1. Experiment and Analysis by Henry Walther Pittsburgh Central Catholic High School Growth Factor Effects on MDSC Populations

2. Brief History of Stem Cells Word stem cell first coined by Alexander Maksimov in 1908 1992- stem cells first cultured successfully in vitro, a major step for future treatments of many disorders. 2006- Led by Yamanaka, IPS or Induced Pluripotent Stem cells are discovered

3. 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.

4. 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

5. Growth Factors Hepatocyte growth factor (HGF) is a paracrine cellular growth, motility and morphogenic factor. Vascular endothelial growth factor (VEGF) is a chemical signal produced by cells that stimulates the growth of new blood vessels. Stem Cell Factor (SCF) a cytokine plays an important role in hematopoiesis (formation of blood cells), spermatogenesis, and melanogenesis. Epidermal growth factor (EGF) plays an important role in the regulation of cell growth, proliferation, and differentiation by binding to its receptor EGFR. Fibroblast growth factors (FGF) are a family of growth factors involved in angiogenesis, wound healing, and embryonic development.

6. Propose To see the effects of different growth factors on MDSCs

7. Hypothesis Null Hypothesis: The effects of the different growth factors on MDSC growth will not be significant outside of chance

8. Materials T-75 Flasks MDSC Cells Cell incubator.25% trypsin PBS Colormetric solution Plate reader 96 well cell culture plate Hemocytometer HGF VEGF SCF EGF FGF Human cell culture hood Pippets Dulbecco’s modified eagle’s medium (DMEM) Fetal bovine serum (FBS) Horse serum (HS) Chicken embryo exact (CEE) Penicillin-streptomycin (PS) Hank’s balance salt solution (HBBSS) Centrifuge

9. Procedure 1. Proliferation media was made by adding 400ml DMEM, 50ml FBS, 50ml HS, 5ml PS. And 2.5ml CEE. 2. The proliferation media was filtered through a.22 um cellulose acetate 3. Cells were rinsed twice with PBS (basic saline solution ) 4..25% trypsin was added to remove cells 5. Cells where incubated at 37c three to five minutes 6. Proliferation media was added to stop trypsin 7. Cells and media were put into a 15ml conical tube and spun down at 2000rpm 5mins 8. Supernatant was removed and cells were resuspended in 10ml of proliferation media and put into 3 t- 75 flasks and left over night at 37c 9. Cells were counted using a hemocytometer 10. Cells were put into 21 conical tubes at 5000 cells per tube 11. Media was removed and cells centrifuged 12. A series of serial dilutions were performed for each growth factor 13. Cells were resuspended in 1ml of corresponding diluted growth factor 14. Cells were resuspended in 1ml of proliferation media to serve as control 15. Then 600ul was removed from each the 21 conical tubes and put into three wells of a 96 well cell culture plate 16. Cells were incubated for 48 hours at 37c 17. Media was removed and 100ul of fresh DMEM was added 18. 20ul of colormetric solution was added to each well 19. Incubated for 2 hours at 37c 20. Plate was read at 490nm by a plate reader

10. Dose 1 (ng/ml) Dose 2 (ng/ml) Dose 3 (ng/ml) Dose 4 (ng/ml) SCF0.66200/8003.3400/80010320/1280502/2000 FGF0.16200/8000.3 100/150 05320/1280252/2000 VEGF0.25200/8000.5300/9002320/1280102/2000 EGF0.2200/8000.448/115210320/1280502/2000 HGF1.75200/8003600/6006320/1280102/2000 final volume approximately 1ml initially put in 2ul

12. Conclusion With the p-value being greater than the.05 confidence cut off, the null hypothesis can be rejected. Thus none of the growth factors significantly affected MDSC populations

13. Extensions/Improvements As in the human body, a cocktail of various growth factors would be used Different growth factors would be used More replicates would be made

14. Reference Burhan Gharaibeh, Ph.D., Research Assistant Professor, University of Pittsburgh Mark Krotec, PTEI Arthroscopy. 2010 Feb;26(2):269-278., “The Use of Platelet-Rich Plasma in Arthroscopy and Sports Medicine: Optimizing the Healing Environment.” Lopez-Vidriero E, Goulding KA, Simon DA, Sanchez M, Johnson DH. Sports Med. 2009;39(5):345-54. doi: 10.2165/00007256-200939050-00002. “Platelet-rich therapies in the treatment of orthopaedic sport injuries.” Sánchez M, Anitua E, Orive G, Mujika I, Andia I. J Bone Joint Surg Br. 2009 Aug;91(8):987-96. “The biology of platelet-rich plasma and its application in trauma and orthopedic surgery: a review of the literature.” Alsousou J, Thompson M, Hulley P, Noble A, Willett K.