejection always present

• Regenerative Medicine -- stem cells implanted to create new, functioning, “normal” tissue that does not induce immune reaction

• Stem cells influenced by implant environment• Oxidative stress may be influencing factor 3

Oxidative Stress• Free radicals of the reactive oxygen species (ROS) can

damage cells in a process called oxidative stress• Free radicals formed as part of natural metabolization

process and also influenced by external factors• Beneficial effects: cell signaling, slow aging• Can cause atherosclerosis, Parkinson's disease, Heart

Failure, Myocardial Infarction, Alzheimer's disease, Fragile X Syndrome and chronic fatigue syndrome

• Hydrogen Peroxide (H2O2) used to cause oxidative stress

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Antioxidants

• Antioxidants: the body’s defense against oxidative stress– Vitamin E, Vitamin C, beta carotene– Neutralize free radicals

• Damaging effects at high concentrations• Vitamin C (Ascorbic Acid -- C6H8O6) used to

combat oxidative stress effects

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Objective • Investigate the main effects and interaction effect

of oxidative stress (Hydrogen Peroxide) and antioxidants (Vitamin C) on the survivorship, proliferation, and differentiation of murine (mouse) myoblastic stem cell line (C2C12).– Survivorship & Proliferation: Effect measured by

counting number of surviving stem cells after exposure to different concentrations of treatment products

– Differentiation: quantitatively measured by counting the number of myosin positive nuclei out of total nuclei in cell photomicrograph

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• As oxidative stress increases, the number of surviving cells decrease when no antioxidant is present

• Unknown antioxidant effect on number of surviving cells when no oxidative stress is present

Hypothesis

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Oxidative Stress Increasing

Antio

xida

nt In

crea

sing

0 +0

+

1

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Survivorship decreasesMediation Effect

Unknow

n Effect

• As oxidative stress levels increase, increasing antioxidant levels have a moderating effect – survivorship will increase as higher concentration of antioxidants counteract the toxic effect

• Differentiation will show similar effects

1

2

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• Two Experiments (Toxicity/Proliferation and Differentiation) :

0.0 µM H2O2 0.1 µM H2O2 1.0 µM H2O2 10. µM H2O2

0.0 µM

Vit. C

0.0 µM H2O2 0.1 µM H2O2 1.0 µM H2O2 10. µM H2O2

0.0 µM Vit. C 0.0 µM Vit. C 0.0 µM Vit. C 0.0 µM Vit. C

1.0 µM

Vit. C

0.0 µM H2O2 0.1 µM H2O2 1.0 µM H2O2 10. µM H2O2

1.0 µM Vit. C 1.0 µM Vit. C 1.0 µM Vit. C 1.0 µM Vit. C

10. µM

Vit. C

0.0 µM H2O2 0.1 µM H2O2 1.0 µM H2O2 10. µM H2O2

10. µM Vit. C 10. µM Vit. C 10. µM Vit. C 10. µM Vit. C

Experimental Design

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Oxidative Stress – H2O2 Concentrations

Antio

xida

nt –

Vit.

C

Con

cent

ratio

ns

+ + + +

+ + + +

+ + + +

Materials and Apparatus• 3% concentrated H2O2

• __% concentrated Ascorbic Acid (C6H8O6)

• C2C12 murine myoblastic stem cells • Deionized sterile water• 100 mL graduated cylinder• Test tube rack• Incubator (37.0°C)• Macropippette with tips• 100 - 1000 µL pipette• 0.1 – 1 mL pipette• 1 – 10 mL pipette• 70% Ethanol (for sterilization)• Felt-tip marker• 15 mL sterile conical tubes• 3 24-well plates• DMEM media (10% calf serum & 1% calf serum)

contains salts, amino acids, vitamins, & glucose• Sterile pipette tips• 0.22 micron syringe filters + 10 mL syringe

• 200 g scales• 75 mL culture flasks• 25 cm2 culture flasks• 50 mL Trypsin-EDTA• 32 mL PBS saline• 32 mL 100% ice-cold ethanol• Penn Strep Solution• 2 Hemocytometers• Light microscope• Inverted microscope (with imaging capabilities)• Class II Biosafety hood• Labcoats, Eye Protection, Disposible Gloves• Anti Myo D stain• DAPI nuclear stain• Vortexor• Delicate task wipes

• Counter

• Aluminum foil

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ProcedurePreparation of Treatment Materials1. 113 µL 3% H2O2 diluted with 9.89 mL sterilized deionized

water to yield 10 mM concentration of H2O22. 182 µL __% ascorbic acid diluted with 9.82 mL sterilized

deionized water to 10 mM concentration of ascorbic acid

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Stem Cell Line Culture1. 1 mL aliquot of C2C12 cells from a cryotank was used to

inoculate 30 mL of 10% serum DMEM media in a 75mL culture flask yielding a cell density of approximately 106 to 2*106 cells

2. Media changed with 15 mL fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO2) for 2 days until a cell density of approximately 4*106 to 5*106 cells/mL was reached

3. The culture was passed into 3 75 mL culture flasks in preparation for experiment (48 hours before)

Procedure (contd.)Treatment Application (Proliferation and

Differentiation: Day 0)1. 36 25 cm2 culture flasks were labeled - 24 for

proliferation/toxicity, 12 for differentiation2. Treatment materials and other materials pipetted into each

of 12 flasks in biosafety hood then left to incubate for 24 hours (see table)

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0.0 µM H2O2 0.1 µM H2O2 1.0 µM H2O2 10. µM H2O2

0.0 µM

Vit. C

H2O2 0.0 µL 0.1 µL 1.0 µL 10. µL

Vit. C 0.0 µL 0.0 µL 0.0 µL 0.0 µL

Water 100 µL 99.5 µL 95 µL 50 µL

Media 3.9 mL 3.9 mL 3.9 mL 3.9 mL

Cells 1.0 mL 1.0 mL 1.0 mL 1.0 mL

1.0 µM

Vit. C

H2O2 0.0 µL 0.1 µL 1.0 µL 10. µL

Vit. C 5.0 µL 5.0 µL 5.0 µL 5.0 µL

Water 95 µL 94.5 µL 90 µL 45 µL

Media 3.9 mL 3.9 mL 3.9 mL 3.9 mL

Cells 1.0 mL 1.0 mL 1.0 mL 1.0 mL

10. µM

Vit. C

H2O2 0.0 µL 0.1 µL 1.0 µL 10. µL

Vit. C 50 µL 50 µL 50 µL 50 µL

Water 50 µL 49.5 µL 45 µL 0 µL

Media 3.9 mL 3.9 mL 3.9 mL 3.9 mL

Cells 1.0 mL 1.0 mL 1.0 mL 1.0 mL

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Antio

xida

nt –

Vit.

C

Con

cent

ratio

nOxidative Stress – H2O2 Concentration

0.0 µM H2O2 0.1 µM H2O2 1.0 µM H2O2 10. µM H2O2

0.0 µM

Vit. C

0.0 µM H2O2 0.1 µM H2O2 1.0 µM H2O2 10. µM H2O2

0.0 µM Vit. C 0.0 µM Vit. C 0.0 µM Vit. C 0.0 µM Vit. C

1.0 µM

Vit. C

0.0 µM H2O2 0.1 µM H2O2 1.0 µM H2O2 10. µM H2O2

1.0 µM Vit. C 1.0 µM Vit. C 1.0 µM Vit. C 1.0 µM Vit. C

10. µM

Vit. C

0.0 µM H2O2 0.1 µM H2O2 1.0 µM H2O2 10. µM H2O2

10. µM Vit. C 10. µM Vit. C 10. µM Vit. C 10. µM Vit. C

Stem Cell Line

Proliferation Differentiation

Procedure (contd.)

Cell count taken from replicates on Day 1 and Day 3

Stained cells fixed Day 4, serum starved Day 2

Experiment

12 Groups

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Oxidative Stress – H2O2 Concentrations

Antio

xida

nt –

Vit.

C

Con

cent

ratio

ns

0.0 µM H2O2 0.1 µM H2O2 1.0 µM H2O2 10. µM H2O2

0.0 µM

Vit. C

0.0 µM H2O2 0.1 µM H2O2 1.0 µM H2O2 10. µM H2O2

0.0 µM Vit. C 0.0 µM Vit. C 0.0 µM Vit. C 0.0 µM Vit. C

1.0 µM

Vit. C

0.0 µM H2O2 0.1 µM H2O2 1.0 µM H2O2 10. µM H2O2

1.0 µM Vit. C 1.0 µM Vit. C 1.0 µM Vit. C 1.0 µM Vit. C

10. µM

Vit. C

0.0 µM H2O2 0.1 µM H2O2 1.0 µM H2O2 10. µM H2O2

10. µM Vit. C 10. µM Vit. C 10. µM Vit. C 10. µM Vit. C

12 Groups

Oxidative Stress – H2O2 Concentrations

Antio

xida

nt –

Vit.

C

Con

cent

ratio

nsx2replicates

Procedure (contd.)Cell Counting (Proliferation: Day 1 and 3)1. For proliferation assay, aspirated off current media2. Added 2mL trypsin and aspirate immediately3. Added 1mL trypsin and incubate for 4 minutes4. Smacked side of flasks hard twice to detach cells from flask

bottom5. Transfered 1 mL of cells to 1 mL tubes in rack using pipette6. Cleaned hemocytometer using 70% ethanol and delicate task

wipes7. Inserted 25 µL of cell solution into each end of

hemocytometer making sure solution wicks across hemocytometer face in an even coating

8. Gently placed cover slip on hemocytometer and examined hemocytometer grid

9. Using the counter, counted and recorded the number of cells in the 3 mm by 3 mm grid

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Procedure (contd.)Media Replacement (Proliferation: Day 2)1. For all cells, aspirated off current media in the process

removing cell waste products2. Added 3.9 mL of media and appropriate concentration of

treatment materials to each group as specified in the previous table,

3. Gently shook to spread cells and left cells in incubator for 24 hours

Serum Starvation (Differentiation: Day 2)4. Aspirated off media5. Added 3.9 mL of 1% serum media to cells in flask6. Added appropriate concentration of degradation materials to

each group, gently shook to spread cells, and left cells in incubator for 12 hours

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Procedure (contd.)Well Plate Transfer (Differentiation: Day 2)1. For differentiation assay cells, repeated steps 1 through 4 of

cell counting (using trypsin to detach cells from flask wall)2. For each group, labeled three wells on the 24-well plates3. Pippetted 0.4 mL of cell solution from each flask to each of

the three corresponding wells4. Added 1.6 mL of 1% serum media to each well and

appropriate concentration of treatment materials to each group in a 2/5 ratio to the volumes specified in the previous table

5. Gently shook to spread cells and left cells in incubator for 36 hours

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Procedure (contd.)Cell Fixing and Myosin and DAPI Staining (Differentiation)1. Poured off media from all wells into a 1 liter beaker2. Pipetted 2 mL of PBS saline into each well3. Swirled each well for 2 seconds and dump out into beaker4. Pipetted 2 mL of ice-cold ethanol into each well5. Swirled wells for 2 seconds and dumped out into beaker6. Let excess ethanol evaporate at room temperature for 5

minutes7. Repeated PBS wash (steps 2 & 3) three times8. Pipetted 10% Goat Serum into each well only enough to cover

surface of wells; let sit for one hour9. Repeated PBS wash three times10. Added primary (Mouse anti-human myosin heavy chain) at a

ratio of 1:300 (7 µL primary/well – 2 mL volume); swirled and let it sit for one hour

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Procedure (contd.)

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11. Dumped well contents into beaker and repeated PBS wash three times

12. Added secondary (goat anti-mouse Fitc) at a ratio of 1:300; swirled, covered well plates in foil, and let sit for one hour

13. Repeated PBS wash three times14. Added DAPI stain at a ratio of 1:2000 (1 µL primary/well – 2

mL volume); swirled, covered well plates in foil, and let it sit for one hour

15. Added 1 mL of PBS to keep cells hydrated and kept cells refrigerated until cell photomicrography

Procedure (contd.)Cell Photomicrography (Differentiation)1. Turned on inverted microscope optical imaging system and

connected computer, opened imaging software2. Wiped condensation off lid of well plates with delicate task

wipes3. Adjust focus, white balance, and exposure as necessary4. For each differentiation well took and labeled six

micrographs with attached camera, three with UV light filter to excite DAPI nuclear stain (blue) and three with blue light filter to excite myosin stain (green)

5. Obtained quantitative result by creating ratio of myosin positive nuclei (number of nuclei within green myosin stain) to total nuclei in cell photomicrograph

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20

Days

3

Procedure (contd.)

0

1

2

4Experiment: Survivorship/Proliferation

Treatment Product Preparation and Stem Cell Line cultured (both experiments)

Treatment Application

Treatment Application

Cell Count Taken

Media Replacement

Cell Count Taken

Serum Starvationand Well Transfer

Cells Fixedand Stained

Experiment:Differentiation

Cell Photomicrography

0 0.1 1 100

5

10

15

20

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30Day 1: Cell Survivorship

0110

H2O2 Concentration (mM)

Ave

rage

Cel

l Sur

vivo

rshi

p

Results: Proliferation – Day 1

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Vit. C Concentrations

(mM)

p-value:

Results: Proliferation – Day 3

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0 0.1 1 100

20

40

60

80

100

120

140

160Day 3: Cell Survivorship

0110

H2O2 Concentration (mM)

Ave

rage

Cel

l Sur

vivo

rshi

p

Vit. C Concentrations

(mM)

Limitations

1. Murine stem cells may not have provided accurate representation of human stem cells

2. Constant and direct exposure to only hydrogen peroxide may not accurately represent oxidative stress process in the human body

3. Constant and direct exposure to only Vitamin C may not accurately represent antioxidant remediation process in the human body

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Experiment Extensions1. Use human stem cells instead of murine stem

cells2. Test a wider range of oxidative stressors to

mimic more closely oxidative stress in the human body

3. Test a wider range of antioxidants to mimic more closely antioxidant remediaiton in the human body

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References"Antioxidants and oxidative stress." NetDoctor.co.uk - The UK's leading

independent health website. Web. <http://www.netdoctor.co.uk/focus/nutrition/facts/oxidative_stress/oxidativestress.htm>.

"Genox - What is Oxidative Stress?" Genox - Leading the way in Oxidative Stress research. Web. <http://www.genox.com/what_is_oxidative_stress.html>.

OrganDonor.gov. Web. <http://organdonor.gov/>."Oxidative stress." Wikipedia, the free encyclopedia. Web.

<http://en.wikipedia.org/wiki/Oxidative_stress>. "Stem cell - definition from Biology-Online.org." Life Science Reference - Biology

Online. Web. <http://www.biology-online.org/dictionary/Stem_cells>. "Stem Cell Basics [Stem Cell Information]." NIH Stem Cell Information Home

Page. Web. <http://stemcells.nih.gov/info/basics/>. "Stem cell." Wikipedia, the free encyclopedia. Web.

<http://en.wikipedia.org/wiki/Stem_cell>.

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• Dr. Bridget Deasy• Mr. Jordan Nance• Mr. Mark Krotec• Dr. Phil Cambell• Dr. Conrad Zapanta• Mr. Gregory Schubert• My parents

Thank You!

Acknowledgements

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