1. Yohimbine Influence on a Mammalian Stem Cell Line
Patrick Leech
Pittsburgh Central Catholic High school
Grade 11
PJAS 2011

2. What is Tissue Engineering
An interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ.

3. Principles of Tissue Engineering

4. Stem Cell Overview
unspecialized cells capable of renewing themselves through cell division
under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions
stem cells offer new potentials
for treating diseases such as
diabetes and heart disease

5. C2C12 Stem Cell Line
Subclone of the mus musculus (mouse) myoblast cell line.
Mouse stem cell line is frequently used as a model in tissue engineering experiments.
Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins.
Useful model to study the differentiation of non-muscle cells (stem cells) to skeletal muscle cells.

6. Variable Yohimbine
Corynanthe yohimbine- a psychoactive plant which contains the tryptamine alkaloid yohimbine
main active chemical present in yohimbe bark is yohimbine HCl (indole alkaloid), found in the bark of the Pausinystalia yohimbe tree.
Strong stimulant, effects both norepinephrine and epinephrine pathways and modulates feedback loops.
Several uses such as a weight loss supplement, blood pressure boosting agent, and most importantly a muscle supplement.

7. Purpose
To examine the effects of Yohimbine on the proliferation, differentiation, and survivorship of normal C2C12 cells.

8. Hypothesis
Null Hypothesis: The addition of Yohimbine WILL NOT significantly affect the proliferation, differentiation, and survivorship of C2C12 stem cells.
Alternative Hypothesis: The addition of Yohimbine WILL significantly affect the proliferation, differentiation, and survivorship of C2C12 stem cells.

9. Materials Cryotank 75mm2 tissue culture treated flasks
Twenty 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])
75 mL culture flask
Incubator
Nikon Inverted Compound Optical Scope
Aspirating Vacuum Line
Laminar Flow Hood
Laminar Flow Hood UV Sterilizing Lamp
Labeling Tape
Yohimbine
Hemocytometer
Sterile PBS
Ethanol (70% and 100%)
Distilled water

10. Procedure Stem Cell Preparation
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 106 to 2x106 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.

11. Procedure Day 0
After trypsinization, cells from all of the flasks were pooled into 1 common 75mm2 flask (cell density of approximately 1 million cells/mL).
0.1 mL of the cell suspension was added to mm2 tissue culture treated flasks containing 5 mL of DMEM (com) media, creating a cell density of approximately 105 cells per flask.
The 1 M stock solution of Yohimbine was created using 1 mL of sterile dilution fluid (PBS) and grams of Yohimbine.
The 10-6 M, and 10-8 M concentrations were created from the stock, where 10-8 M is the suggested working concentration of the drug.
The 2 experimental groups and the control group were created by adding:
20 µl of the 10-6 M solution to 1 flask
20 µl of the 10-8 M solution to 1 flask
20 µl of PBS to 1 flask (Control)
The cells were incubated at 37°C, 5% CO2 for the remainder of the study.
Two flasks from each group were used in the Proliferation Experiment and two flasks from each group were used in the Differentiation Experiment.

12. Procedure Proliferation
Day 1 and Day 3
Using one flask from each group, cell densities were determined as follows:
The cells were trypsinized and collected into cell suspension.
25 µl aliquots were transferred to a Hemocytometer for quantification (4 counts per flask).
Using the Nikon Inverted Microscope, images of eight representative areas of each flask were taken.
Procedure (Differentiation Experiment)
Day 1 and Day 6
Using the Nikon Inverted Microscope, images of eight representative areas of each of the flasks were taken.
Day 2
The original media was removed and replaced with 1% DMEM media (serum starvation) to induce myotube differentiation.

13. Result of Proliferation Assay
P-value 5.81E-12

14. Statistical Analysis ANOVA Dunnett’s Test
Compares the variation within groups to variation between groups
If p-value is <0.05 reject the null hypothesis
P-value 5.81E-12 therefore the null hypothesis is rejected
Dunnett’s Test
Compares each individual experimental group back to the control
T-critical in this experiment is 3.03

15. Dunnett’s Test Concentration T- Critical Variation 10^-8 19>3.03
Significant
10^-6
179>3.03

16. Control (Differentiation)
Day Day 6

17. 10^-8 Differentiation
Day Day 6

18. 10^-6 Differentiation
Day Day 6

19. Conclusion
Proliferation
From the ANOVA and the subsequent Dunnett’s test, the addition of Yohimbine induced a statistically significant increase in proliferation in the C2C12 cells in both concentrations.
Differentiation
From the qualitative analysis of the images gathered from the flasks, it appears that the addition of Yohimbine induced myotubule formation.

20. Limitations and Extensions
Differentiation
purely qualitative
Solution- quantitative differentiation assay (anti-body assay)
CyQUANT™ Cell Proliferation Assay
More quantitative than counting from a hemocytometer
Extensions
Use higher quantities of Yohimbine and use different kinds of muscle stimulants similar to it
Trypan blue assay to determine if the cells are alive
Anti-body assay

21. Sources
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