Estradiol Effects on a
Mammalian Stem Cell Line
JACK LEECH
GRADE 12
PITTSBURGH CENTRAL CATHOLIC HIGH
SCHOOL
PJAS 2011
Stem Cells and Tissue Engineering
 Tissue Engineering (TE) is the science concerned
with improving, restoring, or maintaining the quality
of life via artificially created tissues and organs.
 TE has the potential to create new tissues that have
been lost or damaged due to disease, hereditary
defects, trauma, etc.
 With respect to this project, TE technology has been
shown to be heavily involved with muscle growth.
Targets of Tissue Engineering
Cells
ECM
Defection
Hormones
Regeneration
Blood
Supply
C2C12 Stem Cell Line
 Subclone of the mus musculus (mouse) myoblast cell line.
 A model type of stem cell line that was discovered in 1977
through experimentation of murine thigh muscle growth
after a crush injury.
 Differentiates rapidly, forming contractile myotubes and
produces characteristic muscle proteins.
 Expresses muscle proteins and the androgen receptor (AR).

AR- DNA binding transcription factor which regulates
gene expression.
Estradiol
 The variable estradiol is the major sex hormone in
females, and present in smaller amounts in males.
 Accounts for female secondary sex characteristics.
 Steroid hormone (Passes through cellular and
nuclear membranes and affects transcription).
 Estradiol often associated with activation of certain
oncogenes, often promoting uterine or breast
cancer.
Purpose
 The purpose of this study is to observe the effects of
the estrogen estradiol on the proliferation,
differentiation, and survivorship of C2C12 stem cells.
Hypothesis
 Null Hypothesis: The addition of the estrogen
estradiol WILL NOT affect the proliferation,
differentiation, and survivorship of C2C12 stem cells.
 Alternate Hypothesis: The addition of the estrogen
estradiol WILL significantly affect the proliferation,
differentiation, and survivorship of C2C12 stem cells.
Materials
 Cryotank
 75mm2 tissue culture treated flasks
 Twenty 25 mm2 tissue culture treated
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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])
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Estradiol (powder)
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
Procedure (Culturing the Cells)
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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.
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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.
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The culture was passed into 3 flasks in preparation for
experiment and incubated for 2 days at 37° C, 5% CO2.
Procedure (Addition of Variable on 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 stock solution of estradiol (10-3) was created using 10 mL of ethanol
and 0.027 grams of estradiol. This was then used in a serial dilution to
create a 10-4 concentration.
The 10-6 M concentration was created from 50uL of the stock into
5000uL of ethanol, and the 10-8 M concentrations was created from 5uL
of a 10-5 stock into 5000uL of ethanol.
10-6 M is the suggested working concentration of the estrogen.
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 Experiment)
 Day 1
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 (four counts per flask).
 Day 1 and Day 3
 The previous procedure for determining densities was used again,
and a Nikon Inverted Microscope was used to take images of five
representative areas of each flask.
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Procedure (Differentiation Experiment)
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Day 1 and Day 6
 Using the Nikon Inverted Microscope, images of five
representative areas of each of the flasks were taken.
Day 1
 The original media was removed and replaced with 1% DMEM
media (serum starvation) to induce myotube differentiation.
Results of Proliferation Analysis
p-value:
1.08*10^-4
Cell Count (cells/flask)
160000
140000
120000
100000
Day 1
80000
Day2
60000
40000
20000
0
Control
10^-6
Concentration of Variable
10^-8
Statistical Analyses of the Proliferation Results
 ANOVA
 Compares variation within groups to variation between
groups.
 Using the ANOVA, a p-value less than the alpha of .05 was
gathered (significant variation).
 Reject the null hypothesis.
 Dunnett’s test
 Compares each experimental group to control
individually.
 0.05 alpha was used, and the t-value compared to the tcritical value of 3.03
Dunnett’s Test Results (cont.)
Concentration t-value
t-critical
(.05)
t-critical
(.01)
Variation
10-6
7.645
3.03
4.63
Significant
10-8
4.709
3.03
4.63
Significant
Differentiation Analysis
 Control
Day 1
Day 6
Differentiation Analysis
 10-6 Concentration
Day 1
Day 6
Differentiation Analysis
 10-8 Concentration
Day 1
Day 6
Conclusions
 Proliferation:
 Based upon the results gathered from the ANOVA and
Dunnett’s statistical analyses, it appears that the addition of
estradiol significantly affected the proliferation of C2C12 cells.
 Differentiation:
 Based upon the images gathered from the inverted microscope,
although differentiation was observed, the analysis was
qualitative and variance could not be statistically compared.
Speculatively, it appeared that little difference was noticed
between the control and the various concentrations.
Limitations
 Possible sources of error:
 The methods of analysis for the proliferation assay were
qualitative (hemocytometer).
 A solution is to use a fluorescent dye that could be used
quantify cell counts, and a trypan blue exclusion assay could be
used to measure only live cells.
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The methods of analysis for the differentiation assay were also
qualitative (inverted microscope imaging).
A solution is to use a quantitative method of analysis, such as
MyoD tagging.
Extensions (cont.)
 Use a wider range of concentrations.
 Determine a LD 50% concentration of the hormone.
 Use quantitative analyses.
Works Cited
 Dr. Phil Campbell
 Conrad M. Zapanta, Ph.D. Biomedical Engineering
Laboratory, Carnegie Mellon University
 Mark Krotec, PTEI
 www.PTEI.org
 www.tissue-engineering.net