Anabolic Steroid Influence on a Mammal Stem Cell

LineFrank Wolf

Pittsburgh Central Catholic High SchoolGrade 11

PJAS 2010

What is TE?◦ The development and manipulation of artificial implants,

laboratory-grown tissues, genetically engineered cells and/or molecules to replace or support the function of defective or injured parts of the body

Why is TE important?◦ It 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 muscle therapy.

Tissue Engineering

Cells ECM

Hormones BloodSupply

Defect Regeneration

Phil Campbell, Carnegie Mellon

Principles of Tissue Engineering

Cross-Sectional View of Muscle

Direct Physical Trauma◦ Laceration – cutting of tissue◦ Contusion - bruise◦ Strain – “pulling a muscle”

Biological (Inherited Abnormalities)◦ Muscular Dystrophies◦ Neurological◦ Storage Diseases

Types of Muscle Injury

Main male sex hormone Produced in the sex organs and adrenal cortex of the

adrenal gland Steroid hormone of the androgen group

◦ Steroid hormone – passes through cellular and nuclear and affects transcription

◦ Androgen – any hormone that stimulates/maintains male characteristics in vertebrates

Acts by binding to androgen receptor◦ Androgen receptor – transcription factor

Stimulates myoblast formation (cell differentiation) Particular testosterone molecule used: testosterone C-III

◦ Anabolic steroid Drug meant to mimic the effects of bodily-produced testosterone Increases skeletal muscle mass

Testosterone

Subclone of the mus musculus (mouse) myoblast cell line.

Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins.

Mouse stem cell line is used as a model in many tissue engineering experiments.

Useful model to study the differentiation of non-muscle cells (stem cells) to skeletal muscle cells.

Expresses muscle proteins and the androgen receptor (AR).◦ AR- DNA binding transcription factor which regulates gene

expression.

C2C12 Cells

The purpose of this study is observe the effects of the anabolic steroid testosterone C-III on the proliferation, differentiation, and survivorship of C2C12 stem cells.

Purpose

Alternative Hypothesis: The addition of testosterone C-III WILL affect the proliferation, differentiation, and survivorship of C2C12 stem cells.

Null Hypothesis: The addition of testosterone C-III WILL NOT affect the proliferation, differentiation, and survivorship of C2C12 stem cells.

Hypotheses

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])

Testosterone C-III 1 M stock solution

75 mL culture flask Incubator Nikon Inverted Microscope Aspirating Vacuum Line Laminar Flow Hood Laminar Flow Hood UV Sterilizing

Lamp Labeling Tape Hemacytometer Sterile PBS Ethanol (70% and 100%) Distilled water

Procedure (Stem Cell Line Culture) 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.

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 testosterone C-III was created using 1 mL of ethanol and 0.28842 grams of testosterone C-III.

The 10-6 M, 10-7 M, and 10-8 M concentrations were created from the stock, where 10-6 M is the suggested working concentration of the steroid.

The 3 experimental groups and the control group were created by adding:

20 µl of the 10-6 M solution to 5 flasks (Group A) 20 µl of the 10-7 M solution to 5 flasks (Group B) 20 µl of the 10-8 M solution to 5 flasks (Group C) 20 µl of ethanol to 5 flasks (Control)

The cells were incubated at 37°C, 5% CO2 for the remainder of the study.

Three flasks from each group were used in the Proliferation Experiment and two flasks from each group were used in the Differentiation Experiment.

Procedure (Addition of Variable on Day 0)

Day 1 and Day 6◦ Using the Nikon Inverted Microscope, images of two representative

areas of each flask were taken.◦ 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 hemacytometer for quantification (six counts per

flask).

Procedure (Proliferation Experiment)

Procedure (Differentiation Experiment)

Day 1 Using the Nikon Inverted Microscope, images of two 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.

Day 6 Using the Nikon Inverted Microscope, images of eight representative

areas of each of the flasks were taken.

Control 10-6 M 10-7 M 10-8 M0

50000

100000

150000

200000

250000

300000

100000 100000 100000 100000

196000

265000

218000205000

Proliferation Experiment (Avg. Cell Count)

Day 0Day 1

[Steroid]

Cell C

ou

nt

(Cells/fl

ask)

ANOVA◦ p-value: 1.78E-08◦ REJECT NULL◦ Significant variation

Dunnett’s Test◦ 0.05 t-critical value: 2.71

Proliferation Experiment (Data Analyses)

Group t-value Variation

10-6 M 9.82 Significant

10-7 M 3.11 Significant

10-8 M 1.19 Insignificant

Differentiation Experiment – Control

Day 1 Day 6

Differentiation Experiment – Group A

Day 1 Day 6

Differentiation Experiment – Group B

Day 1 Day 6

Differentiation Experiment – Group C

Day 1 Day 6

Differentiation Experiment - Comparison

ControlGroup A

Group CGroup B

Conclusion

Results Consistent with Prior Study

Results Consistent with Prior Study

Proliferation Experiment◦ From the ANOVA and the

subsequent Dunnett’s tests, the addition of testosterone C-III induced a statistically significant increase in proliferation in the C2C12 cells when it is added at its suggested working concentration, 10-

6 M, and one tenth this concentration, 10-7 M.

Differentiation Experiment◦ From the qualitative analysis

of the images gathered from the flasks, it appears that the addition of testosterone C-III induced myotubule formation. This was especially apparent in the 10-

6 M, while the 10-7 M and 10-8 M concentrations showed less dramatic differentiation in comparison.

Proliferation Experiment – some cell clumping occurred during passage, and counts were lower than expected

Differentiation Experiment◦ Evaluation of images – qualitative and imprecise◦ Solution – quantitative differentiation assay, e.g. MyoD

tagging, myosin/nuclear staining ratio, etc.

Evaluate concentrations of testosterone C-III greater than suggested dosage to determine if the steroid is hazardous in excess

Limitations/Extensions

Dr. Phil Campbell Conrad M. Zapanta, Ph.D. Biomedical

Engineering Laboratory, Carnegie Mellon University

Mark Krotec, PTEI 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

Sources and Acknowledgements