Ethanol Effects on Murine Muscle-Derived Stem Cells Experiment and Analysis by Donald Kline, Jr Pittsburgh Central Catholic High School

Brief History of Stem Cells Word stem cell first coined by Alexander Maksimov in stem cells first cultured successfully in vitro, a major step for future treatments of many disorders Led by Yamanaka, IPS or Induced Pluripotent Stem cells are discovered

Stem Cell Overview Basic Definition- cell that can produce lineages more specialized than themselves and can renew itself Spectrum of Stem Cell Behavior: ◦ totipotent embryonic cells ◦ pluripotent cells ◦ multipotent cells ◦ unipotent cells.

chondrocyte muscle cell osteoblast Adult Muscle- Derived Stem Cells (MDSCs) adipocyte hematopoetic/endothelial cells hepatocyte nerve cells/neurons/glial cells

Stem Cell Applications Duchene's Muscular Dystrophy, limb regeneration, cancer research, and many others Results of this experiment could be applied to the effect of alcohol on general muscle regeneration or on the embryos of pregnant mothers

Mice in Experiments Most Common Laboratory animal ◦ Share a relatively large amount of genes with humans ◦ mammals ◦ Genome completely sequenced ◦ Breed rapidly, easy to handle, small, inexpensive First inbred by Clarence Little in 1909, who promoted the use of mice in the laboratory

Muscle-Derived Stem Cells (MDSCs) Adult Stem Cells taken from muscle tissue Isolated using a variety of techniques to remove the non-stem cell populations Confluency needed for stem cells to differentiate in vitro ◦ Confluency allows the cells to fuse and begin forming multinucleated myotubes

Blood Vessels Nerves…

Cross-sectional View of Muscle Sarcomere

Mouse-Derived Stem Cells (Experimental Focus) Mouse cells used were Muscle Derived Stem Cells (strain pp6 ft 15 mouse) ◦ Pp6 is preplate six; the last step before ‘purified’ stem cell culture ◦ f in the ft15 stands for female, while the t 15, also called c, is the specific strain of mouse cells used

The Preplate Technique Muscle biopsy (mice) Pre-plating technique Muscle Derived Stem Cells Enzymatic digestion After 1 hour After 24 h (repeated 4 times) Qu-Petersen et al., Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. The Journal of Cell Biology, (5): p Pre-plate 6

Cell Differentiation Adult Stem Cells found in most animals are said to be pluripotent: able to differentiate into many cell types The Murine pluripotent stem cell differentiation into myotubes was analyzed ◦ Several ways to track this process  Desmin stain (myosin precursor and intermediate filament)  Myosin stain (essential to muscle motility, appears in myotubes near completion of differentiation)  Hoechst stain (nuclear stain)

Ethanol “Pure Alcohol” EtOH Fermentation reaction One of earliest reactions applied by humanity Depressant Health Issues- Fetal alcohol syndrome, alcohol addiction, impairment of nervous system and motor system

Purpose Explore effects of Ethanol on myoblastic stem cell differentiation

Hypothesis Null Hypothesis: Increasing EtOH concentration would not significantly affect: ◦ The Differentiation of the cell, measured by % differentiation ◦ The average number of nuclei in each fusion

Materials Centrifuge Macro Pipets Micro Pipets FTC 15 mMDSCs (pp6) 2% DMEM media Mouse Cell Culture Hood 37 degrees Celsius Incubator 70% Ethanol Solution (sterilization) Sterile Gloves (ethanol) Live Cell Imaging Robot Suction Filtrater Trypsin (remove cells from adhering to flask) Vortex (EtOH concentrating) Absolute Ethyl-Alcohol (EtOH) C Flask Hemocytometer Culture Flasks Conical Tubes Microscope Wells ScienceCell Media 10% DMEM media Hemocytometer

Materials (Special for Differentiation) 100% Methyl Alcohol 10% Goat Serum Mouse Anti-Human Myosin Heavy-Chain Stain (primary) Goat anti-mouse Fitz (Myosin secondary) Hoechst’s stain Monoclonal Anti-Desmin Pro Mouse (Desmin primary) Anti-Mouse IgG (Desmin secondary) Fluorescent Inverted View Microscope with Northern Eclipse Imaging Software

Procedure 1.pp6 mMDSCs were acquired 2.Expand mMDSCs through repeated culturing 1.Pass cells (aspiration, trypsinization, replating) 3.Transfer cell suspension into 15mL conical tube 4.Rinse Flask with 5mL of PBS twice 5.Add the rinse solution to the 15mL conical tube 6.Spin at 150g for 5 min. 7.Supernatant was removed by suction filtration 8.The cell pellet was resuspended in 1mL of DMEM media 9.Density of cell suspension was determined by use of a hemocytometer 10. Cells were re-plated at a density of 5,000 cells/well

Procedure (Differentiation) 1.The cells were grown until they were in sufficient quantity, and then were pipetted into wells at a density 5,000 cells/well. 2.The cells were allowed to continue growing until they began to differentiate. 3.The variable concentrations of Ethanol were then added. 4.After several days, the media was removed and the cells were fixed in 100% Methanol. 5.The following differentiation assays were performed: 1.After performing the methanol fix, the cells were rinsed three times with PBS. 2.The cells were blocked in 10% Goat Serum for one hour, only enough to cover surface of wells. 3.The Block was removed and the cells rinsed 3Xs w/ PBS. 4.For Myosin, the primary (mouse anti-human myosin stain) was added at a ratio of 1 uL primary to 300 uL PBS. It was allowed to sit for one hour. 5.The primary was removed and the cells were washed 3 times with PBS. 6.Myosin secondary was added (Goat anti-mouse Fitz) and allowed to sit for one hour at a ratio of 1:300uL. It was then covered in foil for an hour and every kept in the foil until analysis was completed. 7.Fifteen minutes before the end of the previous step, a Hoechst stain was added at a ratio of 1:500uL (Hoechst stain is the nuclear stain) 8.After sitting in foil for one day, the desmin stain was performed. This process has the same first three steps, so it began at step four 9.For Desmin, the primary (Monoclonal Anti-Desmin antibody produced in mouse) was used at a ratio of 1:300uL for one hour. 10.The primary was removed and the cells were rinse three times with PBS. 11.The secondary (Anti-Mouse IgG fragment of sheep antibody) was added at a ratio of 1:500uL and allowed to sit for one hour. 6.The cells were then analyzed under an inverted fluorescent microscope with Northern Eclipse Imaging Software. 7.Pictures of the fluorescent myotubes were taken and the data was analyzed.

Differentiation Procedure: Flowchart Confluence Add Variables Several days after Fix in 100% Methynol Rinse Block 1 hour Rinse Myosin OR Desmin Add Primary 1 Hour Rinse Add Secondary 1 Hour Hoechst Stain Cover in foil Analysis Using Inverted Microscope and Northern Eclipse Image Software

Examples of Fluorescent Myotubes 1% EtOH Desmin merged with nuclear stain 1% EtOH Nuclear Stain 1% EtOH Myosin Merged with nuclear stain

Desmin Visualization: Percent Differentiation

Desmin Visualization: Nuclei/Myotube P<.05

Dunnett’s Test Results: Desmin ANOVA0.5% EtoH 1% EtOH3% EtOH5% EtOH Percent Differentiation p>.05 p<.05 T values Nuclei/ Myotube p>.05 p<.05 T values T Critical Values: p<.05:3.26 p<.01:4.71

Conclusions: Desmin % Differentiation: Statistically significant at 5% Ethanol (p<.05) Nuclei/Myotube: Statistically significant at 5% Ethanol (p<.05) At the concentration of 5%, the Ethanol does appear to significantly reduce differentiation and average nuclei per myotube. The null hypothesis can be rejected at this concentration.

Myosin Visualization: Percent Differentiation P<.01 P<.05

Myosin Visualization: Nuclei/Myotube P<.05

Dunnett’s Test Results: Myosin ANOVA0.5% EtOH1% EtOH3% EtOH5% EtOH Percent Differentiation p>.05 p<.05p<.01 T Values Nuclei/ Myotube p>.05 p<.05 T Values T Critical Values: p<.05:3.26 p<.01:4.71

Conclusions: Myosin Percent Differentiation was significant at 3% (p<.05) and 5% (p<.01) ethanol concentrations Nuclei Per Myotube: statistically significant at 5% ethanol Concentration The Differentiation of the cells measured using myosin was significantly affected at 3% and above, while 5% significantly affected the amount of nuclei/myotube. The null hypothesis can be rejected at these levels.

Overall Conclusions Under tests of both desmin and myosin, the effects of Ethanol were significant at 5% for nuclei per myotube. With myosin, % differentiation became significant at 3% while for desmin it was at 5%. The quantity of 5% ethanol rejects the null hypothesis for all tests

Limitations/Extensions Cells can vary in viability Cells may not represent single defined population (mMDSC’s lineage not defined) Increase amount of replicates Test effects of other health hazards on stem cells (such as smoking, radiation, etc) Use a fluorometer to quantify data for greater accuracy

Sources/Acknowledgements NIH Science Cummins, James H. An Overview of Current Scientific Research in Muscle Regeneration. Children’s Hospital of Pittsburgh. Special thanks to Dr. Deasy for assisting me with the project and use of the Deasy Lab Special thanks to Pitt undergraduate Jordan Nance for teaching me the majority of procedures Special thanks to John Huard and Dr. Garibeh for use of the Huard lab