1. Effects of Citrus Aurantium on Stem Cell Differentiation and Proliferation By Kelly Hyland Oakland Catholic High School Grade 11 PJAS 2011

2. Stem Cell Overview  Un-specialized cells that have the potential to differentiate into mature body tissues  Can be either pluripotent (adult stem cells) or totipotent (embryonic stem cells)  Have potential to cure diseases such as heart disease, diabetes, and others

3.  Subclone of the mus musculous (mouse myoblast) stem cell line  Differentiate rapidly to form myotubes, the cells that make up the myofibers of muscle tissue.  A useful model to study the differentiation from stem cells to mature skeletal muscle tissue C2C12 Stem Cell Line

4.  Strong herbal stimulant that modulates adrenaline pathways, increasing heart rate  May be implicated in heart rhythm disorders  An additive in many weight loss drugs  Extracted from orange oils  Interferes with enzymatic activity in the cholesterol production pathway, an essential part of the cell membrane (HMG-CoA reductase)  May crystallize when reacting with certain sugars Variable Citrus Aurantium

5.  To test the effect(s) of Citrus Aurantium on C2C12 proliferation and differentiation. Purpose

6.  Null Hypothesis: Citrus Aurantium will not have a significant effect on C2C12 proliferation and differentiation.  Alternative Hypothesis: Citrus Aurantium will have a significant effect on stem cell proliferation and differentiation. Hypothesis

7. Materials  Cryotank  75mm2 tissue culture treated flasks  Six 25 mm2 tissue culture treated flasks  Fetal bovine serum (FBS)  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 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])  Liquid Citrus Aurantium Extract  75 mL culture flask  Incubator  Nikon Inverted Microscope  Aspirating Vacuum Line  Laminar Flow Hood  Laminar Flow Hood UV Sterilizing Lamp  Hemocytometer  Sterile PBS  Ethanol (70% and 100%)

8.  A 1 mL aliquot of C2C12 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in a 75mm2 culture flask yielding a cell density of approximately 2x10 6 cells.  The media was replaced with 15 mL of fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO2) for 2 days until a cell density of approximately 4x106 to 5x106 cells/mL was reached.  The culture was passed into 3 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2. Procedure: Proliferation

9.  Made up stock solution with [10 -2 ] of variable  Seeded 6 flasks with C2C12 cells from the original 3 flasks in 5mL of 10% DMEM media each  Allowed cells to incubate in CO 2 incubator overnight and adhere to bottom of flask  Added 50μL of variable and removed 50μL of media from 2 flasks creating [10 -4 ]  Added 5μL of variable and removed 5μL of media from 2 flasks creating [10 -5 ]  Recommended doses over 10 L of body fluid  2 flasks for control  Allowed flasks to proliferate in incubator overnight Procedure: Proliferation Assay

10.  Next day, aspirated media off of flasks  Rinsed with 1mL of trypsin, aspirated off  Added 1mL trypsin, incubate for 5 minutes  Slap flask and confirm with microscope that cells are no longer adhered to bottom of flask  Quenched reaction with 5mL media. Re-add variable.  Re-suspended cells before taking counts using pipette  Loaded hemocytometer with approx 75uL from flask  Took 4 counts per flask  Counted cells in field of view of hemocytometer under Nikon inverted microscope and multiplied count by 10 4 for total cells/mL  Repeated on Day 3 Procedure: Proliferation Continued

11. Proliferation: Results P Value: 5.89 * 10 -9 T Values: 12.34, 17.33

12.  Seeded 6 well plates with 3mL of C2C12 cells in 3mL 10% DMEM media  Allowed cells to proliferate until reached 80% confluence  Changed media to 1% DMEM to induce differentiation  Added 30μL of stock to and removed 30μL media from 2 plates, creating [10 -4 ]  Added 3μL of stock to and removed 3μL media from 2 plates creating [10 -5 ]  Kept 2 control plates  Took images of plates Day 1, Day 3, and Day 4  Compared number of myotubes formed on each plate Procedure: Differentiation Assay

13. Differentiation Results: Day 1 ControlLow ConcentrationHigh Concentration

14. Differentiation Results: Day 3 ControlLow Concentration High Concentration

15. Differentiation Results: Day 4 ControlLow ConcentrationHigh Concentration

16. Interpretation: Differentiation  The variable significantly effected differentiation of the C2C12 cells  Crystallization observed in images: citrus aurantium may have crystallized with sugars in media and starved cells of nutrients  May have interfered with cholesterol synthesis and thus the cell membrane structure

17.  Stat analysis showed that variation was significant  Must reject null hypothesis  Data supports alternative hypothesis  Results obtained may be due to either the crystallization of citrus aurantium with media sugars or interference with cholesterol synthesis Conclusion

18.  http://www.ars-grin.gov/cgi- bin/npgs/html/taxon.pl?10684 http://www.ars-grin.gov/cgi- bin/npgs/html/taxon.pl?10684  http://www.cmaj.ca/cgi/pmidlookup?view=long&pmid =15497209 http://www.cmaj.ca/cgi/pmidlookup?view=long&pmid =15497209  http://www.nature.com/nature/journal/v270/n5639/ab s/270725a0.html http://www.nature.com/nature/journal/v270/n5639/ab s/270725a0.html  "Dangerous Supplements: Twelve Supplements You Should Avoid" Consumer Reports Magazine, September 2010 Works Cited

19. Appendix

20. Tissue Engineering  What is TE?  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 potential to replace damaged functional tissues in the body  Shows great promise in replacing functional muscle tissue

21.  Repeat experiment to verify results  To test leading hypothesis of crystallization, measure amount of sugars remaining in fluid and compare with pure media Further Research