1. Serotonin Effects on Adult Stem Cell Behavior Alec DiVito Central Catholic High School

2. Question Does serotonin affect adult stem cell differentiation and/or proliferation?

3. Tissue Engineering The development and manipulation of artificial implants, laboratory-grown tissues, and genetically engineered cells and/or molecules to replace or support the function of defective or injured body parts. Has the potential to replace or supplement the function of tissues destroyed or compromised in any variety of ways, including: Inherent design flaws Hereditary/congenital defects or conditions Disease Trauma Damage from an individual’s environment Aging TE has great potential for supplementing muscle tissue.

4. Cells ECM Hormones Blood Supply Defect Regeneration Phil Campbell, Carnegie Mellon Principles of Tissue Engineering

5. Chemical Signaling Cells responsive to signaling molecules and growth factors such as BMP-5 and IGF. Signaling molecules control migration, proliferation, differentiation, attachment, and continuity. This is essential for stem cells. Do therapeutic drugs alter this signal driven cell regulation?

6. Neurotransmitters Endogenous chemicals transmit signals from neurons to target cells across a synapse. Release of neurotransmitters follows arrival of an action potential at the synapse. Synthesized from precursors molecules.

7. Serotonin Neurotransmitter, is made via a unique biochemical conversion process. Can affect the functioning of other organ systems. Imbalance may influence mood in a way that leads to depression. Regeneration of brain cells is regulated by serotonin throughout lifetime. No way to measure its levels in the living brain. Don't know whether the dip in serotonin causes the depression, or the depression causes serotonin levels to drop.

8. Adult Stem Cells Undifferentiated cells multiply to replenish dying cells. Somatic stem cells, can be found in juvenile and adult animals and humans. Self-renew indefinitely, and generate all cell types of the organ from which they originate. They have mainly been studied in humans and model organisms such as mice and rats.

9. C2C12 Subclone of the mus musculus (mouse) myoblast cell line. Mouse stem cell line is frequently used as a model in tissue engineering experiments. 1.Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins. 2.Useful model to study the differentiation of non-muscle cells (stem cells) to skeletal muscle cells. 3.Expresses the androgen receptor (AR). AR- DNA binding transcription factor which regulates gene expression.

10. 3T3 Cell line established from Swiss mouse embryo tissue. Has become the standard fibroblast cell line. Often used in the cultivation of keratinocytes, with the 3T3 cells secreting growth factors favorable to these kinds of cells.

11. Purpose To assess the effects of serotonin on adult stem cell behavior.

12. Null Hypothesis Serotonin will not affect adult stem cell behavior. Alternative Hypothesis Serotonin will decrease adult stem cell proliferation and/or differentiation.

13. Materials Serotonin Solution 75mm 2 tissue culture treated flasks Twenty 25 mm 2 tissue culture treated flasks C2C12 Myoblastic Stem Cell Line 3T3 Fibroblastic 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 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 Nikon Inverted Microscope Aspirating Vacuum Line Laminar Flow Hood Laminar Flow Hood UV Sterilizing Lamp Labeling Tape Hemocytometer Sterile PBS Ethanol (70% and 100%) Distilled water

14. Procedure 1.Five ml of adult stem cells and media were prepared in 12 T25 flasks. One ml of C2C12 cells were also allowed to grow in nine well plates, six of which were 50% cells and 50% media and the other three were 30% cells and 70% media. 2.The serotonin was diluted with PBS into a low (10,000X) and high (1,000X). X = therapeutic dose. 3.One μl of each diluted serotonin was placed into the selected nine wells and one ml of serotonin into each flask. 4.Cells were imaged one and three days after (for C2C12) and one and two days after (for 3T3) exposure. Four images were taken per flask. 5.Cells were trypsinized prior to imaging and counting to release from bottom of flask. 0.5 ml of trypsin was inserted into each flask and allowed to incubate for four minutes and 1.5 ml of media was added to quench the reaction. 6.Twenty-five μl of trypsinized cells were loaded into hemacytometers. Eight cell counts were performed per flask. 7.The well plates of C2C12 cells were used to see how serotonin affected differentiation and the flasks represented proliferation of two different cell lines. 8.Six flasks for the 3T3 line and six for the C2C12 line, three flasks were counted and imaged as a representation for a day.

15. Proliferation of C2C12 P-value = 0.008 Serotonin Concentration

16. Proliferation of 3T3 P-value = 0.0282 Serotonin Concentration

17. Analysis of C2C12 Proliferation T-valueSignificance Low4.42Significant High4.70Significant T-critical = 4.32 Analysis of 3T3 Proliferation T-valueSignificance Low3.34Significant High3.01Significant T-critical = 2.97

18. Differentiation of C2C12 – Day 1 ControlLow High

19. Differentiation of C2C12 – Day 3 ControlLow High

20. Differentiation of C2C12 – Day 5 ControlLow High

21. C2C12 Differentiation Analysis ControlLow Qualitative Analysis – Low concentration enhances myotube formation

22. Conclusion From the ANOVA and Dunnett’s tests, the addition of serotonin induced a statistically significant increase in proliferation in the C2C12 cells when added at one one-hundredth its suggested working concentration, X, and a statistically significant decrease in proliferation in the cells when added at one tenth its suggested working concentration, 10X. From the ANOVA and Dunnett’s tests, the addition of serotonin induced a statistically significant increase in proliferation when added at one tenth and one one- hundredth its suggested working concentration. From the qualitative analysis of the images gathered from the flasks, it appears that the addition of serotonin induced myotubule formation. This was especially apparent in the low concentration, while the high concentration showed less dramatic differentiation in comparison.

23. Limitations/Extensions Evaluation of differentiation images is qualitative and imprecise. A quantitative differentiation assay can be used, e.g. MyoD tagging. CyQUANT™ Cell Proliferation Assay can be used. More quantitative than counting cells on a Hemocytometer. Fluorescent dye binds to nucleic acid in the cell Test suggested working concentration and increased concentrations, to see if it would continue to be toxic to cells.

24. Sources and Acknowledgments Dr. Phil Campbell Conrad M. Zapanta, Ph.D. Biomedical Engineering Laboratory, Carnegie Mellon University Mark Krotec, PTEI Mr. Scott Windham, MSW, LCSW C2C12 myoblastoma cell differentiation and proliferation is stimulated by androgens and associated with a modulation of myostatin and Pax7 expression – German Sport University, Cologne, Germany Chen Y, Zajac JD Maclean HE 2005 Androgen regulation of satellite cell function. Journal of Endocrinology 186 21-31. Sinha-Hikim I, Taylor WE, Gonzalez-Cadavid NF, Zheng W and Bhasin S 2004 Androgen receptor in human skeletal muscle and cultured muscle satellite cells: up-regulation by androgen treatment. Journal of Clinical Endocrinology and Metabolism 89 5245-5255. "Bone Morphogenetic Protein-2 Converts the Differentiation Pathway of C2C12 Myoblasts into the Osteoblast Lineage." The Journal of Cell Biology. Web. 31 Jan. 2012..