Substrate Elasticity Affects Bovine Satellite Cell Behavior In Vitro


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Substrate Elasticity Affects Bovine Satellite Cell Behavior In Vitro A Possible Model to Control Quiescence
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Lapin,Marni R
University of Florida
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Gainesville, Fla.
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Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Animal Sciences
Committee Chair:
Johnson, Sally
Committee Members:
Dahl, Geoffrey E.
Wohlgemuth, Stephanie


Subjects / Keywords:
aging -- cell -- elasticity -- extracellular -- matrix -- modulus -- muscle -- photocrosslinker -- polyacrylamide -- quiescence -- satellite -- stem -- youngs
Animal Sciences -- Dissertations, Academic -- UF
Animal Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
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Satellite cells, also called muscle stem cells, are responsible for muscle hypertrophy and repair in animals postnatally. Upon activation by various stimuli, they can proliferate, differentiate, and fuse with existing myofibers, contributing cytoplasm and myonuclei. Satellite cell regenerative capacity is compromised with age as aged muscle tissue becomes weak and loses mass. Age differences in satellite cells have been studied in vitro and in vivo in previous literature to better understand the decreased function in cell activity. The elasticity of muscle increases with age and usage, and is an important factor that affects satellite cell behavior in vitro. Semimembranosus (SM) and longissimus dorsi (LD) muscles from young and adult cattle showed differing elasticities with age and muscle type, and these values were mimicked using polyacrylamide gels. Satellite cells isolated from young bovine muscle and cultured on gels of varying elasticities show a 24 hour delayed expression of proliferating cell nuclear antigen (PCNA), an indicator of activation. Cell populations that attach to muscle-like substrates also exhibit approximately 50-70% Pax7/Myf5 co-expression, identified as muscle progenitor cells that can proliferate or return to quiescence. Addition of HGF increases the number of PCNA expressing cells on gels, emphasizing cell ability to become activated on soft substrates with appropriate stimulus. Cells cultured on gels for 6 days exhibit delayed proliferation followed by a decrease in cell number. Results suggest that softer, tissue-like substrates have a significant effect on bovine satellite cell behavior in culture and may maintain satellite cell quiescence through recreation of the microenvironment experienced in vivo.
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by Marni R Lapin.
Thesis (M.S.)--University of Florida, 2011.
Adviser: Johnson, Sally.

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2 2011 Marni Rose Lapin


3 To my family, friends, and Joey, t hank you for your love and support, celebrating the good and getting me through the bad.


4 ACKNOWL EDGMENTS and bringing me into the lab. Thank you for having faith in me and trusting me to experiment with challenging tasks that had not been done in the lab before. I learned some very valuable lessons working with you that have made me grow tremendously as a professional and as a person Through your encouragement and positive attitude you have helped me to improve my independence as a researcher and intellectual. T he skills that I have obtained working in your lab are so valuable and have prepared me well for a professional career in the sciences. You have constantly challenged me to work towards perfection, encouraging me to always continue to improve. You have bee n an excellent mentor as well, guiding me in the right direction and always having my best interest at heart. Our long conversations about life, politics, and the Midwest made me feel right at home and I have been so lucky to have worked under someone that has treated me as a peer as well as a colleague. Through your positive influence I will Thank you for your invaluable lessons and providing me with such an extraordinary e xperience. Thank you to my supervisory committee members, Dr. Dahl and Dr. Wohlgemuth, for taking the time to help me along with this process. Thank you for offering help when needed and posing questions that I had not considered. Thank you for taking the time and effort to look painstaking ly through my thesis and help me to finish a successful project. Thank you to my lab mates, Ju Li, Christy, and John, for being so supportive and helpful in everything I have done. Ju Li, thank you for teaching me just ab out every lab technique I have used. Your patience with me was outstanding and I really appreciated all the time you spent answering questions and teaching me lab techniques when you were so busy with your own work. Christy, thank you for being so wonderfu lly supportive and helpful. You joined the lab at


5 the busiest time during my project and were so patient and understanding w hen I was stressed. to the lab and have survived without you in the lab. Although you were quite the bully, you always had my back when I needed you. Both the lab and I were blessed when you decided to retur n. Thank you for h elping me through countless hurd les and always being available whenever I needed help for anything. Whether it was something you knew a lot or very little about, you were always there to try it out with me and lead me to success. You are irreplaceable and I am not only lucky to have had your help but to have had you as my friend. You brightened my days and made me look forward to going into work. You made the lab atmosphere more comfortable and fun to be in. You made me come out of my shel l and I could finally act like myself once you came along. I am forever grateful for having you as my partner in crime. Thank you for making my experience so enjoyable and successful! To my Florida friends thank you for being so wonderful and welcoming as I moved to Florida. I am so lucky to have had such a wonderful group of friends who were so supportive and wonderful colleagues, classmates, and friends. Thank you for listening to me vent, making me laugh, and introducing me to the greatest parts of Gain esville. I am forever grateful for you welcoming me into your lives Sara and John, I am SO lucky that you asked me to watch the dogs while you were in Spain because it sparked a truly special friendship that I will always cherish. Thank you for welcoming me into your lives and home and for always being supportive of everything that I did. I love you two very much and thank you for being like family to me John, thank you for being such a fun and wonderful friend and always initiating adventurous activities Sara, I am so lucky to have met John because he brought me to you. I have loved


6 sharing so many memorable moments with you and am so grateful for your wonderful friendship. Y ou are a truly wonderful person, I am so lucky to have you in my life. Thank you for being there to support me in all my endeavors and to give me advice along the way. I am truly inspired by you and lucky to have you as a role model and friend. To Mom, D ad, Ashley, and Joel thank you for being such wonderful and supportive family mem bers with anything I do. I am so lucky to have such a wonderful and caring family. Thank you for always believing in me and getting me through difficult times. Thank you for taking the time to visit me and to always get me over my waves of homesickness. Th ank you for getting me to this point, supporting me in all of my decisions, and constantly making yourselves available anytime I needed you. successful in everything I do. I love you and would not have succeeded without any of you. And finally, to Joey. Thank you for moving all the way to Florida with me and to have stayed through the good and the bad. Thank you for your patience with my unpredictable schedule and accompanying me with everything I had to do Thank you for always celebrating my successes and providing an ear to listen or a shoulder to cry on. Your constant presence and support was exactly what I needed and I am more than grateful to have you in my life. Thank you for all of the unfor gettable adventures and for encouraging me to try new things. Thank you for being the best roommate, boyfriend, and friend. You made the fun times more enjoyable and the rough times survivable. I love you.


7 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF FIGURES ................................ ................................ ................................ ......................... 9 ABSTRACT ................................ ................................ ................................ ................................ ... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 12 2 MATERIALS AND METHODS ................................ ................................ ........................... 14 Bovine Satellite Cell Isolation ................................ ................................ ................................ 14 Cell Culture ................................ ................................ ................................ ............................. 14 Preparation of Matrix Protein and Photocrosslinker Attachment ................................ ........... 15 Polyacrylamide Substrate Prepa ration ................................ ................................ .................... 16 ............................ 16 Immunocytochemistry ................................ ................................ ................................ ............ 17 Statistical Analysis ................................ ................................ ................................ .................. 19 3 LITERATURE REVIEW ................................ ................................ ................................ ....... 20 Defining the Satellite Cell ................................ ................................ ................................ ...... 20 Pax7 ................................ ................................ ................................ ................................ 21 Satellite Cell Quiescence ................................ ................................ ................................ ........ 22 Satellite Cell Activation ................................ ................................ ................................ .......... 23 HGF and Activation ................................ ................................ ................................ ........ 25 Nitric Oxide and Activation ................................ ................................ ............................ 30 Age and Activation ................................ ................................ ................................ .......... 31 Satellite Cell Self Renewal: Asymmetric Division ................................ ................................ 34 Satellite Cells as Stem Cells ................................ ................................ ................................ ... 38 Attachment Matrix Elasticity Affects Cell Behavior ................................ .............................. 40 4 RESULTS ................................ ................................ ................................ ............................... 45 Muscle Elasticity Differs with Age an d Can Be Mimicked in Polyacrylamide Gels ............. 45 Cell Attachment on Polyacrylamide Gels Requires Covalently Linked ECM Proteins ......... 46 Sa tellite Cell Proliferation is a Function of Age ................................ ................................ ..... 47 Satellite Cell Activation Varies with Substrate Stiffness ................................ ....................... 48 Summary ................................ ................................ ................................ ................................ 50 5 DISCUSSION ................................ ................................ ................................ ......................... 64 Muscle Elasticity Varies with Age and Muscle Type ................................ ............................ 64


8 Proliferation Is Delayed in Adult Satellite Cells in vitro ................................ ........................ 68 Satellite Cells Respond to Elasticity in vitro ................................ ................................ .......... 69 Conclusions ................................ ................................ ................................ ............................. 72 AP P ENDIX : PROTOCOLS USED ................................ ................................ ............................... 73 Bovine Satellite Cell Isolation ................................ ................................ ................................ 73 EC L Photocrosslinking on Polyacrylamide Gels ................................ ................................ ... 74 Immunofluorescence Staining ................................ ................................ ................................ 77 Elasticity Measurements Using the Instron for Gels and Tissue ................................ ............ 78 LIST OF REFERENCES ................................ ................................ ................................ ............... 83 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ......... 94


9 LIST OF FIGURES Figure page 4 1 Elastic modulus, E measurements for isolated semimembranosus (SM) and longissimus dorsi (LD) muscle tissues from young and adult cattle ................................ 52 4 2 Graph of standardized elastic modulus, E measurements for varying concentrations of acrylamide. ................................ ................................ ................................ .................... 53 4 3 Representative images for C2C12 cells grown on various substrates at 24 hours (left column) and 48 hours (right column). ................................ ................................ ............... 54 4 4 Representative images of C2C12 cells (A,B) and bovine satellite cells (C,D) cultured on acrylamide gels with photocrosslinked ECL ................................ ............................... 55 4 5 Effect of ECL and ECL crosslinker on C2C12 and BSC attachment and proliferation .... 56 4 6 Developing satellite cells exhib it a lag phase in proliferation compared with young cells. ................................ ................................ ................................ ................................ ... 57 4 7 Comparison of EdU incorporation and PCNA expression in C2C12 myoblasts (A) and young (<5d) BSC (B). ................................ ................................ ................................ 58 4 8 Representative graphs (n = 1) of bovine satellite cells (<5d) grown on acrylamide gels of varying elastic modulii over a six day period ................................ ........................ 59 4 9 Pax7 and Myf5 expression at 24 hours post plating in bovine satellite cells (<5d) grown on soft substrates ................................ ................................ ................................ ..... 60 4 10 Cells on soft substrates exhibit three groups of myogenic expression, Pax7 only c ells, Myf5 only cells, and Pax7 / Myf5 cells. ................................ ................................ ...... 61 4 11 Percent of bovine satellite cells (<5d) positive for proliferating cell nuclear antigen (PCNA) after immunostaining ................................ ................................ ........................... 63


10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science SUBSTRATE ELASTICITY AFFECTS BOVINE SATELLITE CELL BEHAVIOR IN VIT RO : A POSSIBLE MODEL TO CONTROL QUIESCENCE By Marni Lapin August 2011 Chair: Sally E. Johnson Major: Animal Sciences Satellite cells, also called muscle stem cells, are responsible for muscle hypertrophy and repair in animals postnatally. Upon activatio n by various stimuli, they can proliferate, differentiate, and fuse with existing myofibers contributing cytoplasm and myonuclei Satellite cell regenerative capacity is compromised with age as aged muscle tissue becomes weak and loses mass. Age differenc es in satellite cells have been studied in vitro and in vivo in previous literature to better understand the decreased function in cell activity. The elasticity of muscle increases with age and usage, and is an important factor that affects satellite cell behavior in vitro. Semimembranosus (SM) and longissimus dorsi (LD) muscles from young and adult cattle showed differing elasticities with age and muscle type, and these values were mimicked using polyacrylamide gels. Satellite cells isolated from young bov ine muscle and cultured on gels of varying elasticities show a 24 hour delayed expression of proliferating cell nuclear antigen (PCNA), an indicator of activation. Cell populations that attach to muscle like substrates also exhibit approximately 50 70% Pax 7 / Myf5 co expression, identified as muscle progenitor cells that can proliferate or return to quiescence. Addition of HGF increases the number of PCNA expressing cells on gels, emphasizing cell ability to become activated on soft substrates with appropriat e stimulus. Cells cultu red on gels for 6 days exhibit delayed proliferation followed by


11 a decrease in cell number. Results suggest that softer, tissue like substrates have a significant effect on bovine satellite cell behavior in culture and may maintain s atellite cell quiescence through recreation of the microenvironment experienced in vivo.


12 CHAPTER 1 INTRODUCTION Satellite cells are muscle stem cells that are responsible for muscle hypertrophy postnatally. In adult muscle they reside in a quiescent stat e between the sarcolemma and the basal lamina of the myofiber, making up a small percentage of total nuclei in the muscle. They become activated in adult muscle via environmental and intrinsic cues that cause them to proliferate, diff erentiate, and eventua lly fuse with the myofiber. Satellite cells are considered adult muscle stem cells because of their ability to self renew and replenish the quiescent population of cells. The y contribute to the myonuclei pool upon differentiation, maintaining the amount of cytoplasmic material that each nuclei controls while increasing the overall myofibrillar area. Studying satellite cells i n vitr o is not indicative of in vivo behavior in the animal, as isolated cells become spontaneously activated within 24 hours post pla ting. Past studies have demonstrated that the elasticity of the substrate to which a cell attaches can affect cell behavior and cells with anchorage attachments such as epithelial cells and mesenchymal stem cells, can sense the elasticity of the material through projected forces (Engler et al., 2004 a,b, 2006; Pelham and Wang, 1997; Peyton and Putnam, 2005). S atellite cells grown on softer substrates that mimic muscle elasticity exhibit improved myotube development and regenerative capacity both in culture and when transplanted to injured muscle (Engler et al., 2004b; Gilbert et al., 2010). Satellite cells exhibit different behavior in aging animals as muscle weakens and loses mass. It is not fully understood why the regenerative ability of a satellite cell is compromised with age, but various changes in the satellite cell environment could be the cause. One such


13 change is increased tensile strength in muscle with age which is a direct result of increased muscle development and connective tissue composition The objectives of the current study were to (1) determine muscle elasticity changes with muscle type and age and (2) define how satellite cells respond to differing elasticities in vitro by culturing them on pliable substrates that mimic elasticity of m uscle from different age groups.


14 CHAPTER 2 MATERIALS AND METHOD S Bovine Satellite Cell Isolation The semimembranosus (SM) was excised from neonates (<5 days; n=4) and developing (2 3 months, n=2) Holstein bull calves and subsequently trimmed of connecti ve tissue and fat and ground in a conventional meat grinder. Tissue was digested with protease (Sigma Aldrich, St. Louis, MO) at 1.25mg/g tissue in phosphate buffer ed saline solution (PBS) at a 1:1 (w/v) ratio for 45 minutes at 37C (physiological temperat ure) with gentle swirling at 10 minute intervals. Protease was washed from the tissue by addition of PBS and centrifugation at 1500 X g for 5 minutes and supernatant discarded, repeated for a total of three consecutive washes. The tissue was then re suspe nded in PBS, shaken vigorously, and centrifuged at 500 X g for 10 minutes. The supernatant containing satellite cells was collected and the process was repeated for a total of four times to extract maximal satellite cell number from the tissue The collect ed supernatant was centrifuged at 1500 X g for 10 minutes and the remaining pellet containing the satellite cells was re suspended in PBS and consecutively filtered through 70m and 40m cell strainers (Fisher Scientific, Pittsburgh, PA). Filtered c ells in PBS were centrifuged at 1500 X g for 5 minutes and the resulting pellet was re suspended in freezing media containing low glucose MEM; Invitrogen, Carlsbad, CA) supplemented with 10% v/v horse serum (Invitrogen), 1% v/v penicillin/streptomycin (Invitrogen), 0.1% v/v gentamicin (Invitrogen), and 10% v/v dimethyl sulfoxide (DMSO; Sigma Aldrich). Cells were stored in liquid nitrogen vapor until needed. Cell Culture 1) Cell line: Stock C2C12 myoblasts were passaged at 70% co nfluency on 10cm polystyrene p lates pre coa ted with 0.1% w/v gelatin. C2C12 myoblast s were cultured in high


15 glucose D MEM supplemented with 10% v/v fetal bovine serum, 1% v/v penicillin/st reptomycin, and 0.1% v/v gentami cin reag ent solution. 2) Primary bov ine satellite cells (BSC): Media for BSC in cluded low glucose D MEM supplemented with 10% v/v horse serum, 1% v/v penicillin/st reptomycin, and 0.1% v/v gentami cin reagent solut ion on entactin collagen type IV laminin (ECL; Millipore, Billerica, MA) coated plates Treatment media containing recombinant human hepatocyte growth factor (rh HGF ) was used for satellite cell activation and consisted of low glucose D MEM supplemented with 2% v/v horse serum, 1% v/v penicilli n/streptomycin, 0.1% v/v gentami cin, and 1 0 ng/mL (w/v) of rhHGF ( R&D Systems, Minneapolis, MN ) Both cell types were cultured at a density of 1,000 cells per cm 2 All cells were cultured at 37C in 5% CO 2 Consecutive trials were defined as individual cell cultures representing each trial. All ex periments using cells were repeated for at least three times trials and all experiments involving isolated BSC used a new animal for each repeated trial. Preparation of Matrix Protein and Photocrosslinker Attachment Entactin collagen type IV laminin (ECL) was used as the in vitro protein matrix for cell attachment. Phenol R ed was removed from the ECL concentrate through a buffer exchange with PBS ( Zeba TM Spin Desalting Columns Thermo Fisher Scientific, Waltham, MA) The ECL was mixed with 10 mM of Sulfo NHS Diazarine Photocrosslinker (Sulfo SDA ; Thermo Scientific ) at a 1:1 v/v ratio to allow for optimal protein:crosslinker interactions and allowed to react for 30 minutes. The reaction was stopped by adding sterile 1 HCL, pH 8.0 ( Fisher Scientific ) quenching buffer to a final buffer concentration of 100 mM. The mixture was allowe d to sit for 5 minutes before the T ris buffer was removed ( Zeba TM Spin Desalting Column ) The ECL protein concentration was measured ( Micro BCA Protein Assay Kit ; Thermo Sci entific) and subsequently diluted to 20 g/mL with sterile PBS. The ECL Sulfo SDA compound mixture was


16 immediately photocrosslinked on to polyacrylamide gels or u sed to coat 48 well plates or glass bottom plates. Polyacrylamide Substrate Preparation Polyacr ylamide gels were made as d escribed in Chadhuri et al., 2010 and Peyton and Putnam, 2005 Acrylamide and N methylene bis acrylamide (Bio Rad, Hercules, CA) solutions were made to their desired concentrations in water and 1% v/v HEPES (Fi sher Scientific) To the acrylamide solution, 1% of a 10% ammonium persulfate solution (APS, 1/100 v/v; Bio Rad) and 0.1% of TEMED (1/1,000 v/v; Bio Rad) were quickly added and thoroughly mixed to start the polyacrylamide polymerization process The solution was poured be tween glass plates separated by 0.75 mm spac ers to ensure uniform thickness and was left to p olymerize for two hours. Gel discs were manually punched from the polyacrylamide sheets in a sterile environment using an 8 mm diameter steel punch and placed insi de a sterile multi well plate cover. Gel discs were coated with the covalently attached ECL Sulfo SDA mixture at a 20ug/mL concentration (w/v) of ECL in sterile PBS (pH 7.4). ECL was covalently attached to the gels via the photocrosslinking reactivity of t he Sulfo SDA Photocrosslinker with UV light irradiation at 365 nm ( UVP, Upland, CA ). The UV light source was placed on top of the multi well plate cover, and g els were exposed to UV light for 15 minutes, with a 1 cm distance between the gels and the light source Gels were subsequently pla ced in 48 well plates (Corning Lowell, MA ) and rehydrated with PBS until needed. ssue were quantified with an Instron 3343 ( Instron, Norwood, MA ). Gels were polymerized for 2 hours as sheets between glass plates and were then cut into s as descr ibed in Peyton and Putnam, 2005, with dimensions as follows (length x height ): two 2x5cm rectangles connected by


17 a 4x1cm rectangle, all with 0.75mm thickness. Gels were hydrated in PBS until needed, and were attached to the Instron load cell by clamping both 2x5cm rectangles and leaving the 4x1cm rectangle unclamped. Semimembr anos us (SM) and longissimus dorsi (LD) muscles were removed from the bone in young (<2 week) Holstein bull calves (n=3) and adult (estimated age 24 to 36 months) Holstein females (n=4) at slaughter. Muscle tissue samples were kept in warm D MEM post harvesting and subsequently cut into strips approximately 10 cm for width and thickness and 20 cm in length. Muscle samples were attached to the Instron load cell and the working length (unclamped region) was noted. Samples were subjected to stretching at a rate of 5 mm/min until the specimen broke or showed a significant decrease in force output. Data output calculated from the data output as follows: city, E = stress/strain = F* L 0 A 0 where F is force (N), L 0 is the origi nal length of the specimen (mm), ange in distance stretched (mm), and A 0 is the original cross sectional area (mm 2 ) Units were given in N/mm 2 which can be converted t o kPa with 1 kPa = 1 N/m 2 and t he higher the elasticity value, the more stiff/rigid the tissue. Immunocytochemistry Cells were immunostained for proliferating cell nuclear antigen (PCNA) expression, a marker for satellite cell activation, or co stained fo r Pax7 expression, a satellite cell marker, and Myf5 expression, an early myogenic marker. C ells on either gel substrates or glass/ polystyrene plates were washed with PBS to remove media and fixed with either 4% v/v paraformaldehyde in PBS for 10 minutes a t room temperature (for Pax7 and Myf5 ) or 90% v/v methanol in PBS for 10 minutes at 4C (for PCNA). Cells were subsequently washed with PBS. Cell membranes were permeabilized and non specific antigen binding sites were blocked by incubation with blocking s olution ( 10% v/v horse serum and 0.1% v/v TritonX 100 in PBS ) for 60 minutes at room


18 temperature. Primary antibodies used wer Pax7 mouse monoclonal IgG 2a cultured supernatant Myf5 rabbit polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:100, and PCNA (c 20) goat polyclonal IgG (Santa Cruz Biotechnolo gy) diluted 1:100. Primary antibodies, either individually (PCNA) or in combination ( Pax7 and Myf5 ) were diluted in 10 % v/v blocking solution in PBS Cells were incubated in primary antibody solution overnight at 4C. Primary antibody was then removed and cells were washed for 5 minutes three times with PBS. Secondary antibodies used were goat anti mouse Alexa Fluor 488, donkey anti goat Alexa Fluor 488 and donkey anti rabbit Alexa Fluor 568 ( Invitrogen ) diluted 1:150 in 10% v/v blocking s olution alo ng with Hoechst dye ( Invitrogen) at 10 g/ml Secondary antibody mixtures were added to cells for 60 minutes. Proliferation was measured using a Click iT EdU Alexa Fluor 594 Imaging Kit ( I nvitrogen ) This kit could not be used on polyacrylamide gels beca use of interference with imaging dyes, and therefore was only used on polystyrene or glass plates. 5 ethynyl deoxyuridine (EdU) is a nucleoside analogue to thymidine and is incorporated into DNA during DNA replication and was used as an indicator for ce ll proliferation. The EdU (10 mM final) was added to live cells and incubated at 37C in 5% CO 2 for 2 hours. Cells were washed with PBS to remove media and fixed with 4% v/v paraformaldehyde for 15 minutes at room temperature. Cells were s ubsequently washe d with 3% w/v b ovine s erum a lbumin ( BSA; Fisher Scientific ) in PBS twice and permeabilized with 0.5% v/v TritonX 100 in PBS for 20 minutes at room temperature. Cells were again washed twice with 3% BSA wash solution and a reaction cocktail, made according to the imaging kit manual, was added to the cells and incubated for 30 minutes at room temperature. Cells were washed once with the wash solution and once with PBS, and


19 Hoechst dye diluted at 1:1000 in PBS was added to cells and incubated for 30 minutes at room temperature. Fluorescence signaling was viewed with an Eclipse TE 2000 U stage microscope (Nikon, Lewisville, TX) equipped with an X Cite 120 epifluorescence illumination system (EXFO, Mississauga, Ontario, Canada). Images were captured with a Photom etrics CoolSnap EF digital camera and analyzed with the NIS Elements software (Nikon). Statistical Analysis All data were analyzed to measure mixed effect models using the PROC MIXED statement in SAS and a one way or two way ANOVA (SAS Institute, Inc., Car y, NC 2008) Comparisons of the least square means were assessed using the LSMEANS statement and differences were determined by the PDIFF option with P < 0.05 considered significant.


20 CHAPTER 3 LITERATURE REVIEW Defining the Satellite Cell Skeletal muscl e function is specialized to produce movement. Muscles are composed of muscle fibers grouped into fascicles by a connective tissue called the perimysium The muscle fiber, also considered the muscle cell, is a multinucleated cell containing an array of fil aments involved in contractile movement of the cell. The diameter of a muscle fiber, determined by the number of filaments, determines the strength of the fiber. The cytoplasmic volume is maintained by the nuclei of the muscle fiber. E ach nucleus controls protein synthesis within the cell. Myogenesis is the formation of m uscle tissue. It occurs prenatally via muscle hyperplasia, which is an increase in the number of muscle fibers. Following birth, muscle tissue grows through protein accretion within existin g fibers, or muscle hypertrophy. At this point, skeletal muscle growth occurs in two stages: a rapid, intense growth phase that occurs during perinatal and postnatal life and a second phase of muscle maintenance and repair during adult life (Knapp et al., 2006). The result of muscle hypertrophy and repair facilitated by muscle stem cells, called satellite cells. Postnatal muscle growth and repair is dependent upon the actions of satellite cells, a population of adult stem cells interspersed within the muscl e fibers. Satellite cells were named They make up 2 7% of the adult muscle fiber, but this number can vary with age and muscle type (Snow, 2007; Tatsumi, 2010). Satellite cells were first identified with electron microscopy by Alex ander Mauro in 1 961 as mononucleated cells that run along the periphery of the frog myofiber. They reside in indentations between the sarcolemma and the basal lamina of the muscle fiber (Muir et al., 1965). Featur es that identify satellite cell populations include a higher nuclear to cytoplasmic


21 ratio with few organelles, small nuclear size compared to adjacent myofibrillar nuclei, and an increase in the amount of nuclear heteroch romatin compared with myonuclei (re viewed in Schultz, 1994). More recently, ma r kers specific for satellite cells, such as Pax7 have been used to identify the unique myogenic population. Pax7 Pax7 the paired box transcription factor, is one of the most prominent markers for satellite cells It is expressed in both quiescent and activated satellite cells. Using cell culture and electron microscopy, Seale et al., (2000) identified Pax7 as a crucial factor for satellite cell specification. Muscle fibers isolated from Pax7 null mice had a compl ete absence of satellite cells but did not have any defects in patterning or structure of the already differentiated fiber. Pax7 is co expressed with its homologue Pax3 during embryonic skeletal myogenesis, after which Pax7 becomes the sole transcription f actor expressed by most, but not all, satellite cells. Pax3 activates MyoD and can be replaced by Pax7 for this function (Relaix et al., 2004). Pax3 is co expressed with Pax7 in some adult satellite cells but cannot compensate for Pax7 function. Furthermor e, Relaix et al. (2006) showed that Pax7 in vivo has an anti apoptotic function that is absent in Pax3 suggesting that when Pax7 is absent satellite cells may be lost to apoptosis causing muscles to lose their regenerative capabilities in adulthood. Pax7 is regulated by the expression of myogenic regulatory factors ( MRFs ) and other local factors as satellite cells become activated. Pax7 is down regulated upon myogenic differentiation with the up regulation of myogenic markers such as MyoD and myogenin (Zam mit et al., 2004). inhibitory interactions between Pax7 and MRFs, suggesting a possible function for Pax7 to regulate the decision of an activated cell to proliferate or return to quiescence. Furthermore, ove rexpression of Pax7 causes cells to exit the cell cycle even under proliferative conditions. Specifically, Pax7 overexpression causes down


22 regulation of MyoD prevents myogenin induction, and blocks MyoD induced commitment to myogenic lineage (Olguin et al ., 2004). The role for Pax7 in adult satellite cells has been put in to question more recently. Lepper et al., (2009) established in vivo using inducible Cre/loxP lineage tracing and Pax7 conditional gene inactivation that when Pax7 is inactivated in adult mice, mutant satellite cell regenerative capabilities are not compromised. By looking at multiple time points of inactivation they also determined that Pax7 is required only in juvenile mice to cause progenitor cells to return to a quiescent state. These r esults further support the idea that an important function for Pax7 in adult cells may be for self renewal purposes to replenish the quiescent satellite cell population, thereby maximizing regenerative capacity of adult muscle. Conversely, Mitchell et al. (2010) showed evidence for a group of progenitor cells that express PW1 a cell stress mediator that is also expressed in satellite cells, but not Pax7 and yet are able to commit to a myogenic lineage in vitro and contribute to muscle regeneration in vivo However, when isolated from Pax7 mutant mice, unlike Pax7 mutant satellite cells, these cells were unable to commit to a myogenic lineage. Therefore although not necessarily required in adult satellite cells, Pax7 is essential for other local progenitor cell s to be able to eventually commit to a myogenic lineage in adult animals Satellite Cell Quiescence Satellite cells in adult muscle fibers remain in a quiescent, or non mitotically active, state. At this point they are in the G 0 phase of the cell cycle or are considered to have exited the cell cycle in a dormant state. Unlike myotubes that have exited the cell cycle without being able to re enter it, satellite cells are able to re enter the cell cycle upon activation. Quiescent satel lite cells divide i nfrequently but although characterized as dormant, continue transcription of


23 various protein markers. In fact, quiescent cells have been described to be under active transcriptional control as opposed to an inactive state of dormancy (Dhawan and Rando, 2 005). Satellite cell expression of various genes regulates quiescence. Microarray data from mouse satellite cells identified a number of genes highly up regulated in quiescent satellite cells (Fukada et al., 2007). Quiescent cells showed preferential expre ssion of genes involved in cell to cell adhesions, regulation of cell growth, formation of extracellular matrix, copper and iron homeostasis, and lipid transportation, all of which are important in intact satellite cell environments. Of these genes, calcit onin receptor ( CTR ) was expressed exclusively in quiescent cells and exogenous calcitonin stimulation delayed satellite cell activation. Gene profiling analysis in vivo has also demonstrated high expression in quiescent cells of transcripts that inhibit ex pression of proteinases expressed for extracellular matrix remodeling in activated cells (Pallafacchina et al., 2010). Satellite cell regulation and maintenance of an intact environment may therefore signal quiescence. Quiescent satellite cells also expres s the inhibiting factor Sprouty1 ( Spr y1 ), a negative regulator of tyrosine kinase signaling receptors whose ligands are potent satellite cell activators. Several studies have recently determined that Spry1 is expressed in quiescent satellite cells of uninj ured muscle down regulated upon injury, and is required to maintain or re establish satellite cell quiescence (Abou Khalil and Brack, 2010; Shea et al., 2010). Satellite Cell Activation Satellite cells become activated from their quiescent state by vario us stimuli and re enter 1 phase of the cell cycle from the G 0 phase, or quiescence. Cells become activated by injury or stimulus for muscle growth and begin to proliferate and become muscle progenitor cells. Once activated, cel ls can either undergo myogenic differentiation or contribute to self renewal of the satellite cell pool. Cells that follow


24 a regenerative pathway eventually differentiate and fuse together or with existing myofibers to become muscle fibers. In vitro analysis of activated satellite cells is difficult because damage to muscle fibers from the satellite cell isolation inevitably triggers cell activation (Zammit et al., 2004). However, many factors have been identified as pro moting or inhibiting activation in vitro and in vivo studies have allowed us to better understand how activation is initiated and regulated. There are several ways to mark satellite cell activation Bromodeoxyuridine (BrdU) is a synthetic nucleoside and a nalog of the nucleoside thymidine. It can substitute for thymidine during DNA replication in the S phase of the cell cycle, marking cell proliferation and is therefore a good indicator for satellite cell activation. S atellite cells, however, do not begin t o proliferate immediately upon activation, and therefore other markers are necessary to determine the exact point of activation. Proliferating cell nuclear antigen (PCNA) is a pro tein cyclin that clamps DNA polymerase during DNA replication. It is maximal expression in the S phase of the cell cycle but expression is also evident in the G 1 phase as well Johnson and Allen (1993) identified PCNA as a good marker for initial satellite cell activation by showing that satellite cells isolated from young and adu lt rats both showed expression of PCNA before they began to divide. PCNA can be used as a primary indicator for cell activation prior to proliferation and differentiation, allowing for better analysis of factors that trigger activation. Satellite cells exp ress various transcription factors upon activation and proliferation prior to differentiation. These different transcription factors can serve as markers for satellite cell status. Muscle progenitor cells are specified to become muscle lineage committed my oblasts through the action of basic helix loop heli x (bHLH) transcription factors, or MRFs Quiescent satellite cells do not up regulate MRFs, suggesting a stem cell lineage distinct from myoblasts (Cornelison et al., 1997). When a satellite cell is activa ted, it begins to express the primary


25 MRFs, Myf5 and MyoD Myf5 is expressed in both quiescent and activated satellite cells and is the earliest marker of myogenic commitment (Beauchamp et al., 2000). MyoD expression appears to follow Myf5 expression upon satellite cell activation (Hirsinger et al., 2001), and Cornelison and Wold (1997) found that Myf5 and MyoD were expressed individually or as a combination upon activation. An important part of satellite cell activity involves the environment in which it r esides, called the stem cell niche. A stem cell niche is a microenvironment that houses the stem cells and is supported by surrounding cells within the niche that secrete factors affecting stem cell behavior. These supporting cells must help to maintain st em cell quiescence in the absence of external signaling cues as well as promote activation, self renewal, and commitment to a particular lineage as needed. The primary supporting cells for satellite cells are the myofibers associated to them through anatom ical location and by secretion of paracrine factors. Secondary supporting factors include the basal lamina that surrounds the satellite cell and myofiber and that contains matrix proteins that interact with membrane proteins on the satellite cells. The bas al lamina also contains various growth factors that can be released upon signals for activation (Tatsumi et al., 2004). HGF and Activation Bischoff (1986) demonstrated that there were endogenous activation factors in muscle tissue by exposing quiescent sa tellite cells associated with muscle fibers to a phosphate buffered saline (PBS) extract from lightly compressed or crushed muscle tissue and observing some cells becoming activated after exposure. The active factor within the extract was later identified as hepatocyte growth factor (HGF) that could be released from the extracellular matrix of muscle fibers by crushing (Allen et al., 1995; Tatsumi et al., 1998).


26 Allen et al. (1995) was the first group to identify HGF as a mitogenic factor for satellite cell s. By using satellite cells isolated from 9 month old rats that experienced a lag phase prior to division, they were able to show that with the addition of HGF the cells experienced a dose dependent increase in proliferation suggesting that it caused an e arlier onset of proliferation in cells They were also able to show PCNA expression earlier in treated cells compared to controls. Tatsumi et al., (1998) replicated this work in vivo using B rdU incorporation to confirm proliferating cells after treatment w ith HGF. HGF is synthesized as a n inactive 90 kDa single chain protein, and cleaved by an HGF activator into a 60 kDa chain and a 30 kDa chain that are linked by disulfide bonds to become activated (Naka et al., 1992). Whether HGF is activated as needed in muscle tissue or previously activated prior to release is important information to understand the satellite cell activation process. Tatsumi and Allen (2004) determined that HGF is tethered to the extracellular matrix of uninjured muscle in its active, he terodimeric form, suggesting easy release upon injury and absence of a rate limiting activation step prior to release. S imple d amage to the muscle fiber due to injury is one way to cause the already activated HGF to be released from its tethered position a nd act upon local satellite cells. HGF is not only expressed by muscle tissue, but by satellite cells as well. Anastasi et al. (1997) determined in mouse C2C12 myoblasts co expression of HGF and its receptor, Met, and both were down regulated as prolifera tion stopped and myoblasts began to differentiate. I n an effort to determine an autocrine role i n muscle tissue, Sheehan et al. (2000) determined using reverse transcriptase polymerase chain reaction (RT PCR) that HGF mRNA was present in adult satellite ce lls 12 hours post plating prior to documented activation Expression increased between 36 and 48 hours, and HGF protein was detectable at 48 hours using localized


27 immunofluorescence. Western blot analysis determined HGF associated with the cell surface an d inside cells. A ddition of HGF neutralizing antibodies at 42 90 hours (a proliferating state) reduced cell proliferation. HGF has also been shown to be expressed by spleen and liver cells in response to muscle injury, increasing HGF serum levels that are transported to the site of injury and made available to further increase the satellite cell activation (Suzuki et al., 2002). These results indicate that HGF works in an autocrine fashion expresse d in satellite cells, a para crine fashion by expression in m uscle tissue, and an endocrine fashion by expression in non myogenic cells in response to injury. Furthermore, local administration of HGF to injured muscle increases myoblast proliferation, establishing that the source of HGF does not affect its mitogenic ability (Miller et al., 2000). HGF functions through signaling pathways after binding to the c M et receptor tyrosine kinase proto oncogene which is expressed in both quiescent and activated cells Cornelison and Wold (1997) determined through single cell RT PCR analysis that c M et is expressed by all myofiber associated satellite cells from the time of explant all the way through activation, proliferation, and differentiation. Upon binding of HGF to the c M et receptor, multiple signaling cascades can occu r, including the Mitogen activated protein kinase (MAPK) pathway and the phosphotidyl inositol 3 kinase (PI3K) pathway. Once bound, the activated c met receptor signals the transcription of growth and proliferation genes within the cell nucleus, and the ac tivated cell begins to proliferate (Furge et al., 2000). The binding of HGF to the c M et receptor can also be determined by the subsequent expression of MyoD and Myf5 (Tatsumi et al., 1998). As cell motility is important in activated satellite cel ls, HGF i nduces dissociation and cell mo vement, and HG F/c M et signaling becomes important in response to injury at a given site and for cell


28 migration during embryogenesis (reviewed in Matsumoto and Nakamura, 1996; Bladt et al., 1995) HGF regulation in muscle rege neration and growth is not well understood. While it is known that HGF is released upon injury, HGF and HGF activator (HGFA) levels in human muscle tissue and serum increase significantly after single bouts of lengthening contraction. Moreover, HGF A inhibitors 1 and 2 increased in tissue and serum levels by 24 hours thereafter, indicating a mechanism for tightly regulating HGF activation post response. Another proposed regulator of HGF involves the Janus kinase (JAK)/signa l transducer and activator of transcription (STAT) pathway. While a JAK 1/STAT 1/STAT 3 signaling pathway has been shown to promote myoblast proliferation and prevent differentiation (Sun et al., 2007), an alternate pathway consisting of JAK 2/STAT 2/STAT 3 caused decreased expression of HGF, promoting differentiation (Wang et al., 2008). High concentrations of HGF appear to regulate myogenic differentiation as well. E xcess exogenous HGF in the presence of satellite cells has been shown to attenuate differe ntiation by inhibiting transcription of myogenic regulatory factors such as MyoD and myogenin (Gal Levi et al., 1998). Anastasi et al. (1997) mimicked the effect of Met receptors fully saturated with HGF ligand in C2C12 cells by stable expression of the co nstitutively activated receptor catalytic domain which prevented myoblast differentiation with a corresponding absence of MyoD and myogenin expression HGF was shown to prevent differentiation in vivo as local administration of HGF to injured mouse muscle increased myoblast proliferation, but after 4 days of treatment, when normally myoblasts begin to differentiate and fuse, the exogenous HGF appeared to prevent myoblast differentiation. Inhibition of differentiation was reversed, however, with cessation o f the exogenous HGF (Miller et al., 2000).


29 HGF mediated prevention of differentiation has been explored in depth A proposed molecular mechanism for effects on differentiation suggests that HGF temporarily prevents differentiation by down regulating genes involved in cell cycle arrest in order to delay cell withdrawal from the cell cycle and entrance into differentiation (Leshem et al., 2000). HGF was shown to regulate the expression of Twist, a bHLH transcription factor and inhibitor of MyoD and MEF2 trans cription factors, and p27, a cdk inhibitor that promotes withdrawal from the cell cycle upon differentiation. Specifically, HGF promotes cell proliferation by up regulating Twist protein and down regulating p27. As p27 levels slowly increase in response to cell proliferation, they reach a threshold at which point cells exit the cell cycle, down regulate Twist, and begin to differentiate. Increased exogenous concentrations of HGF added to mouse primary satellite cells and myoblasts inhibited proliferation an d caused cells to return to the G 0 phase of the cell cycle (Li et al., 2009). In the same study it was determined that the protein tyrosine phosphatase SHP2, a mediator for Met receptor activation, mediates the inhibitory effects of excess HGF and these ef fects are reversed when SHP2 is absent. Further evidence for HGF having a role in quiescence is apparent as varying doses of HGF added to activated/proliferating rat satelli te cells in culture resulted in decreased BrdU incorporation with increasing HGF do sage ( Yamada et al. 2009) Cells were reactivated with decreased HGF treatment, and dual methods of RT PCR and Western b lot revealed the up regulation of myostatin mRNA expression and protein expression with high HGF concentrations. These results suggest a threshold concentration for HGF in the local satellite cell environment, above which HGF reverses its mitogenic effect on satellite cells and prevents differentiation by inducing cell return to quiescence.


30 Nitric Oxide and Activation Nitric oxide (NO) i s a short lived free radical that is freely diffusible and ubiquitous throughout muscle tissue. It is produced by nitric oxide synthases, with neuronal nitric oxide (NOS I) specific to muscle tissue and localized to the sarcolemma of muscle fibers (Silvag no et al., 1996). NO is synthesized by NOS I as a product from the conversion of L arginine to citrulline. It is normally produced at low levels in resting muscle tissue and increases with exercise, load, or injury (Tidball et al., 1998). Anderson (2000) was one of the first to suggest that the production of NO radical is a key signal responsible for satellite cell activation, hypertrophy and detachment from the adjacent muscle fiber following injury. After determining that activation occurred in the mouse after one minute following induced injury in vivo, nitro L arginine methyl ester (L NAME), resulted in prevention of immediate cell release post injury and delayed muscle regeneration. Further evidence from in vitro experiments using single fiber culture models support th e idea that NO is a significant signal for satellite cell activation post injury or stretch (Anderson and Philipowicz, 2002; Wozniak and Anderson, 2007). Supplementation with a NO donor compound or the NOS substrate, L arginine, results in increased satel lite cell activation, establishing the importance in NO bioavailability for satellite cell activity (Betters et al., 2008). In fact, an increase in NO concentration during functional muscle unloading prevented atrophy and increased satellite cell numbers ( Kartashkina et al., 2010). R ecently a new drug was developed, guaifenesin dinitrate (GDN), as a NO radical donor to be delivered to muscle (Wang et al., 2009). Results using this drug revealed a strong increase in satellite cell activation and proliferatio n 24 hours after oral administration in adult mice. Western blot and immunohistochemical analysis revealed increased expression of myogenic markers myf5, myogenin, and follistatin.


31 NO production upon injury or stretch is related t o HGF release and binding to c M et receptors in satellite cell activation. In fact, NO is a key factor in satellite cell proliferation following stretch induced activation (Soltow et al., 2010). Interestingly, when the NOS inhibitor L NAME was administered to rats subjected to mus cle overloading, NOS inhibition did not seem to affect HGF protein content. However, addition of L NAME inhibited myonuclear addition in animals under basal conditions compared to animals without L NAME administration, suggesting the importance for NO unde r normal conditions (Gordon et al., 2007). Experiments ked with L NAME incorporation and it can be liberated when unperturbed satellite cells are incubated with NO donors. They also use d stretch cultures in the presence of L NAME and observed that stretch activation of satellite cells was inhibited by the NOS inhibitor in a dose dependent manner, and that the addition of HGF restored the activation response. Further analysis of the L NAM E conditioned medium confirmed the absence of HGF that could have been released from cells, validating that exogenous HGF was the cause of activation. These results suggest that activation by HGF is dependent upon NO. The inhibitor L NAME does not inhibit HGF but instead acts upstream of it, indicating that HGF release is dependent upon NO radical production that is a result of mechanical stretch or injury. Age and Activation Age plays an important role in satellite cell activation. It has been very apparen t in in vitro studies that satellite cells isolated from aged animals experience a delay in proliferation Satellite cells isolated from young (3 weeks to 3 months) versus adult rats (9 30 months) showed that the cells of young tissue begin proliferating a fter 24 hours in culture, whereas cells from adult tissue experienced a lag phase of 48 hours before proliferating (Schultz et al., 1982). Johnson and Allen (1993) determined using PCNA incorporation that satellite cells isolated from young rats (3 4


32 weeks ) increased PCNA expression 24 and 30 hours post plating, while cells from adult rats (9 12 months) did not increase PCNA expression until 42 to 48 hours post plating. This lag period was also shown in vivo by McGeachie and Gro unds (1995) using radiograph te ch n ologies. Induced injury in mice caused activation in both young and old cells around 18 24 hours, but the peak proliferative activity for old cells occurred 26 36 hours later than young cells. The lag phase observed in cells from adult tissue was sug gested to be the amount of time required for quiescent cells to activate and proliferate in response to a stimulus. Cells of younger, developing muscle tissue, on the other hand, may have not yet exited the cell cycle and therefore may be the reason for th e faster activation observed (Tatusmi et al., 1998). However, Li et al. (2011) showed through Pax7 / Myf5 expression that the percentage of muscle stem cells ( Pax7 + / Myf5 ) and muscle progenitor cells ( Pax7 + / Myf5 + ) were roughly equal in bovine satellite cells from adult and young animals after 24 hours post plating. Only after 24 hours was there a larger decline in muscle progenitor cells of young animals likely to progenitor cell commitment to differentiation. This would argue that the delay is not due to the initial status of the cell. Satellite cell number decreases with age in muscle tissue and older satellite cells show a delayed response to activation stimuli in vitro Several studies have shown that exposing old muscle (Carlson and Faulkner, 1989) or ind ividual satellite cells (Conboy et al., 2005) into a young environment results in increased regenerative capacity, but exposing young muscle or satellite cells to an old environment elicits no change. Recently Hall et al. (2010) was able to show that trans planting donor satellite cells from adult mice with their associated myofibers into adult and aged hosts with induced muscle injury resulted in increased regeneration by donor cells. Brack et al. (2007) also showed that sy stemic exposure of old tissue to y oung serum


33 resulted in improved regeneration, whereas systemic exposure o f young tissue to old serum resulted in increased collagen deposition in regenerating areas. This would indicate that changes in the systemic environment surrounding satellite cells g reatly affect their regenerative capabilities and that old satellite cells maintain regenerative capability but are inhibited by the environment. Several changes occur in the satellite cell microenvironment that can affect satellite cell activity. Changes in the basal lamina due to aging include increased formation of connective tissue. Excess connective tissue was first reported by Snow (1977) the formation of which surrounded the myofiber of aged mouse and rat muscles. Goldspink et al. (1994) suggested th at the increase in fibrosis in normal aged muscle is not a result of increased connective tissue expression but a decrease in connective tis sue degradation associated with tissue homeostasis. Extra formation of connective tissue could result in the impairm ent of chemotaxis for growth factors and cytokines that promote satellite cell activation. Furthermore, collagen fibers have been shown to undergo increased crosslinking with age, which is a direct effect of the Maillard reaction, a non catalyzed reaction that results in polycyclic compounds that are efficient in crosslinking proteins and nucleic acids (Robert and Labat Robert, 2000). Crosslinked collagen is insoluble and thus difficult for normal degradation as part of tissue turn over, therefore permanent ly changing the basal lamina construction. Other age related changes in transient cytokines and serum components that are involved in satellite cell regulation can explain the resulting effects from aged muscle. Aged satellite cells from mice showed eviden ce for increased Wnt signaling, of which the Wnt 1 and Wnt7a pathways are important in normal muscle regeneration but have been shown to be associated with myogenic conversion to fibrogenic lineage (Brack et al., 2007). Cells of the immune system


34 are impor tant for tissue regeneration, including macrophage dependent removal of damaged tissue and deposition of chemoattractants for satellite cell activation. Analysis of total macrophage composition in young and old human muscle biopsies pre and post exercise revealed lower macrophage levels in the aged muscle at both time points (Przybyla et al., 2006). Although evidence supports the idea that age affects satellite cell ability, satellite cell populations from aged animals show some potential for regeneration. The number of progeny produced by aged satellite cells is reduced and partly responsible for the overall reduction in satellite cell number with age (Day et al., 2010) However, the remaining cells still show regenerative capacity. When myofibers of aged muscle were isolated and cultured, although the fibers were depleted of a large number of Pax7 expressing cells, a small population of satellite cells were able to generate larger progeny of both differentiated cells and new quiescent satellite cells chara cteristic of self rene wal (Collins et al., 2007). Furthermore, t hese progeny were able to regenerate when grafted into mdx nude mice, a strain of mice that have dystrophic muscle due to the loss of the protein dystrophin There is also evidence of increase d satellite cell progeny in aged rat muscle with endurance exercise (Shefer et al., 2010) further providing evidence for aged satellite cell ability. The potential in aged satellite cells emphasizes the effect that an aged environment has on satellite cell activity and satellite cell ability to overcome interfering factors in the aged niche is an important topic to explore in future studies. Satellite Cell Self Renewal: Asymmetric Division Satellite cell replenishment is an important part in growing and ad ult muscle as the satellite cell environment is constantly changing and satellite cells are recruited for muscle repair and regeneration Using BrdU incorporation, Moss and Leblond (1970) confirmed that satellite cells undergo cell division following activ ation that give rise to increased fiber associated nuclei. Radioisotope labeling of satellite cells in growing muscle in vivo showed that half of the


35 daughter cells differentiate into myonuclei while the other half remains as continual ly dividing satellite cells (Moss and Leblond, 1971). Cell cycle time analysis of satellite cells in growing rats in vivo revealed that 80 % of cells divide within a 32 hour cycle duration while 20% divide slowly (Schultz, 1996). The slower division rate may be due to those cel ls returning to quiescence between cycles and this group may give rise to the faster dividing group of cells. Two different models for cell division have been proposed to explain satellite cell homeostasis: the asymmetric model and the stochastic model. I n the asymmetric model cells divide asymmetrically giving rise to one daughter cell that maintains satellite cell identity and the other going on to differentiate. In the stochastic model, symmetric cell division gives rise to two identical daughter cells that eventually follow different lineage fates. A modified stochastic model has also been proposed that su ggests a cell initially divides asymmetrically and t he two daughter cells continue on to divide symmetrically amongst their differing cell fates (revi ewed in Kuang et al., 2008). The dynamics of Pax7 MyoD and myogenin expression found in vitro in proliferating and differentiating myoblasts supports the stochastic model for satellite cell division. Upon activation, satellite cells induce MyoD expressio n and proliferating satellite cells express both Pax7 and MyoD Most of these cells begin to down regulate Pax7 maintain MyoD and up regulate myogenin as they commit to differentiation. However, some cells are able to down regulate MyoD up regulate Pax7 and eventually withdraw from the cell cycle and return to quiescence (Olguin et al., 2004; Zammit et al., 2004). This population of cells observed to down regulate myogenic markers and retu rn to a quiescent state suggests a signal upon cellular division t o maintain the satellite cell pool.


36 The asymmetric model also contains significant supporting evidence from both in vitro and in vivo experiments. Tracking cell division using labeled DNA by pulse BrdU incorporation, cells displayed selective DNA template strand segregatio n during division Furthermore, Numb, the Notch signaling antagonist protein involved in inhibiting activation, was observed to segregate selectively during division to the daughter cell that receives the older DNA template and express es P ax7 suggesting that Numb is also involved in the self renewal process (Shinin et al., 2006). Numb was also observed to be localized asymmetrically towards the basal lamina as the cell divides in an apical basal orientation (Venters et al., 2005). Addition ally, myogenic marker expression is important in determining cell history and cell fate to understand the division process. Kuang et al. (2007) found in vivo using Myf5 Cre and ROSA26 YFP Cre reporter alleles that 10% of sublaminar Pax7 expressing cells ha v e never expressed Myf5 denoted Pax7 + / Myf5 This finding opposes the stochastic model that would suggest self renewed cells were once activated before returning to quiescence In addition, Pax7 + / Myf5 cells gave rise to a committed Pax7 + / Myf5 + daughter i n the apical position and a Pax7 + / Myf5 non committed daughter in the basal position through apical basal oriented division. Isolation and transplantation of these cells to injured muscle resulted in committed cells contributing to regeneration and non com mitted cells contributing to the satellite cell pool. These results not only suggest asymmetrical division in self renewal, but also infer that satellite cells consist of a heterogeneous population of non committed and committed satellite cells through cel l division. Satellite cell populations may include both the stochastic and asymmetric forms of division, as research has provided evidence for both models. It is very possible that satellite cells use a variety of mechanisms in order to maintain homeostasi s of a constantly changing


37 microenvironment. As evidence previously mentioned indicates that satellite cells make up a heterogeneous population of cells expressing varying myogenic markers, it can also be inferred that the satellite cell pool is generally heterogeneous F rom the differences amongst individual cells to differences in subpopulations of cells, it is clear that various methods are used within a population in order to self renew t he existing satellite cell pool. Thus the satellite cell pool can be considered a heterogeneous population consisting of differing fates, myogenic expression, and cellular division mechanisms. There is evidence of satellite cell heterogeneity a t multiple levels Fiber types differ from muscle to muscle as well as within the same muscle (reviewed in Biressi and Rando, 2010) G lycolytic, or fast twitch (type II) fibers and oxidative, or slow tw itch (type I) fibers determine strength and endurance respectively, of the muscle tissue. Moreover cells within a single populatio n of satellite cells experience a variety of genetic expression and different activities. The observed heterogeneity appears to be important considering the constant change observed in the local satellite cell environment as muscle tissue is constantly str etched and in use. Satellite cells from differing fiber types exhibit gene expression inherent of their particular origins. Isolation and in vitro culturing of murine satellite cells identified a pool of cells forming myotubes expressing slow (type I) myos in heavy chain that was preferentially associated with individual slow muscle fibers ( Rosenblatt et al., 1996 ). Additionally, satellite cells isolated from the semimembranosus proprius (100% type I fibers) and the semimembranosus accessorius (100% type II fibers) of 3 month old rabbit s and cultured in vitro revealed differing behaviors from cells of the two fiber types. Specifically, cells isolated from the slow fiber type proliferated faster, fused earlier into more numerous myotubes, and mature d more rapi dly into striated muscle than cells isolated from f ast fibers. MyoD and myogenin expression were delayed 2 and 4


38 days, respectively, in cells from fast twi tch fibers compared to those of slow fibers (Barjot et al., 1995). Cell origin does not, however, det ermine ultimate cell fate. Snoj Cvetko et al. (1996) isolated cells from slow twitch muscle and transplanted them into injured muscle of fast twitch origin. After 3 months the presence of fibers expressing slow myosin heavy chain were observed, but were ab sent after 6 months. This would suggest that the slow phenotype in the transplanted cells was stable but not irreversible. E xtrinsic factors such as innervations can therefore override i ntrinsic expression in the cell, emphasizing the plasticity that satel lite cells exhibit to meet environmental condition requirements. Satellite Cells as Stem Cells Following development, many tissues establish populations of pliable stem cells that will last throughout the remainder of the adult life. An adult stem cell is defined by its amount of plasticity, slow cycling, and ability to respond to environmental factors that cause it to either self renew or differentiate (reviewed in Fuchs and Segre, 2000). Adult s atellite cells are considered stem cel ls based on being the s ource for muscle hypertrophy and regeneration in adult animals as well as being able to self renew Postnatal satellite cells isolated from mice that were cultured and expanded over 200 population doublings maintained the stem cell markers Sca 1 and CD34, providing evidence for stem cell characteristics as well as self renewing capability to replenish the satellite cell pool (Deasy et al., 2005). Quiescent satellite cells are characterized by their slow dividing behavior, as they only begin to divide upon activation. Although primarily recruited for myogenic lineage, satellite cell plasticity has been observed as well in vitro further qualifying satellite cells as having stem cell characteristics. Evidence of satellite cell plasticity in vitro has been sho wn as well with induction media. For instance, satellite cells have been shown to tran sdifferentiate into adi pocytes from adult mice (Grimaldi et al., 1997 ; Asakura et al., 2001; Sarig et al., 2006 ) and pigs (Singh et al., 2007) with


39 adi pogenic media or in hibition of the Wnt 1 signaling pathway (Ross et al., 2000) and into osteocytes from adult mice with bone morphogenic protein s 2 and 4 (BMP 2 and BMP4 ) induction media (Asakura et al., 2001; Sarig et al., 2006). The ability for these cells to differentiate into other mesodermal lineages is clearly affected by the surrounding environment. Several studies, however, have reported spontaneous satellite cell transdifferentiation in single fiber cultures without the addition of other lineage induction factors. Fib er a ssociated satellite cells exposed to oxygen concentrations higher than physiological levels resulted in increased adipogenic expressing cells, thought to have originated from satellite cells (Cseste et al., 2001). Satellite cells on isolated mouse fibe rs exhibited clones that resulted in variable myogenic to non myogenic lineage ratios from fiber to fiber, suggesting that satellite cells were randomly able to transdifferentiate to a non myogenic fate (Shefer et al., 2004). Satellite cells on isolated fi bers cultured in Matrigel also showed spontaneous transdifferentiation compared to isolated primary myoblasts that did not (Asakura et al., 2001). Stark ey et al. (2011) challenged these findings with permanently labeled mouse satellite cells in single fibe r and clonal cultures and found that cells of adipogenic lineage were not of satellite cell origin. Furthermore, satellite cells grown in adipogenic induced media accumulated cytoplasmic lipid but never fully differentiated into adipocytes, maintaining myo genic protein expression. This group suggested that the cells that were suspected of transdifferentiation were in fact non myogenic progenitors from the muscle interstitium that were co purified with the fibers. Furthermore, an additional group of progenit or cells was recently identified within the muscle interstitium that do not express the Pax7 satellite cell marker but contribute to myogenesis during muscle regeneration (Mitchell et al 2010) and could thus contribute to the transdifferentiation observ ed in previous studies S atellite cells therefore show plasticity by expressing other


40 mesodermal lineage markers under lineage specific induction media but at the same time show evidence for commitment to a myogenic fate. They demonstrate characteristics defined under an adult stem cell, but it is yet to be determined whether they actually contribute to other lineages in vivo Attachment Matrix Elasticity Affects Cell Behavior The interaction between cells and the extracellular matrix is important for cell regulation and behavior. Not only do the specific ligand receptor binding complexes create a cellular response, but cells have been shown to respond to forces exerted through attachment mediums as well (Girad et al., 1995; Wang et al., 1993). The tuning m odel proposes that several parameters concurrently control how a cell attaches and migrates, the combination of which impacts the Cells can detect rigidity, or stiffness, of a matrix u sing integrin mediated adhesion and mechanosensor signaling, which makes it important to explore how differing stiffness regulate s cellular behavior in vivo. Pelham and Wang (1997) were one of the first groups to explore substrate stiffness and how it affects cellular attachment and subsequ ent behavior. For a viable substrate, they used polyacrylamide gels with variable bis acrylamide cross linker concentrations to create a variety of substrate elasticity. The gels were coated with collagen to provide available ligands for cell attachment, an d NRK 52E endoth elial cells and 3T3 fibroblast cells were cultured on the substrates as well as on plastic for comparison. Both cell types exhibited differing morphologies, with less spreading and more irregular shapes observed from cells on softer, more f lexible substrates, and increased spreading with stiffer, more rigid substrates. Cells on stiffer substrates also exhibited increased tyrosine phosphorylation and actin myosin cytoskeletal forces generated, leading to more stable focal adhesions. These res ults indicate that cells sense differences in substrate flexibility by active pushing and pulling of their integrin receptors


41 through the associated cytoskeleton, resulting in changes in tyrosine phosphorylation. In addition, the myosin motor may be involv ed in generating the force to probe substrate mechanical properties and initiate signaling to constitute cellular action. Various efforts have been made to investigate substrate stiffness effects on cell activity using different kinds of flexible substrate s. Different techniques have been used to analyze various aspects of cellular behavior upon attachment, such as cell motility (Beningo et al., 2001; Lo et al., 2000; Peyton et al., 2005), optimal ligand density (Engler et al., 2004a), and cell signaling an d function (Polte et al., 2004; Putnam et al., 2003; Wang et al., 2000). Results from these studies show substantial evidence that cells grown on a substrate with a more tissue like stiffness respond very differently than those grown on plastic or glass. T his would suggest that stiffness is a major factor in creating an in vitro environment that closer mimic s cellular microenvironments in vivo. Engler et al., (2006) explored how substrate elasticity affects lineage commitment made by mesenchymal stem cells (MSCs), marrow derived stem cells that have been shown to differentiate into various lineages. T hree different soft tissue elasticities were compared, brain, muscle and bone (measured from murine tissues in Flanagan et al., 2002, Engler et al., 2004b, and the present study respectively) and replicated in collagen coated polyacrylamide gels (as in Pelham and Wang, 1997). Morphology analysis showed that MSC attachment with varying stiffness resulted in morphological characteristics similar to those of cells from each tissue type: cells on brain like substrates exhibited filopodia rich branching seen in neurons, cells on muscle like substrates exhibited spindle shapes comparable to cultured C2C12 myoblasts, and cells on osteoid like substrates exhibited polygo nal shapes observed in osteoblasts. Use of RNA profiling confirmed the presence of tissue specific transcripts within MSCs plated on the various elastic


42 substrates (Engler et al., 2006). Also, detection of cytoskeletal markers validated that MSCs on varyin g elastic substrates begin to commit to the lineage of cells particular to each tissue, highlighting the importance of stiffness alone in determining cell behavior in vitro. In depth examination of satellite cell response to softer substrates in culture ha s become an important area of interest The satellite cell niche is very difficult to reproduce in vitro partly because of the 3 dimensional location in which the cell lays, between the myofiber and the basal lamina. Transplantation of satellite cells when incorporated with their original myofibers yield higher regenerative and self renewal capacity than transplantation of isolated cells, inferring a clear dependence the satellite cell has for its natural microenvironment (Collins et al., 2009). Substrate e lasticity is one part to incorporate in the attempt to recreate the satellite cell niche and the corresponding effect it has on cell activity has yet to be determined. One of the first studies to specifically explore myogenesis on an elastic substrate used mouse C2C12 myoblasts and determined an optimal elastic stiffness for myotube formati on (Engler et al., 2004b). Mouse muscle tissue elasticity was determined using atomic force microscopy (AFM) techniques and collagen coated polyacrylamide gels were ta ilo red to convey the resulting AFM muscle elasticity measurement While myotubes formed on both gels and glass surfaces, myosin and actin striations were observed only on gel substrates that mimic normal adult muscle tissue. Furthermore, cells plated on a mon olayer of C2C12s that had been plated on glass earlier and allowed to form myotubes showed striations on the top layer but not on the bottom layer, further supporting the idea of softer, 3 dimensional conditions needed for optimal differentiation. In addit ion, cell adhesion strength on substrates increased with stiffness, the strongest observed on glass surface. These results have important implications for the difference in satellite cell behavior in vitro compared to behavior observed in vivo.


43 Satellite c ell attachment is another important consideration that affects mechanosensing of the substrate. Cells attach to the basal lamina through a network of collagen IV and laminin proteins interconnected by the protein entactin. These networks also serve as a re servoir for growth factors, such as HGF, that regulates satellite cell activity. In vitro studies using rat and mouse skeletal myoblasts revealed that availability of these extracellular matrix proteins for attachment promoted proliferation and differentia tion (Grossi et al., 2007; Macfelda et al., 2007). Cell attachment to matrix proteins are subsequently affected by the stiffness of the substrate, as in vitro culture conditions on plastic or glass do not embody the flexibility apparent on a myofiber. Boon en et al. (2009) tested satellite cell dependence on substrate stiffness and matrix protein availability. Isolated mouse primary satellite cells were cultured on varying polyacrylamide gel elasticities and varying protein coatings. Results showed that prol iferation and differentiation increased with increasing elasticity Optimal g el elasticity was observed at 21 kPa compared with 3, 14, 48, and 80 kPa gels, higher than the 12 kPa previously report ed for mouse muscle tissue Satellite cells did, however, ex hibit significantly different behavior on gels relative to glass, such as slower proliferation and an absence of cross striations or contractions in differentiated m yotubes. Cells had improved differentiation patterns on gel substrates and glass when attac hed to matrix proteins compared with pure protein coatings such as collagen or laminin. Primary satellite cells displayed different behavior than observed in C2C12 cells from give more ins ight into normal activity in vivo U sing freshly isolated mouse satellite cells Gilbert et al. (2010) explored how substrate stiffness regulates preservation of satellite cell stem like features Mouse muscle tissue elasticity was determined and laminin c oated polyethylene glycol hydrogels were developed to


44 portray this elasticity as well as those of brain and cartilage tissues. Culturing the satellite cells on hydrogels of variable elasticity demonstrated that cells grown on substrates softer than rigid p lastic surfaces are associated with decreased cell death and an overall increase in cell number. Softer substrates also prevented cells from differentiating, as determined by an absence of myogenin after one week in culture. To assess regenerative capacit y in vivo from mice expressing firefly luciferase on the varying elastic substrates or plastic for 7 days and subsequently transplanted into the tibialis anterior of immunodeficient mice depleted of satellite cells. Bioluminescence imaging analysis revealed that cells cultured on hydrogels with elasticity that mimicked muscle elasticity exhibited the highest percentage of satellite cell engraftment. Satellite cells grown on muscle like hydrogels also returned to thei r native satellite cell niche beneath the basal lamina and atop myofibers Cells isolated from mice expressing LacZ under regulation of Myf5 were cultured on variable substrates and transplanted into injured muscle tissue. Tissue was subsequently harvested and histological analysis revealed that the transplanted cells grown on soft substrates and expressing Myf5 had returned to their native location on the myofiber. The previously described data suggest s that cells grown on pliable substrates are able to ma intain satellite cell characteristics post transplantation, meaning that not all transplanted cells immediately differentiate when transplanted but could also contribute to the satellite cell pool. These results have implications for therapeutic treatment of transplanted satellite cells grown on pliable substrates. They also support the idea that mimicking micronevironmental conditions in vitro supports native satellite cell behavior, which can lead to improved understanding of satellite cell activity in fu ture studies.


45 CHAPTER 4 RESULTS Muscle Elasticity Differs with Age and Can Be Mimicked in Polyacrylamide Gels The surface tension, or elasticity, of a substrate directly impacts the lineage decisions of multipotent stem cells. As a first step toward exami ning the effects of matrix elasticity on satellite cell behavior, the elasticity of both young and adult skeletal muscle was measured. The right longissimus dorsi (LD) and semimembranosus (SM) were removed from young and adult cattle at slaughter. A subpor tion of the intact muscle was attached to a load cell and tensile force young muscle tissue was approximately 8 kPa, in dependent of muscle type (Figure 4 1). The calcu lated elasticity for the SM did not differ as a function of age. However, a significant increase ( p = 0.0115 ) in elasticity was noted in the adult LD (14.57 1.64 kPa) compared with the immature calf (8.12 1.74 kPa). A difference was also noted between the adult LD and SM tissue ( p < 0.0081), whereas no significant difference was observed between the two muscle types from young tissue. The higher value of elasticity, E measured in the adult LD tissue indicates a more rigid less elastic tissue Tissue e lasticity measurements can be mimicked in vitro by developing polyacrylamide gels of varying acrylami de composition (Pelham and Wang, 1997). As the next step toward examining matrix elasticity on satellite cell behavior, a standard curve for elasticity was developed for polyacrylamide gels of varying acrylam ide concentrations This curve was used to construct polyacrylam ide gels with elastic moduli values analogous to the above determined muscle elasticity. Gels were made with acrylamide c oncentrations of 5 8, 10, 12, o r 15% and cut into shapes with standard dimensions as described in Peyton and Putnam (2005). Gels were attached to a load cell and tensile force was measured in the same manner as the ex vivo muscle


46 tissue. Elastic modulus was calculated from the tensile force measurements and fit to a linear curve (R 2 = 0.99; Figure 4 2). The linear equation of the curve was used to determine acrylamide concentrations that mimicked the elasticity of young and adult tissue. These methods allow for development of substrates that re present muscle tissue elasticites that may be detected as such by satellite cells in vitro Cell Attachment on Polyacrylamide Gels Requires Covalently Linked ECM Proteins Satellite cells attach to myofibers via membrane integrins that form linkages with proteins contained in the extracellular matrix (ECM) of the fiber. I n vitro satellite cells show improved attachment and formation of myofibers upon differentiation when cultured on plates coated with Matrigel, containing ECM proteins, a nd laminin (Rooney et al., 2009; Wilschut et al., 2010). Previous work using softer substrates such as polyacrylamide gels have used a chemical cross linker to covalently attach ECM proteins to the substrate (Engler et al., 2004a; Gilbert et al., 2010; Pelh am and Wang, 1997). To test whether a chemical cross linker was required, 2% w/v gelatin or ECL were added to the gel solution prior to polymerization, or 2% w/v ECL was coated on already polymerized gels. C2C12 myoblasts were plated on all treatments and m onitored at 24 and 48 hours post plating for attachment and proliferation. For all three treatments, cells exhibited clumping indicativ e of cells that were unable to attach At 48 hours the clumping increased and was comparable to the negative control (gel without protein incorporation, not shown). This behavior differs from that of C2C12 cells plated on gelatin coated plates (Figure 4 3A), suggesting that the cells had not attached to the substrate and that ECM proteins were not available on the surface of the gel for cells to attach properly. In contrast, both C2C12 cells and BSC plated on gels with ECL covalently attached via a chemical cross linker showed improved attachment over 48 hours (Figure 4 4)


47 Incorporating a chemical cross linker that is not natu rally occurring on the myofiber prese nts the possibility for toxic effects on satellite cell attachment and normal activity. Crosslinker toxicity was tested for possible interference with attachment at 24 hours using C2C12 myoblasts and BSC, and for interf erence with proliferation at 48 hours using C2C12 myoblasts. Cell attachment on ECL coated glass plates was compared to ECL+Crosslinker (ECL XL) coated glass plates. Glass plates serve as a control and provide successful C2C12 and BSC attachment with the a ddition of ECL coating. They were therefore used to observe effects of a chemical cross linker added to the ECL protein network. Differing concentrations of ECL and ECL XL were also compared to test for interference in protein availability with the addition of a cross linker The chemical cross linker proved to have no significant effect on C2C12 or BSC attachment as well as C2C12 proliferation (Figure 4 5). Furthermore, the C2C12 cells plated on the lower concentration of ECL XL coating showing significantly higher proliferation than the higher ECL XL concentration and both concentrations of ECL alone. Satellite Cell Proliferation is a Function of Age Satellite cells from adult animals experience a lag phase prior to proliferation compared to cells from young animals, the reason for which is not well understood (Johnson and Allen, 1993; Li et al., 2011; McGe achie et al., 1995). Whether this delay is due to a delayed activation or proliferation is unknown This was examined here using incorporation of PCNA and E dU as markers of activation and proliferation BSC isolated from neonatal (< 5d) and developing (2 3m) animals were plated under normal growth conditions on glass plates coated with ECL and analyzed at 24, 48 and 72 hours post plating for the percentage of EdU and PCNA positive cells (Fi gures 4 6 and 4 7 ). These results were compared to C2C12 myoblast incorporation of EdU and PCNA after 24 hours, of which 90% of cells incorporated both (Figure 4 7 ). Young BSC began to proliferate at 48 hours with 3.73 0. 16% incorporating EdU whereas 0% of developing


48 animals incorporated EdU at this time point ( p = 0.0430). At 72 hours both age groups had proliferating cells, however the amount of young cells incorporating EdU was significantly higher than the amount from developing animals (38.85 3.73% and 20.5 2.78%, respectively, p < 0.0001). PCNA incorporation however, began at 24 hours for both ages and was maintained throughout by 72 hours, the percentage of which did not differ significantly between a ges for eac h day. Satellite Cell Activation Varies with Substrate Stiffness Satellite cells grown on substrates that mimic muscle tissue elasticity exhibit different behavior compared with cells grown on plastic or glass plates, including morphology, myogenic marker expression, and regenerative ability (Collins et al., 2009; Engler et al., 2004a; Gilbert et al., 2010). As muscle tissue elasticity measured in the current study differs significantly between young and adult animals, satellite cells from young and adult a nimals may also exhibit differences in proliferative behavior and regenerative capacity. The possibility for matrix elasticity affecting satellite cell behavior was therefore examined. Satellite cells were grown on a range of elasticiti es to examine the re lationship between elasticity and satellite cell identity, activation and proliferation. Polyacrylamide gel of 6, 16, and 32 kPa were made the elasticity val ues of which resembled young (6 kPa) and adult (16 kPa) tissue, as well as a stiff extreme (32 kPa ) to observe the effect of changing elasticity on satellite cells BSC were cultured on the polyacrylamide of above mentioned elasticities and cell number was counted over a period of 6 days. Although the concentration of cells plated remained constant thr oughout all trials, the number of cells attached on day 1 varied widely among trials. However, a similar growth trend w as observed for all three elasticities up to day 3, after which the stiffest elasticity (32 kPa) differed from the lower two (6 and 16 kP a) Cell n umbers remained the same for 2 days and then significantly dropped at day 3 and remained constant thereafter (Figure 4 8 ). Cells grown on gels


49 with an elasticity of 32 kPa showed a similar trend for the first 4 days after which cell number began to steadily increase. The d ecrease in cell number was a result of cells lifting off of the gel, as cells were also observed attached to the bottom of the wells in which the gels were placed (not shown). However, subsequent transfer of the gels to new wells each day did not change cell loss with no apparent cell attachment to the plastic bottom of the wells. The variation in cell attachment number observed brought into question the identities of the individual cells attaching and lifting, and was therefore e xamined more closely. It was recently shown that BSC populations are het erogeneous, including subpopulations with different expression patterns of myogenic marker s in vitro (Li et al., 2011). These populations include Pax7 + / Myf5 muscle stem cells (which a re quiescent) Pax7 + / Myf5 + muscle progenitor cells (which are activatable) and Pax7 / Myf5 + myoblasts. Pax7 / Myf5 expression was analyzed after 24 hours and assessed. On each type of substrate all three subpopulations were observed (Figures 4 9 and 4 10 ). N umbers of Pax7 + / Myf5 and Pax7 / Myf5 + cells were small and did not differ significantly among substrates. Pax7 + / Myf5 + cells represented the largest subpopulation for each substrate consistent with pre vious results (Li et al., 2011). I nterestingly there we re significantly smaller numbers of these cells ( p = 0.0043) on the softest 6 kPa substrate, representing young tissue, compared to the st iffer 16 and 32 kPa substrates. This corresponded to an increased number of Pax7 / Myf5 null cells on the 6 kPa and 16 k Pa substrates compared with the 32 kPa substrate ( p = 0.013 and p = 0.040 respectively). Satellite cells become spontaneously activated in culture under normal growth conditions (Schultz et al., 1982). Proliferating cell nuclear antigen (PCNA) is a good i ndicator of satellite cell activation prior to proliferation (Johnson and Allen, 1993), and was used to determine the amount of activation on soft substrates compared to plastic. PCNA expression was monitored at


50 24 and 48 hours in BSC grown on gels with fo ur elasticities, 6, 16, 32 kPa, and on plastic ( ~10 6 kPa cited in Gilbert et a l., 2010) After 24 hours, the 6 16 and 32 kPa substrates showed significantly smaller percentages of PCNA positive cells ( p < 0.0001 p < 0.0001 and p < 0.0312 respectively ) compared with the 10 6 kPa plastic surface. The number of PCNA positive cells was also significantly different with comparison to all three gel substrates ( 6 and 16kPa, 6 and 32 kPa, and 16 and 32 kPa were significantly different with p < 0.0001, Figure 4 1 1 A). After 48 hours, t he 6 and 16 kPa substrates continued to show significantly fewer PCNA positive cells ( p < 0.0001 and p = 0.0230 respectively) compared to the 32 and 10 6 kPa s urfaces while the latter two were not statistically different Addition of hepatocyte growth factor (HGF) a potent activator of satellite cell s, increased PCNA positive cells on 6, 16 and 32 kPa substrates ( p < 0.0001, p < 0.0001, and p < 0. 0 263 respectively) after 24 hours post plating compared with cells wi thout addition of HG F (Figure 4 11 B). On the plastic surface, the number of PCNA positive cells with the addition of HGF was not significantly different from cells without HGF. Summary Experiments from the current study revealed that bovine satellite cells respond to the subs trate on which they are attached in terms of elasticity. Elasticity was measured in muscle tissue from young and adult cattle, and the values were different between muscle types as well as age, showing increased stiffness with increasing value. These value s were used to assemble polyacrylamide gels with varying elasticity. A standard curve was developed by varying the concentration of acrylamide and the resulting linear equation was used to determine acrylamide concentrations that correspond to a particular elasticity value. Polyacrylamide gels were fabricated to represent the elastic values measured for muscle tissue, and extracellular matrix proteins were tested for cell attachment. A chemical cross linker was incorporated for successful


51 C2C12 and BSC attac hment, and was proven to have no interfering effects on cell attachment and proliferation. BSC isolated from young (<5d) and developing (60d) cattle were analyzed for proliferation and activation, and cells from developing animals showed a delay in prolife ration but a similarity in the number of activated cells compared to cells from young animals. BSC isolated from young animals were cultured on substrates of soft (6 kPa), moderate (16 kPa) and stiff (32 kPa) and observed for proliferation over six days. T hese cells showed delayed proliferation and decreased cell number after three days. Cell identity was determined with Pax7 and Myf5 expression in cells on these three substrates after 24 hours, and cells on the softest substrate showed a decreased number o f progenitors ( Pax7 + / Myf5 + ) with a corresponding increase in Pax7 / Myf5 null cells. Activation was monitored by analyzing PCNA expression and showed an overall decrease in the number of activated cells with decreasing stiffness, but was reversed with the ad dition of HGF. These results show that age and elasticity have an overall effect on BSC in vitro


52 Figure 4 1 Elastic modulus E measurements for isolated semimembranosus (SM) and longissimus dorsi (LD) muscle tissues from young an d adult cattle Elastic modulus, E = stress /strain and is measured in units of pressure (kPa), where stress = force F (N) / cross sectional area, A (mm 2 length, L 0 (mm). (*Denotes P < 0.05 with respect to other measurements )


53 Figure 4 2 Graph of standardized elastic modulus, E measurements for varying concentrations of acrylamide Linear relationship represents the standard curve; r 2 =0.99.


54 A B C D E F G Figure 4 3 Representative images for C2C12 cells grown on various substrates at 24 hours (left column) and 48 hours (right column). A) Cells on gelatin coated plastic ( control) B,C) Pure ECL added to acrylamide gels prior to polymerization. D,E) P olymerize d gels coated with pure ECL. F,G) G elatin added to acrylamide gels prior to polymerization


55 A B C D Figure 4 4 Representat ive images of C2C12 cells (A,B) and bovine satellite cells (C,D ) cultured on acrylamide gels with photocrosslinked ECL. Ima ges were take n at 24 hours (left column) and 48 hours (right column ).


56 A B Figure 4 5 Effect of ECL and ECL cross linker on C2C12 and BS C attachment and proliferation. A) ECL cross linker mixture and ECL coatings with two different concentrations of ECL were compared for C2C12 attachment and prolifera tion at 24 and 48 hours. B) BSC attachment was compared for ECL and ECL cross linker coatings at 24 hours. (*Denotes P < 0.05 with respect to other measurements.)


57 A B Figure 4 6 Developing satellite cells exhibit a lag phase in proliferation compared with young cells. BSC isolated from developing (60d) and young (<5d) cattle were plated on glass plates and analyzed proliferation with EdU incorporation (A) and activation with PCNA expression (B) over 72 hours. (*Denotes P < 0.05 with respect to measurements within a given time period.


58 A B Figure 4 7 Comparison of EdU incorporation and PCNA expression in C2C12 myoblasts (A) and young (<5d) BSC (B). Hoechst dye was used for nuclei visualization. Cells were incubated with EdU for 2 hours prior to fixing and with primary PCNA overnight post fixation for immun ofluorescence detection. Box indicates cells that are positive for PCNA and EdU, triangle indicates cells that are positive for PCNA and negative for EdU, and arrowhead indicates cells that are negative for both PCNA and EdU. Cells were photographed at 10x magnification and magnified for visualization. Hoechst PCNA EdU Hoechst PCNA EdU


59 A B C D Figure 4 8 Representative g raphs (n = 1) of bovine sat ellite cells (<5d) grown on acrylamide gels of varying elastic moduli over a six day period A) Gel representing calf elastic modulu s (<5d). B) Gel repre senting adult elastic modulus C) Gel representing a stiff extreme elastic modulus. D) Graphs of the three gels combined to highlight growth trend.


60 Figure 4 9 Pax7 and Myf5 expression at 24 hours post plating in bovine satellite cells (<5d) grown on soft substrates. Three groups were observed, a Pax7 (+) Myf5 ( ) group of muscle stem cells, a Pax 7 ( ) Myf5 (+) group of myoblasts, and a Pax7 (+) Myf5 (+) group of progenitor cells. A Pax 7 ( ) Myf5 ( ) group was also observed. (*Denotes P < 0.05 with r espect to comparis ons denoted by brackets .)


61 A Figure 4 10 Cells on soft substrates exhibit three groups of myogenic expression, Pax7 only cells, Myf5 only cells, and Pax7 / Myf5 cells. BSC (<5d) were co stained for both antibodies at 24 hours post plating. R epresentative images were taken at 20x magnification. A) Highlights Pax7 only cells. B) Highlights Myf5 only cells Arrowhead indicate s Pax7 only cells, triangle indicate s Myf5 only cells, and box indicates Pax7 / Myf5 expressing cells. Merge photos repres ent all three stains overlapped. Hoechst Pax7 Myf5 Merge


62 B Figure 4 10 Continued Pax7 Hoechst Myf5 Merge


63 A B Figure 4 11 Percent of bovin e satellite cells (<5d) positive for proliferating cell nuclear a ntigen (PCNA) after immunostain ing. Positive cells represent cells that have entered the DNA synthase phase of the cell cyc le and are mitotically active. A) Cells grown on gels of varying elasticities analyzed at 24 and 48 hours post plating. ( a g Mean percentages in the same time period wit h different letters are significantly different P < 0.05 ) B) Cells grown on gels of varying elasticities treated with or without hepatocyte growth factor (HGF) analyzed at 24 hours post plating (*Denotes P < 0.05 with respect to ( ) HGF measurements ).


64 CHAPTER 5 DISCUSSION Muscle Elasticity Varies with Age and Muscle Type Elasticity, a factor contributing to the microenvironment of a cell, affects mesenc h ymal stem cell ability to commit to a particular lineage in vitro (Engler et al., 2006). The use of an elastic substrate analogous to muscle tissue has been shown to improve satellite cell regenerative ability (Boonen et al., 2009; Gilbert et al., 2010). A m uscle elasticity value of 12 kPa has previously been determined for a dult mouse muscle (Engler et al., 2004b; Gilber t et al., 2010). This value was presumed to be representative for mouse muscle tissue in general. However, it may not be the same for all muscle types or muscle from other species The use of a particular muscle contributes to postnatal m uscle development, and rodents undergo d ifferent locomotion and strain on their muscles compared to that of a larger animal or human The difference in strain on particular muscles may ultimately affect muscle elasticity, making the generalized mouse model a mis representation for muscle from larger species. In this study t wo different types of muscle tissue from Holstein cattle were investigated. The SM is a muscle of locomotion, and the LD is a muscle that supports lateral movement and posture The results presented here revealed that both muscles had elasticity values that were a) different from mouse muscle and b) different between each other. We therefore conclude that for the study of satellite cells isolated from cattle, the muscle elasticity values of mouse muscle is not applicable. The discovery of different species and muscle types exhibiting differing elasticities could be important with consideration to the animal model used as well as in the development of tissue gra fts for muscle therapies. Sate llite cell activation is affected by age, which may be a consequence of a changing microenvironment that includes fiber elasticity. Muscle elasticity ( E ), or stiffness, was therefore hypothesized to increase with age as a result of increased collagen depos it that has been shown to


65 increase with age (Robert and Labat Robert, 2000). Muscle elasticity was measured and compared between calves and adult cattle to represent young and adult tissue. Results indicate a significant increase in elasticity for adult LD tissue compared to young LD tissue, confirming that elasticity or stiffness, increases with age. Surprisingly, no significant difference was observed in young and adult SM tissue, whereas the SM in adult beef cattle is widely recognized as a muscle produ cing steaks of tougher palata bility, which could be related to muscle elasticity measurements (Senaratne et al., 2010). Muscle elasticity contributes to tenderness palatability and age is associated with decreased muscle tenderness (Smith et al., 1982). Th erefore one would expect the SM to increase in elasticity with age and to exceed that of the adult LD tissue, a mu scle producing some of the most tender steaks in beef cattle. An explanation for these unexpected results may involve the method by which mus cle elasticity was assessed Due to the size of the harvested whole muscle and limitations of the loading cell, subsections of muscle tissue were cut and used as representative samples. Although results revealed values with minimal variability, these subse ctions may not have been representative of the entire muscle elasticity as muscle is a heterogeneous tissue containing different fiber types and fiber sizes. Measurement of single fibers may be more indicative of the elasticity satellite cells are experien cing; however size and distribution of overlapping fibers limit isolation and measurement of a single fiber in its entirety. Another option for future studies could explore multiple isolated subsections or whole single fibers from one muscle type to see if these values would differ from the results in the current study. In addition, one aspect that was not considered in the current study is the differences in skeletal muscle elasticity with regards to gender Male neonatal calves and adult female cattle wer e used in the current study based on availability. However, differences in adult female and male muscle tissue are very apparent with


66 regards to fiber type, size, and chemical properties (Novk et al., 2010 Patten et al., 2008 ). Differences between neonat al male and female muscle have also been observed in mice with regard to kinase expression levels involved in cellular energy metabolism (Desai et al., 2009). An important factor to consider for future studies would be to compare gender effects on muscle e lasticity as well and how these differences might have a gender specific impact satellite cell activity. The values obtained for young and adult muscle tissue were applied to creating a substrate that can mimic these values in vitro Mouse satellite cells show increased striation formation and regenerative ability when grown on substrat es with elasticity equal to measured muscle tissue compared with cells grown on glass (Engler et al., 2004b; Gilbert et al., 2010). As muscle elasticity was shown to differ w ith age in one muscle type, and as young and adult BSC showed differing prol iferative behavior in culture, we developed a range of elastic substrates from soft (young, 6 kPa), moderate (adult, 16 kPa) to stiff (32 kPa) elasticities based on bovine muscle e lasticity measurements to observe how change in elasticity affects satellite cell behavior in vitro. Polyacrylamide gels were fabricated using the standard curve developed to determine elasticities corresponding to specific acrylamide concentrations. Polya crylamide was specifically chosen because its elasticity is easily tunable, its formula does not contain any interfering substances, and its porous nature resembles physiological conditions of the network of proteins that make up the extracellular matrix ( Pelham and Wang, 1997). It also does not swell or change shape when hydrated, therefore maintaining its surface area while cells are attached. It was established in the current study that BSC require covalently attached proteins in order to successfully at tach to the polyacrylamide substrate, as other methods to polymerize or coat proteins with the gels resulted in cell failure to attach (Figure 4 3) Extracellular matrix proteins


67 were covalently attached to the gels using a chemical crosslinker that was sh own to have no interfering effects on satellite cell attachment or proliferation Cell activity could therefore only be in response to elastic prop erties and not outlying factors such as interference from the use of a chemical cross linker or polyacrylamide Cell attachment and pro liferation were monitored on all three substrate elasticities and showed a common trend of delayed growth followed by a significant decrease in cell number after two days (Figure 4 8) Representative graphs were shown for each subs trate because the percentage of cells that attached to the substrates ( out of the total cells attached to glass ) was different for each trial. However, the same trend for all trials was observed, which, considering that BSC are a heter ogeneous population ( Li et al., 2011), could mean that cells are being separated by identity when attaching to the various substrates. This was explored later by staining for Pax7 and Myf5 antibodies. The lack in proliferation could lie in apical basal asymmetric cell division Activated satellite cells isolated from rodents have been shown to divide in this manner producing a proliferating cell and a cell that returns to quiescence (Kuang et al., 2007; Shinin et al., 2006; Venters et al., 2005). This type of division could be the cause of the observed delay in proliferation after two days as the proliferating cell may lift off of the gel while the cell that returns to quiescence remains. Evidence of apical basal division was shown in BSC cultures (Li et al., 2011), t he manner o f which could also be a cause for the dividing cell to lift off of the substrate with its daughter cell, accounting for the resulting decrease in cell number at day three This decrease was also suspected to be linked to the identity of the cells, which le d to the investigation of Pax7 and Myf5 expression, discussed later Finally while all substrates experienced a decrease in cell number, only the stiffest 32 kPa substrate experienced an increase


68 in cell number following the decrease Boonen et al. (2009) established that mouse satellite cells show increased attachment and growth with increasing stiffness. Stiffer substrates may therefore be a signal for cell proliferation, whereas softer substrates may prevent the cell from proliferating. This result appe ars contrary to the effects of aging on satellite cell activity, and was further explored using markers for cell identity and activation. Proliferation I s Delayed in Adult Satellite Cells in vitro Quiescent adult and aged satellite cells undergo a lag phas e prior to cell proliferation. (Johnson and Allen, 1993; Li et al., 2011; McGeachie et al., 1995; Tatsumi et al., 1998). It has been previously suggested that the lag phase is a result of an increased amount of satellite cells in adult tissue that have ret urned to quiescence after being activated and cycling through the cell cycle (Tatsumi et al., 1998). PCNA is used to detect early activation, and satellite cells isolated from 9 12 month old rats showed a 24 hour delay in increased PCNA expression compared to cells from 3 4 week old rats with addition of a stimulus (Johnson and Allen, 1993). Upon induced injury, mu s cle satellite cells in both young and old mice became activated after 24 hours, but a lag in peak proliferation was exhibited in satellite cells of old mice (McGeachie and Grounds, 1995). Adult BSC also exhibit a lag in peak proliferative activity (Li et al., 2011), therefore early detection of activation was explored in developing BSC. Activation status using PCNA expression was compared to proli feration analyzed via EdU incorporation for BSC isolated from young (<5d) and developing (60d) cattle on glass plates D eveloping BSC began to incorporate Ed U 24 hours later than young BSC, evidence of delayed proliferation. In terestingly, the percentage o f PCNA positive cells was about equal for both developing and young cells, representing roughly 85% of the total cell population, and did not change significantly for either age group over a 72 hour period. This would suggest that both age groups contained a majority of activated cells 24 hours post plating prior to proliferation, and


69 that a 24 hour lag experienced by developing cells was not due to a lower percentage of activated cells. The lag experienced in developing BSC is therefore a result of delayed proliferation and not extended quiescence as shown in rats. This lag appears to be due to intrinsic factors in the cells as culture conditions remained the same for both age groups Interestingly, these intrinsic signals seem to change from the young cal f to the developing calf, although both animals are undergoing muscle growth. It may be that something intrinsically slows the proliferation of activated cells somewhere between birth and the developing calf, which may be a technique to slow muscle growth as the animal approaches mature size. These intrinsic factors can be overridden in vitro with the addition of HGF (Allen et al., 1995; Tatsumi et al., 1998) or exposure to serum from young animals (Conboy et al., 2005), suggesting an interplay between sign als from the environment and innate properties of the cell. It may be that satellite cells in developing muscle have a larger subpopulation of slow dividing cells resulting from asymmetric division during satellite cell self renewal (Hashimoto et al., 2004 ; Li et al., 2011; Schultz, 1996). Satellite cell populations contain subpopulations of satellite cells that asymmetrically divide and return to quiescence to replenish the satellite cell pool. As the animal ages, this number of self renewing satellite cel ls may increase while the number of regenerative cells decreases leading to a concentrated population of cells destined to replenish the satellite cell pool and thus divide and proliferate slowly. Further investigation of developing and adult satellite ce ll behavior is required to understand the reasoning behind this slow dividing group. Satellite Cells Respond to Elasticity in vitro Satellite cell populations are heterogeneous and consist of subpopulations that represent stem, progenitor, and myoblast cel ls. BSC grown on polyacrylamide substrates of varying elasticities exhibited a decrease in cell number after three days in culture. This was speculated to


70 be due to asymmetrical division, but another contributing factor could be that only a certain subpopu lation of BSC attaches and another subpopulation lifts off. Cells lifting off of the gels were suspected to be a part of a subpopulation of activated cells that are not supported to proliferate on softer substrates. There was no evidence of cells attaching to the well after daily transfer of each gel to a new well (data not shown), which made it difficult to characterize the cells that had lifted. Therefore, cells were characterized by Pax7 and Myf5 expression 24 hours post plating to determine the status o f initially attached cells (Figure 4 9) Results revealed that Pax7 + / Myf5 + progenitor cells made up the largest subpopulation of attached cells on all three substrate elascticities, similar to that observed in satellite cells on a plastic surface (data not shown). Smaller subgroups of Pax7 only and Myf5 only cells were also observed on all three substrates with no signific ant differences. Another group of Pax7/Myf5 null cells were evident and significantly higher in cells on the softest (6 kPa) and moderate ly soft (1 6 kPa ) substrate s compared with the stiff substrate (32 kPa) This could be indicative of Pax3 expressing cells, the homologue to Pax7 that is expressed by satellite cells during embryogenesis and expressed in addition to Pax7 by some satellite c ells postnatally. Pax3 however, is difficult to monitor du e to lack of available antibody. The Pax7/Myf5 null cells could also be a myogenic derived side population of stem cells previously reported in mouse cultures (Asakura et al., 2002; Benchaouir et a l., 2004; Kallestad and McLoon, 2010). Cells on the softest substrate did, however, show a significantly smaller population of progenitor cells corresponding to an increased population of Pax7/Myf5 null cells which could be indicative of the softest subst rate being the more preferred surface for the subpopulation of muscle stem cells to attach Both quiescent and activated satellite cells express Myf5 (Beauchamp et al., 2000), so it is unknown whether a progenitor cell is quiescent or activated. However, a s the softer substrates seem to have a negative effect on cell


71 proliferation (Figure 4 8) it may be possible that the quiescent progenitor cells attach while the activated cells do not on the softest substrate. The presence of Myf5 only myoblasts on the s oftest substrate would argue against this, however progenitor cells appear to make up the largest subpopulation of isolated satellite cells and brings to question why less attach to the softest substrate. The analysis of PCNA expression therefore became im portant in determining if softer substrates select for quiescent cells by having fewer PCNA expressing cells. The largest Pax7 + / Myf5 + subgroup of cells found in the satellite cell pool represents progenitor cells that have committed to a myogenic lineage, but does not indicate quiescence or activation. The first signs of satellite cell activation can be monitored using the proliferating cell nuclear antigen (PCNA) antibody (Johnson and Allen, 1993). BSC activation was therefore monitored over two days post plating on all three substrates and compared to BSC on plastic surface. After 24 hours post plating all three gel substrates exhibited significantly smaller percentages of PCNA incorporating cells than BSC on plastic as well as significantly differing from each other. A gradual increase in PCNA incorporating cells was observed with increased stiffness similar to results from Boonen et al. (2009). HGF is a potent mitogen that has been shown to activate satellite cells both in vitro and in vivo (Allen et al., 1995; Tatsumi et al., 1998). Addition of HGF to BSC on all substrates resulted in a significant increase in PCNA positive cells compared to cells without HGF, which showed that HGF is able to reverse the effects of the softer substrates on satellite cell activation and that the elasticity is in fact the cause for the observed difference in activated cells Furthermore, cells on the plastic surface with HGF were not significantly different from cells without HGF, inferring that cells on a hard surface alrea dy contain a majority of activated cells after 24 hours post plating. This would suggest that substrate


72 elasticity has an effect on cell activation, possibly causing progenitor cells to return to a quiescent state on softer substrates Con c lusions The effe cts of substrate elasticity on BSC behavior in vitro were significant, although not as expected. BSC appear to increase in activation and proliferation with increasing stiffness. It is known, however, that aging corresponds to reduced satellite cell activi ty, with a stiffer elasticity representing an aged muscle. Exposing young mouse satellite cells to an aged microenvironment resulted in increased collagen deposits and reduced satellite cell activity (Brack et al., 2007). Therefore it was expected that yo ung BSC exposed to an elasticity characteristic of aged muscle would also result in reduced activity. The results showed the opposite to be true, with decreased proliferation (Figure 4 8), decreased progenitors (Figure 4 9), and decreased PCNA expression ( Figure 4 11) indicating that change in elasticity may not be a cause for age related effects observed in satellite cells. Furthermore, results comparing young and developing BSC under normal growth conditions on plastic resulted in a lag in proliferation from already activated cells in the developing BSC, indicating that possible intrinsic factors that may be associated with age delay the growth of these cells even on the most optimal surface. Nonetheless it is worth noting the significant decrease in acti vated cells with decreasing stiffness (Figure 4 11) It is possible that decreasing elasticity may be a negative signal for satellite cell activation in vitro causing cells to return to a quiescent state. Further investigation is required to verify that t he cells are in fact quiescent, and in doing so softer substrates may be an important technique in laboratory methods to control satellite cell activity and allow for improved investigation into satellite cell behavior.


73 APPENDIX PROTOCOLS USED Bovine Sa tellite Cell Isolation 1. Prior to isolation, all utensils used that com e in to contact with tissue should be covered and autoclaved for sterilizati on. The following utensils are used and autoclaved for the isolation: surgical pan, 250mL wide mouth bottles, m eat grinder accessories, surgical utensils, and two knives. Prepare f our liters of 1x PBS (pH 7.4), divide int o 500mL bottles and autoclave for sterilization. PBS should be kept warm at 37C prior to use. Prepare b ovine satellit e cell growth media with Low G l e Media (GIBCO ; cat. no. 11885 084 ), 10% horse serum ( GIBCO; cat. no. 16050), 1% Pen Strep (GIBCO; cat. no. 15070 063), and 0.2% gentamicin (GIBCO; cat. no. 15710 064 ). Growth media should be kept warm at 37C prior to use. 2. Harvest muscle tissue from >2 week old Holstein bull calves using a sterile knife and place tissue in a sterile surgical pan for transfer. Place tissue in a lamin ar flow culture hood and trim connective tissue, fat, and hair using a new sterile knife and surgical utensils. All outer tissue layer exposed to a non sterile environment should be removed. 3. Place trimmed tissue in a meat grinder with a larg e plate attachment and collect in a sterile dish. Load ground tissue into 250mL wide m outh bottles and bala nce by weight (approximately 40 60g tissue per bottle). 4. Prepare protease (Sigma Aldrich) at 1.25mg/g tissue and dissolve in warm PBS at a 1:1 weight to volume ratio. Gently mix t issue with protease mixture by gently swirling bottles. Incubate b ot tles at 37 C for 45 minutes, swirling every 10 minutes. 5. Fill bottles with 60mL of PBS, gently swirl, and centrifuge at 1500xg for 4 minutes. Discard s upernatant and repeat two more times.


74 6. Fill bottles with 50mL of PBS, shake vigorously, and centrif uge at 500xg for 1 0 minutes. Carefully collect s up ernatant in a clean 250mL bottle, making sure not to collect any floating fat or connective tissue and incubate at 37C. Repeat three more times for a total of four supernatant collections 7. Balance all collected supernatant between multiple clean 250mL bottles and centrifuge at 1500xg for 10 minute s. During the centrifugation, prepare freezing med ia with 10% DMSO (Sigma; cat. no. D8418 ) in growth media at a volume that allows for 4g of tissue per mL of freezing media. Place freezing media on ice until needed. After centrifugation, discard res ulting supernatant and add 10mL of PBS to one bottle Re suspend the pellet in the PBS and add this mixture to each subsequent pellet, re suspending all pellets in a total 10mL of PBS. 8. T ransfer to one or two 50mL conical tube s and filter twice through a 70m cell strainer (Fisher; cat. no 22 363 548 ) and then twice through a 40m cell strainer (Fisher; cat. no 22 363 547 ) 9. Centrifuge the resulting filtrate at 1500xg for 5 minutes and di scard resul ting supernatant. 10. Re suspend r esulting pellet in th e freezing media and distribute into freezing tubes (Fisher; cat. no 12 565 167N ) at 1mL per tube (4g tissue per tube). Place t u bes in 80C for 12 18 hours and then place in liquid nitrogen f or at least 24 hours before plating. ECL Photocrosslinking on Polyacrylamide Gels 1. All steps involving ECL and cell culture take place under sterile conditions in a laminar flow culture hood. Prior to beginning the crosslinking process, a buffe r exchange i s completed for Enta ctin Collagen IV Laminin (ECL) Cell Attachment Matrix ( Millipore ;


75 cat. no. 08 110 ) to r emove phenol red that interferes with the photocrosslinking process later on. a. A Zeba TM Desalt Spin Column (Thermo Scientific ; cat. no. 89891) is used for the buffer exchange Place column in a 15mL conical tube and remove storage buffer by centrifuging a column at 1000xg for 2 minutes. b. Discard 15mL tube and place column in a new, sterile 15mL tube. Run sterile 1x PBS through the column three times via centrifu gation at 1000xg for 2 minutes. c. Discard 15mL tube and place column in a new, sterile 15mL tube. Add ECL to the column and centrifuge at 1000xg for 2 minutes to obtain pure ECL with phenol red removed. 2. Bring t he photocrosslinker, Sulf o NHS Diazirine (Sulfo SDA; Thermo Scientific; cat. no. 26173) to room temperature and p rotect from light. Prepare a 10mM concentration of Sulfo S DA by dissolving 2mg of Sulfo SDA in 611l of sterile PBS. Add solution immediately to the pure ECL at a 1:1 vo lume to volum e ratio and incubate in the dark for 30 minutes at room temperature 3. A dd sterile quenching buffer to the solution for a final concentration of 100mM incubate for 5 minutes at room temperature 4. Place a new Zeba TM Desalt Spi n Column in a 15mL tube and remove storage buffer by spinning at 1000xg for 2 minutes. Discard tube, place column in a new, sterile 15mL tube and slowly add ECL cross linker (ECL XL) mixture to the column. Centrifuge column at 1000xg for 2 minutes to remove the quenching buffer.


76 5. Test t he resulting ECL XL for protein concentration by performing a protein assay using the Micro BCA Protein Assay Kit (Thermo Scientific; cat. no. 23235) and a 1:10 dilution of the ECL XL solution. 6. During the 2 hour incubation time required for the Micro BCA protein assay, prepare polyac rylamide solutions with 5, 7, or 10% acrylamide ( Bio Rad; cat. no. 101 0140 ), 0.2% bis acrylamide (Bio Rad; cat. no. 101 0142) 1% 1M HEPES (pH 8.0), and water. (HEPES buffer should be prepared prior and stored in one time use aliquots at 20C, covered from light.) Polymerize solutions by adding 10% ammonium persulfate ( APS; Bio Rad; cat no. 161 0700) and TEMED ( Bio Rad; cat. no. 161 0801) at a 1:10 ratio of APS:TEMED Pour solution between two glass plates separated by 0.75mm spacers to ensure uniform thickness. Allow to poly merize for 2 hours to ensure maximal polymerization. 7. Once protein concentration is determined via the protein assay, prepare a 2% concentration of ECL XL in sterile PBS (20g ECL XL per 1mL PBS). Coat control wells in a 48 well plate with 2% ECL XL and inc ubate at 37C for 1 hour. 8. Cut acrylamide gels disks of diameter 8mm and place on 4 glass microslides. Place microslides inside of a 48 well plate cover. Place 50l of 2% ECL XL o n top of one gel and sandwich a second gel on top. Separate the two gels and place adjacent to one another in the small, leftover puddle of ECL XL to stay hydrated, the sides of each gel that were in contact with the ECL XL facing up. 9. Once gels are placed maximally on microslides (about 6 gels can fit per slide), place UV light to rest on top of the plate cover leaving ~1cm distance between the gel and the light


77 source Expose the gel disks to 15 minutes of UV light to allow for the ECL to photocrosslink to the gel via the attached cross linker 10. At this point, gels may be slightly dried out. Carefully place gels in one by one in the wells of a 48 well plate using a spatula and/or tweezers to ensure that the photocrosslinked side is facing up. Gently add ~500 l of PBS to wells ensuring that the vigorously). 11. When ready to plate cells, remove PBS from wells by gently sucking off with a P1000 micropipette in the bottom corner of the well, ensuring that the gels are not disturbed. 12. Plate cells at a density of 10,000 cells per gel in warmed growth media (Low glucose DMEM, 10% horse serum, 1% Pen Strep, and 0.2% gentamicin) and allow to attach overnight. 13. When washing gels after att achment or performing any other protocol, use a P1000 micropipette to gently remove liquid, so as not to disturb gels. Immunofluorescence Staining 1. Remove media from wells with gentle pipetting Wash wells to remove any remaining media with 1x PBS 2. Fix cell s with 90% methanol in PBS (Fisher; cat. no. 67 56 1) for 10 minutes at 4C or on ice. 3. Remove methanol promptly and wash wells once with PBS. If staining on polyacrylamide gels, gels will shrivel from methanol. Handle carefully when washing and allow gels to rehydrate in PBS until normal shape is achieved.


78 4. Prepare fresh blocking solution with 10% horse serum (GIBCO; cat. no. 16050) and 0.1% Triton X 100 () in PBS. Add enough blocking solution to wells to cover cells and block for 1 hour at room temperature 5. Prepare primary antibody by diluting goat polyclonal IgG PCNA (C 20) (Santa Cruz; cat. no. sc 9857) at a 1:100 dilution in 10% blocking solution (prepared in step 4). Add enough primary antibody solution to cover cells. Incubate overnight at 4C. Primary antibody should be used sparingly. Calculate the minimum volume required to cover the cells in each well and multiply this value by the total number of wells This will be the minimum volume needed to dilute antibody. 6. Remove primary antibody and wash with PBS, allowing PBS to incubate for 5 minutes at room temperature. Repeat two more times for a total of three 5 minute washes. 7. Prepare secondary antibody solution by diluting anti goat 488 (Invitrogen; cat. no. A11055) at a 1:150 dilution and Hoechst 33342 ( Invitrogen; cat. no. H3570) at a 1:1000 dilution in 10% blocking solution. This should be done in the dark. Secondary antibody should be used sparingly. Follow directions from 5a to determine minimum volume needed. 8. Add enough secondary antibody solution to cover cells and incubate for 1 hour at room temperature in the dark. 9. Remove secondary antibody solution and wash once with PBS. Analyze fluorescence using UV light for the Hoechst stain and blue light for the secondary antibody. Elasticity Measurements U sing the Instron for Gels and Tissue 1. Parameters for the Instron Bluehill program should be under a Tension Test Method using Extension Control, and set at a speed of 5mm/minute. Output should be set up to


79 export as an Excel worksheet that should be saved t o a known folder all raw data is needed for data analysis. Test parameters can be saved as a test to reopen for future experiments. a. Instron setup should incl ude two clamps that will connect to the Instron loading cell and are used for the stretch test. b. D etermine the minimum and maximum amount of stretch by adjusting the setting s on the side of the machine. Th ese do not need to be a specific setting they simply need to allow enough room to load and stretch the sample. These are for safety purposes the p rogram will immediately stop if one of these limits is reached. 2. If testing muscle tissue, fill several 250mL bottles halfway with either Krebs Ringer no serum added (GIBCO; cat. no. 11885 084). Incubate bottles in 37C until needed. a. Obtain harvested muscle tiss ue and immediately cut into large sections, making sure to cut parallel to the muscle fibers, and place in warm buffer/media. Keep incubated at 37C until ready to use b. Cut tissue into thin strips along the muscle fibers. Suggested dimensions are a width and thickness of about 10mm and a minimum length along the fibers of 20mm. Measurements may differ from those suggested, however it is important to make sure there is en ough length to load the sample between the two clamps and a thin enough strip to fit between the clamp holders. All dimensions should be recorded f or every single sample measured they will be used to analyze the data.


80 3. If testing polyacrylamide gels, gels should be made as normal with working acrylamide/bis acrylamide percentages, allowing to polymerize between two glass plates with a thickness of 0.75mm for 2 hours. a. sided T shape on a piece of saran wrap with a permanent marker Suggested dimensions for this shape are two 2cm x 5cm rectangles separated by a 4cm x 1cm rectangle, the two rectangles 4cm apart and the 5cm sides facing each other. b. Place the stencil over the gel and cut out multip le dog bone shape s using a single edge razor blade. The 2cm x 5cm rectangles should be connected by the 4cm x 1cm rectangle Cover gels with wet paper towels until needed. 4. Once samples are prepared and dimensions recorded, load the sample in the clamps, be a. Muscle samples should be loaded so that ~1 2cm is clamped. After sample is loaded, the distance of the sample leng th that is unclamped should be recorded this will be the original length. b. Gels should be loaded so that the 2cm x 5cm rectangles are completely clamped, and the full 4cm x 1cm rectangle is unclamped. The recorded dimensions for gels should be 4cm length, 1cm width, and 0.75mm thickness. 5. until the load is ~0 .00 once the load has stabilized. 6. Monitor the sample and its output as it is stretched.


81 a. If measuring muscl e samples, allow the test to c ontinue until there is a small break or drop on the curve, or until the slope has changed drastically. There will most likely not be a definitive breaking point for the sample. b. If measuring polyacrylamide gels, allow the gel to be stretched until it breaks 7. simply loading another sample and running the test so that the outputs overlay each other. Samples should never be run twice; after a sample is stretched, it will not go back to its exact original dimensions. 8. individual Excel worksheets for each sample and grouped by test. The program will give the option to run more samples u nder the same test parameters click yes if continuing to measure more samples, or no if finished. 9. To analyze data, open the Excel worksheets with raw data. Title two columns next to the he original length, dF is the change in force, and A is the cross sectional area. Create equations within these columns that divides each distance data point by the original length of the particular cross sectional area (thickness x width). Graph these new columns using a scatter plot with unconnected data points. Determine the linear equation for the curve using the linear regression option in Excel. The slope of this equation multiplied by 10 3 give When analyzing the slope for the muscle tissue samples, sometimes the curve changes slope over ranges of distance. When this happens, try to determine the range of distance of the initial slope before it begins to change and r e graph the data points ending at this limit.


82 value.


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94 BIOGRAPHICAL SKE TCH Ma rni Rose Lapin was born in Evanston, Illinois and grew up in Deerfield, Illinois, a suburb on the north shore of Chicago. Growing up she was involved in many extracurricular activities including music, dance, softball, and swimming as well as forming an interest in math and science through honors and AP courses. In the fall of 2005, Marni left Chicago to explore Washington, D.C. at The George Washington University. There, she focused her academic career in chemistry and continued to fulfill her intere st in the arts and athletics She graduated with a Bachelor of Science in Chemistry in May of 2009, after which moved to Gainesville, Florida to pursue a Master of Science in Animal Sciences at the University of Florida. She worked as a graduate assistant for two years under Dr. Sally Johnson in muscle biology. In the fall of 2011, Marni will be returning to her home state to attend the University of Illinoi s in pursuit of a Doctorate of Veterinary Medicine in 2015.