Effects of three-dimensional culture conditions on skeletal muscle myoblasts

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dc.contributor.advisorMarguerite Sognieren_US
dc.contributor.committeeMemberRobert Leonarden_US
dc.contributor.committeeMemberMary Moslenen_US
dc.contributor.committeeMemberJeff Rabeken_US
dc.contributor.committeeMemberBrian Hashemien_US
dc.creatorMichele Lynn Marquetteen_US
dc.date.accessioned2011-12-20T16:04:34Z
dc.date.accessioned2014-02-19T22:05:03Z
dc.date.available2009-06-09en_US
dc.date.available2011-12-20T16:04:34Z
dc.date.available2014-02-19T22:05:03Z
dc.date.created2007-04-02en_US
dc.date.issued2007-03-30en_US
dc.description.abstractEffects of Three-dimensional Culture Conditions on Skeletal Muscle Myoblasts\r\n\r\nPublication No._____________\r\n\r\n\r\nMichele Lynn Marquette, Ph.D.\r\nThe University of Texas Medical Branch, 2007\r\n\r\nSupervisor: Marguerite A. Sognier\r\n\r\nThe objectives of this research were to: 1) develop a three-dimensional in vitro model; and 2) subsequently, utilize this model to investigate mechanisms of myoblast adhesion, fusion, and differentiation. C2C12 cells were examined as pre-aggregated single cells and multicellular aggregates in the Rotary Cell Culture System (RCCS). At the time intervals tested, RCCS cultured cells maintained viability and did not exhibit increased apoptosis markers such as Caspase 3 (activated) and phosphatydylserine. In contrast, increases in cell death and apoptotic markers were noted in suspension culture (SC) control cells. RCCS cultured cells fused to form multinucleated syncitia and expressed sarcomeric myosin heavy chain (MHC) in significantly higher levels than SC aggregates after cultivation for 3 and 6 days. This occurred in the presence of mitogens without exogenous matrix or support structures. Myoblast fusion was inhibited by exposure to soluble anti-Neural-cadherin antibody, but this treatment increased MHC levels assessed using immunohistochemistry.\r\nDuring early RCCS culture, myoblasts exhibited numerous cytoplasmic protrusions (podia). Microscopic examination of cells cultured in RCCS and SC revealed significantly more and slightly longer podia in the RCCS at 3, 6, and 9-hours. Podia were F-actin dependent as shown by exposure to an F-actin depolymerizing agent, Latrunculin A. Podia were inhibited, but recovered upon Latrunculin A removal. \r\nPodia were postulated to play a role in cell-cell adhesion in conjunction with Neural Cadherin (N-cadherin), an adhesion molecule important in myoblast differentiation. To determine if N-cadherin was critical to cell-cell adhesion, RCCS cultured cells were examined for the presence of N-cadherin at both the podia and membrane using confocal microscopy. N-cadherin levels decreased at the podia and membrane of RCCS cultured cells but not in SC cells at 3, 6, and 9-hours. \r\nIn summary, these results revealed: 1) podia formation is F-actin dependent but N-cadherin independent; 2) N-cadherin is critical for myoblast maturation; 3) synctia formation and differentiation can occur with mitogens present, without exogenous substrates in the RCCS; 4) this novel myoblast model test system is suitable for defining muscle development/regeneration processes, identification of molecular targets for development of therapies, and potential regenerative medicine applications.\r\n \r\nen_US
dc.format.mediumelectronicen_US
dc.identifier.otheretd-04022007-090813en_US
dc.identifier.urihttp://hdl.handle.net/2152.3/86
dc.language.isoengen_US
dc.rightsCopyright © is held by the author. Presentation of this material on the TDL web site by The University of Texas Medical Branch at Galveston was made possible under a limited license grant from the author who has retained all copyrights in the works.en_US
dc.subjectRCCSen_US
dc.subjectpodiaen_US
dc.subjectN-cadherinen_US
dc.subjectfusionen_US
dc.subjectF-actinen_US
dc.subjectdifferentiationen_US
dc.titleEffects of three-dimensional culture conditions on skeletal muscle myoblastsen_US
dc.type.genredissertationen_US
dc.type.materialtexten_US
thesis.degree.departmentCell Biologyen_US
thesis.degree.grantorThe University of Texas Medical Branchen_US
thesis.degree.levelDoctoralen_US
thesis.degree.namePhDen_US

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