Yohimbine Influence on
a Mammalian Stem Cell
Line
Patrick Leech
Pittsburgh Central Catholic High school
Grade 11
PJAS 2011
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.
Principles of Tissue
Engineering
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
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.
1. Differentiates rapidly, forming contractile myotubes
and produces characteristic muscle proteins.
2. Useful model to study the differentiation of nonmuscle cells (stem cells) to skeletal muscle cells.
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.
•
Purpose
• To examine the effects of Yohimbine on
the proliferation, differentiation, and
survivorship of normal C2C12 cells.
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.
Materials
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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
Procedure Stem Cell
Preparation
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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.
Procedure Day 0
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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 20 25 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 0.28842 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:
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20 µl of the 10-6 M solution to 1 flask
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20 µl of the 10-8 M solution to 1 flask
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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.
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).
• Day 1 and Day 3
• Using the Nikon Inverted Microscope, images of eight representative areas of
each flask were taken.
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.
Result of Proliferation Assay
P-value 5.81E-12
2500000
Cell Count (Cells/flask)
2000000
1500000
Day 1
Day 3
1000000
500000
0
Control
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10^6 conc
Concentrations
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10^8 conc
Statistical Analysis
• ANOVA
• 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
Dunnett’s Test
Concentration
T- Critical
Variation
10^-8
19>3.03
Significant
10^-6
179>3.03
Significant
Control (Differentiation)
• Day 1
Day 6
10^-8 Differentiation
• Day 1
Day 6
10^-6 Differentiation
• Day 1
Day 6
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.
Limitations and Extensions
• Limitations
• 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
Sources
• Becker AJ, McCulloch EA, Till JE (1963). "Cytological demonstration of the clonal nature of
spleen colonies derived from transplanted mouse marrow cells". Nature 197: 452–4.
doi:10.1038/197452a0. PMID 13970094.
• Ulloa-Montoya F, Verfaillie CM, Hu WS (Jul 2005). "Culture systems for pluripotent stem
cells". J Biosci Bioeng
• Adewumi O, Aflatoonian B, Ahrlund-Richter L, et al. (2007). "Characterization of human
embryonic stem cell lines by the International Stem Cell Initiative”
• Rosengren, A. H.; Jokubka, R.; Tojjar, D.; Granhall, C.; Hansson, O.; Li, D.-Q.; Nagaraj, V.;
Reinbothe, T. M. et al. (2009). "Overexpression of Alpha2A-Adrenergic Receptors
Contributes to Type 2 Diabetes". Science 327 (5962): 217–20
• Yonezawa A, Yoshizumii M, Ebiko M, Amano T, Kimura Y, Sakurada S (October 2005). "Longlasting effects of yohimbine on the ejaculatory function in male dogs". Biomedical Research
26 (5): 201–6.
• Millan MJ, Newman-Tancredi A, Audinot V, et al. (February 2000). "Agonist and antagonist
actions of yohimbine as compared to fluparoxan at alpha(2)-adrenergic receptors (AR)s,
serotonin (5-HT)(1A), 5-HT(1B), 5-HT(1D) and dopamine D(2) and D(3) receptors.
Significance for the modulation of frontocortical monoaminergic transmission and
depressive states". Synapse 35
• Kaumann AJ (June 1983). "Yohimbine and rauwolscine inhibit 5-hydroxytryptamine-induced
contraction of large coronary arteries of calf through blockade of 5 HT2 receptors". NaunynSchmiedeberg's Archives of Pharmacology 323