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Anabolic Steroid Influence on a Mammal Stem Cell Line

This study explores the effects of the anabolic steroid testosterone C-III on the proliferation, differentiation, and survivorship of C2C12 stem cells. The goal is to understand the potential impact of this steroid on tissue engineering and muscle tissue supplementation.

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Anabolic Steroid Influence on a Mammal Stem Cell Line

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  1. Anabolic Steroid Influence on a Mammal Stem Cell Line Frank Wolf Pittsburgh Central Catholic High School Grade 11 PJAS 2010

  2. 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 • 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 supplementing muscle tissue.

  3. Principles of Tissue Engineering Cells ECM Defect Regeneration Hormones Blood Supply PhilCampbell, Carnegie Mellon

  4. Cross-Sectional View of Muscle

  5. Types of Muscle Injury • Direct Physical Trauma • Laceration – cutting/tearing of tissue • Contusion - bruise • Strain – “pulling a muscle” • Biological (Inherited Abnormalities) • Muscular Dystrophies (DMD, BMD) • Neurological Disorders (multiple sclerosis, Cerebral palsy) • Storage Diseases (mucopolysaccharidoses, lipidoses)

  6. Testosterone • Male sex hormone produced in the sex organs and adrenal cortex of the adrenal gland • Androgen • Any hormone that stimulates/maintains male characteristics in vertebrates • Steroid hormone • Passes through cellular and nuclear membranes and affects transcription • 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

  7. C2C12 Cells • 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. • Expresses the androgen receptor (AR). • AR- DNA binding transcription factor which regulates gene expression.

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

  9. Hypotheses • 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.

  10. 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 (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 Materials

  11. Procedure (Stem Cell Line Culture) • A 1 mL aliquot ofC2C12 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.

  12. Procedure (Addition of Variable on 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 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 • 20 µl of the 10-7 M solution to 5 flasks • 20 µl of the 10-8 M solution to 5 flasks • 20 µl of ethanol to 5 flasks (Control) • The cells were incubated at 37°C, 5% CO2for 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.

  13. 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 (six counts per flask). • Day 1 and Day 6 • 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.

  14. p-value: 1.78E-08

  15. Proliferation Experiment (Data Analyses) • ANOVA • Compares variation within groups to variation between groups • p-value: 1.78E-08 • REJECT NULL • Significant variation • Dunnett’s Test • Compares each experimental group to control individually • 0.05 t-critical value: 2.71

  16. Differentiation Experiment – Control Day 1 Day 6

  17. Differentiation Experiment – 10-8 M Day 1 Day 6

  18. Differentiation Experiment – 10-7 M Day 1 Day 6

  19. Differentiation Experiment – 10-6 M Day 1 Day 6

  20. Differentiation Experiment - Comparison Control 10-6 M Most significant differentiation (qualitative – most dramatic myotubule formation) Relatively comparable degrees of differentiation (qualitative) 10-7 M 10-8 M

  21. Conclusion • 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. Results Consistent with Prior Study Results Consistent with Prior Study

  22. Limitations/Extensions • Differentiation Experiment • Evaluation of images – qualitative and imprecise • Solution – quantitative differentiation assay, e.g. MyoD tagging • CyQUANT™ Cell Proliferation Assay • More quantitative than counting cells on a Hemocytometer • Fluorescent dye binds to nucleic acid in the cell • Evaluate concentrations of testosterone C-III greater than suggested dosage to determine if the steroid is hazardous in excess

  23. Sources and Acknowledgements • 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 • Chen Y, Zajac JD Maclean HE 2005 Androgen regulation of satellite cell function. Journal of Endocrinology 186 21-31. • Sinha-Hikim I, Taylor WE, Gonzalez-Cadavid NF, Zheng W and Bhasin S 2004 Androgen receptor in human skeletal muscle and cultured muscle satellite cells: up-regulation by androgen treatment. Journal of Clinical Endocrinology and Metabolism 89 5245-5255.

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