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 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 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 -- C 6 H 8 O 6 ) 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

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 Hypothesis 7 Oxidative Stress Increasing Antioxidant Increasing Survivorship decreases Mediation Effect Unknown 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 3

Two Experiments (Toxicity/Proliferation and Differentiation) : 0.0 µM H 2 O µM H 2 O µM H 2 O µM H 2 O µM Vit. C 0.0 µM H 2 O µM H 2 O µM H 2 O µM H 2 O µM Vit. C 1.0 µM Vit. C 0.0 µM H 2 O µM H 2 O µM H 2 O µM H 2 O µM Vit. C 10. µM Vit. C 0.0 µM H 2 O µM H 2 O µM H 2 O µM H 2 O µM Vit. C Experimental Design 8 Oxidative Stress – H 2 O 2 Concentrations Antioxidant – Vit. C Concentrations

Materials and Apparatus 3% concentrated H 2 O 2 __% concentrated Ascorbic Acid (C 6 H 8 O 6 ) C2C12 murine myoblastic stem cells Deionized sterile water 100 mL graduated cylinder Test tube rack Incubator (37.0°C) Macropippette with tips 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 cm 2 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 9

Procedure Preparation of Treatment Materials µL 3% H2O2 diluted with 9.89 mL sterilized deionized water to yield 10 mM concentration of H2O µL __% ascorbic acid diluted with 9.82 mL sterilized deionized water to 10 mM concentration of ascorbic acid 10 Stem Cell Line Culture 1.1 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 10 6 to 2*10 6 cells 2.Media changed with 15 mL fresh media to remove cryo- freezing fluid and incubated (37° C, 5% CO 2 ) for 2 days until a cell density of approximately 4*10 6 to 5*10 6 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) cm 2 culture flasks were labeled - 24 for proliferation/toxicity, 12 for differentiation 2.Treatment materials and other materials pipetted into each of 12 flasks in biosafety hood then left to incubate for 24 hours (see table) 11

0.0 µM H 2 O µM H 2 O µM H 2 O µM H 2 O µM Vit. C H2O2H2O2 0.0 µL0.1 µL1.0 µL10. µL Vit. C0.0 µL Water100 µL99.5 µL95 µL50 µL Media3.9 mL Cells1.0 mL 1.0 µM Vit. C H2O2H2O2 0.0 µL0.1 µL1.0 µL10. µL Vit. C5.0 µL Water95 µL94.5 µL90 µL45 µL Media3.9 mL Cells1.0 mL 10. µM Vit. C H2O2H2O2 0.0 µL0.1 µL1.0 µL10. µL Vit. C50 µL Water50 µL49.5 µL45 µL0 µL Media3.9 mL Cells1.0 mL 12 Antioxidant – Vit. C Concentration Oxidative Stress – H 2 O 2 Concentration

0.0 µM H 2 O µM H 2 O µM H 2 O µM H 2 O µM Vit. C 0.0 µM H 2 O µM H 2 O µM H 2 O µM H 2 O µM Vit. C 1.0 µM Vit. C 0.0 µM H 2 O µM H 2 O µM H 2 O µM H 2 O µM Vit. C 10. µM Vit. C 0.0 µM H 2 O µM H 2 O µM H 2 O µM H 2 O µM Vit. C Stem Cell Line ProliferationDifferentiation Procedure (contd.) Stained cells fixed Day 4, serum starved Day 2 Experiment 12 Groups 13 Oxidative Stress – H 2 O 2 Concentrations Antioxidant – Vit. C Concentrations 0.0 µM H 2 O µM H 2 O µM H 2 O µM H 2 O µM Vit. C 0.0 µM H 2 O µM H 2 O µM H 2 O µM H 2 O µM Vit. C 1.0 µM Vit. C 0.0 µM H 2 O µM H 2 O µM H 2 O µM H 2 O µM Vit. C 10. µM Vit. C 0.0 µM H 2 O µM H 2 O µM H 2 O µM H 2 O µM Vit. C 12 Groups Oxidative Stress – H 2 O 2 Concentrations Antioxidant – Vit. C Concentrations

Procedure (contd.) Cell Counting (Proliferation: Day 1 and 3) 1.For proliferation assay, aspirated off current media 2.Added 2mL trypsin and aspirate immediately 3.Added 1mL trypsin and incubate for 4 minutes 4.Smacked side of flasks hard twice to detach cells from flask bottom 5.Transfered 1 mL of cells to 1 mL tubes in rack using pipette 6.Cleaned hemocytometer using 70% ethanol and delicate task wipes 7.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 14

Procedure (contd.) Media Replacement (Proliferation: Day 2) 1.For all cells, aspirated off current media in the process removing cell waste products 2.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) 1.Aspirated off media 2.Added 3.9 mL of 1% serum media to cells in flask 3.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.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 plates 3.Pippetted 0.4 mL of cell solution from each flask to each of the three corresponding wells 4.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 16

Procedure (contd.) Cell Fixing and Myosin and DAPI Staining (Differentiation) 1.Poured off media from all wells into a 1 liter beaker 2.Pipetted 2 mL of PBS saline into each well 3.Swirled each well for 2 seconds and dump out into beaker 4.Pipetted 2 mL of ice-cold ethanol into each well 5.Swirled wells for 2 seconds and dumped out into beaker 6.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 17

Procedure (contd.) 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

Procedure (contd.) Cell Photomicrography (Differentiation) 1.Turned on inverted microscope optical imaging system and connected computer, opened imaging software 2.Wiped condensation off lid of well plates with delicate task wipes 3.Adjust focus, white balance, and exposure as necessary 4.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 19

20 Procedure (contd.) Experiment: Survivorship/Proliferation Treatment Product Preparation and Stem Cell Line cultured (both experiments) Treatment Application Cell Count Taken Media Replacement Cell Count Taken Serum Starvation and Well Transfer Cells Fixed and Stained Experiment: Differentiation Cell Photomicrography

Results: Proliferation – Day 1 21 Vit. C Concentrations (  M) p-value:

Results: Proliferation – Day 3 22 Vit. C Concentrations (  M)

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 23

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 24

References "Antioxidants and oxidative stress." NetDoctor.co.uk - The UK's leading independent health website. Web.. "Genox - What is Oxidative Stress?" Genox - Leading the way in Oxidative Stress research. Web.. OrganDonor.gov. Web.. "Oxidative stress." Wikipedia, the free encyclopedia. Web.. "Stem cell - definition from Biology-Online.org." Life Science Reference - Biology Online. Web.. "Stem Cell Basics [Stem Cell Information]." NIH Stem Cell Information Home Page. Web.. "Stem cell." Wikipedia, the free encyclopedia. Web.. 25

Dr. Bridget Deasy Mr. Jordan Nance Mr. Mark Krotec Dr. Phil Cambell Dr. Conrad Zapanta Mr. Gregory Schubert My parents Acknowledgements 26