Ishan Chatterjee
Grade 10 – Fox Chapel Area High
School
1
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
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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
4
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
5
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
6
Hypothesis
Antioxidant Increasing
Unknown Effect
• As oxidative stress increases, the number of surviving cells
1
decrease when no antioxidant is present
• Unknown antioxidant effect on number of surviving cells when
2
no oxidative stress is present
• As oxidative stress
3
Oxidative Stress Increasing
levels increase,
0
increasing antioxidant
Survivorship decreases
0
levels have a
1
moderating effect –
survivorship will
increase as higher
concentration of
3
2
antioxidants
counteract the toxic
+
effect
• Differentiation will
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+
Experimental Design
• Two Experiments (Toxicity/Proliferation and
Differentiation) :
Antioxidant – Vit. C
Concentrations
Oxidative Stress – H2O2 Concentrations
0.0 µM H2O2
0.1 µM H2O2
1.0 µM H2O2
10. µM H2O2
0.0
µM
Vit. C
0.0 µM H2O2
+
0.0 µM Vit. C
0.1 µM H2O2
+
0.0 µM Vit. C
1.0 µM H2O2
+
0.0 µM Vit. C
10. µM H2O2
+
0.0 µM Vit. C
1.0
µM
Vit. C
0.0 µM H2O2
+
1.0 µM Vit. C
0.1 µM H2O2
+
1.0 µM Vit. C
1.0 µM H2O2
+
1.0 µM Vit. C
10. µM H2O2
+
1.0 µM Vit. C
10.
µM
Vit. C
0.0 µM H2O2
+
10. µM Vit. C
0.1 µM H2O2
+
10. µM Vit. C
1.0 µM H2O2
+
10. µM Vit. C
10. µM H2O2
+
10. µM Vit. C
8
Materials and Apparatus
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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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Procedure
Preparation of Treatment Materials
1.
2.
113 µL 3% H2O2 diluted with 9.89 mL sterilized deionized
water to yield 10 mM concentration of H2O2
182 µL __% ascorbic acid diluted with 9.82 mL sterilized
deionized water to 10 mM concentration of ascorbic acid
Stem Cell Line Culture
1.
2.
3.
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
Media changed with 15 mL fresh media to remove cryofreezing 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
The culture was passed into 3 75 mL culture flasks in
preparation for experiment (48 hours before)
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Procedure (contd.)
Treatment Application (Proliferation and
Differentiation: Day 0)
1.
2.
36 25 cm2 culture flasks were labeled - 24 for
proliferation/toxicity, 12 for differentiation
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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Oxidative Stress – H2O2 Concentration
Antioxidant – Vit. C
Concentration
0.0
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
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
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
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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Procedure (contd.)
Stem Cell Line
Experiment
Proliferation
Differentiation
0.0
µM
Vit. C
Oxidative Stress – H2O2 Concentrations
0.1 µM H2O2
1.0 µM H2O2
10. µM H2O2
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
0.0 µM H2O2
12 Groups
1.0
µM
Vit. C
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
Antioxidant – Vit. C
Concentrations
Antioxidant – Vit. C
Concentrations
Oxidative Stress – H2O2 Concentrations
0.0 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 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
0.0 µM H2O2
12 Groups
1.0
µM
Vit. C
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
Stained cells fixed Day 4, serum
starved Day 2
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Procedure (contd.)
Cell Counting (Proliferation: Day 1 and 3)
1.
2.
3.
4.
5.
6.
7.
8.
9.
For proliferation assay, aspirated off current media
Added 2mL trypsin and aspirate immediately
Added 1mL trypsin and incubate for 4 minutes
Smacked side of flasks hard twice to detach cells from flask
bottom
Transfered 1 mL of cells to 1 mL tubes in rack using pipette
Cleaned hemocytometer using 70% ethanol and delicate task
wipes
Inserted 25 µL of cell solution into each end of
hemocytometer making sure solution wicks across
hemocytometer face in an even coating
Gently placed cover slip on hemocytometer and examined
hemocytometer grid
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.
2.
3.
For all cells, aspirated off current media in the process
removing cell waste products
Added 3.9 mL of media and appropriate concentration of
treatment materials to each group as specified in the previous
table,
Gently shook to spread cells and left cells in incubator for 24
hours
Serum Starvation (Differentiation: Day 2)
1.
2.
3.
Aspirated off media
Added 3.9 mL of 1% serum media to cells in flask
Added appropriate concentration of degradation materials to
each group, gently shook to spread cells, and left cells in
incubator for 12 hours
15
Procedure (contd.)
Well Plate Transfer (Differentiation: Day 2)
1.
2.
3.
4.
5.
For differentiation assay cells, repeated steps 1 through 4 of
cell counting (using trypsin to detach cells from flask wall)
For each group, labeled three wells on the 24-well plates
Pippetted 0.4 mL of cell solution from each flask to each of
the three corresponding wells
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
Gently shook to spread cells and left cells in incubator for
36 hours
16
Procedure (contd.)
Cell Fixing and Myosin and DAPI Staining (Differentiation)
1.
2.
3.
4.
5.
6.
Poured off media from all wells into a 1 liter beaker
Pipetted 2 mL of PBS saline into each well
Swirled each well for 2 seconds and dump out into beaker
Pipetted 2 mL of ice-cold ethanol into each well
Swirled wells for 2 seconds and dumped out into beaker
Let excess ethanol evaporate at room temperature for 5
minutes
7. Repeated PBS wash (steps 2 & 3) three times
8. Pipetted 10% Goat Serum into each well only enough to cover
surface of wells; let sit for one hour
9. Repeated PBS wash three times
10. 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.)
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 times
14. 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
18
Procedure (contd.)
Cell Photomicrography (Differentiation)
1.
2.
3.
4.
5.
Turned on inverted microscope optical imaging system and
connected computer, opened imaging software
Wiped condensation off lid of well plates with delicate task
wipes
Adjust focus, white balance, and exposure as necessary
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)
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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Procedure (contd.)
Experiment:
Survivorship/Proliferation
Cell Count Taken
Cells Fixed
and Stained
Media Replacement
Cell Count Taken
Treatment Application
Serum Starvation
and Well Transfer
Treatment Application
Treatment Product
Preparation and Stem Cell Line
cultured (both experiments)
Cell
Photomicrography
Experiment:
Differentiation
20
Results: Proliferation – Day 1
30
Day 1: Cell Survivorship
Average Cell Survivorship
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p-value:
20
15
Vit. C
1 Concentrations
(mM)
0
10
10
5
0
0
0.1
1
10
H2O2 Concentration (mM)
21
Results: Proliferation – Day 3
160
Day 3: Cell Survivorship
Average Cell Survivorship
140
120
100
80
Vit. C
1 Concentrations
(mM)
0
60
10
40
20
0
0
0.1
1
10
H2O2 Concentration (mM)
22
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 Extensions
1. Use human stem cells instead of murine stem
cells
2. 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/oxida
tivestress.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.biologyonline.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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Acknowledgements
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•
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Dr. Bridget Deasy
Mr. Jordan Nance
Mr. Mark Krotec
Dr. Phil Cambell
Dr. Conrad Zapanta
Mr. Gregory Schubert
My parents
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