1. The Effects of Stress on Post-Natal Stem Cell Proliferation and Differentiation Robert McKinstry Pittsburgh Central Catholic High School

2. Stress Stress: anything that disrupts regular cellular processes and reduces survivorship. - mechanical (violent shaking) - chemical (toxins), - oxidative (UV, X-ray) - ischemic (lack of oxygen)

3. Oxidative Stress X-ray radiation can result in oxidative stress on cells. Increases oxidant production in cells Results in cellular degeneration Could cause various cancers, such as skin cancer Many X-ray procedures expose patients to oxidative stress.

4. Ischemic Stress Caused by a lack of O 2 in the cell Results in the cell’s inability to make appropriate amounts on ATP Apoptosis usually follows Because of the nature of regenerative medicine, the vast majority of TE treatments will stress stem cells ischemically.

5. 3T3 Cells Immortalized cell line Fibroblastic Common cellular model Represents non-stem cells Image of 3T3 cells, taken by presenter

6. C2C12 Post-natal mouse myoblastic stem cell line Immortalized Differentiates quickly, forming myotubes when serum starved at confluence Image of C2C12 cells, taken by presenter

7. Purpose The purpose of this experiment was to determine the effects of oxidative and ischemic stress on post-natal stem cell proliferation and differentiation in comparison to non-stem cells in vitro.

8. Hypotheses Null: Stress would not significantly affect post-natal stem cell proliferation or differentiation. Alternative: Stress would significantly slow post-natal stem cell proliferation and inhibit differentiation.

9. Variable and Control Groups Proliferation Experiment Two flasks for each of the following groups: Control Ischemic Stress – High dose X-ray – High dose Ischemic Stress – Low dose X-ray – Low dose 2x – One for each cell type

10. Variable and Control Groups Differentiation Experiment Four plates for each of the following groups: Control X-ray – High doseX-ray – Low dose 1x – Only C2C12

11. Materials Cryotank 75mm 2 tissue culture treated flasks Twenty 25 mm 2 tissue culture treated flasks Ten glass plates 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 Serum - 1% and 10% (4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate + fetal bovine serum determined by desired serum concentration) Dental X-ray machine N 2 tank 75 mL culture flask Incubator Nikon Inverted Microscope Aspirating Vacuum Line Laminar Flow Hood Labeling Tape Hemocytometer Sterile PBS Ethanol (70%) Distilled water Permanent marker Methanol Primary and secondary antibodies for staining (Desmin, Dapi, and myosin antibodies in suspension) Blocker solution Fluoroscope

12. Doubles/Hour P-value = 4.61E-13 α =.05 2.30 3.89 1.26 3.24

13. Dunnett Test Results VariableT-ValueT-CritSignificance X-ray: Low 2.302.95Insignificant X-ray: High 3.892.95Significant Ischemic: Low 1.262.95Insignificant Ischemic: High3.242.95Significant

14. P-value: 2.9E-3 α =.05 2.16 3.46 3.12 1.83

15. Dunnett Test Results VariableT-ValueT-CritSignificance X-ray: Low 2.162.95Insignificant X-ray: High 3.462.95Significant Ischemic: Low 1.832.95Insignificant Ischemic: High3.122.95Significant

16. Percent Decrease 3T3C2C12 X-ray: High30.571428618.53794Significant Ischemic: High24.07239829.57943Significant

17. α =.05 P-value: 0.5

18. Conclusions High doses of oxidative and ischemic stress do significantly affect the proliferation of both post- natal stem cells and non-stem cells. Post-natal stem cells are significantly more resistant to the affects of oxidative stress on proliferation than non-stem cells. Post-natal stem cells are significantly more resistant to the affects of ischemic stress on proliferation than non-stem cells.

19. Conclusions Oxidative stress does not significantly affect post-natal stem cell differentiation. The null hypothesis was rejected for the proliferation study. The null hypothesis was accepted for the differentiation study.

20. In the next round of testing, a larger sample size will be used. Ischemia effects on differentiation will be studied. Different stresses will be used. Future Studies Limitations Over-trypsinization Hemocytometer accuracy