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Differentiation of Recombinant Myoblasts in Alginate Microcapsules

A cost effective approach to the delivery of therapeutic gene products in vivo is to immunoprotect genetically-engineered, universal, non-autologous cells in biocompatible microcapsules before implantation. Myoblasts may be an ideal cell type for encapsulation due to their inherent ability to differentiate into myotubes, thereby eliminating the problem of cell overgrowth within the capsular space. To evaluate the interaction between the differentiation program and the secretory activity of the myoblasts within the microcapsule environment, we transfected C2C12 myoblasts to express human growth hormone and followed their expression of muscle differentiation markers, such as creatine phosphate kinase (CPK) protein and up-regulation of muscle-specific genes (ie. myosin light chains 2 & 1/3, Troponin I slow, Troponin T, myogenin and MyoD1). As the transfected myoblasts were induced to differentiate for up to two weeks, their myogenic index (i.e. the percentage of multinucleate myoblasts) increased from 0 to ~50%. Concomitantly, up-regulation of differentiation marker RNA levels, and as much as a 23-fold increase in CPK activity, were observed. After encapsulation and the induction of differentiation, the myoblasts showed a lag phase of ~3 days before an increase in CPK was observed, although the level of CPK activity increased by as much as 63-fold. The myogenic index of the encapsulated cells remained at zero. The rate of human growth hormone secretion was relatively constant throughout the two-week differentiation period, at an average of 7.78 x 10^-2 ng hGH per hour per (mu)g protein, however, human growth hormone secretion was slightly decreased by about twofold during the differentiation of encapsulated myoblasts. In conclusion, the differentiation of myoblasts into myotubes is retarded after encapsulation while the secretion of a recombinant product is slightly reduced. Further studies are necessary to elucidate the cause of this atypical differentiation pattern such that the proliferation and differentiation of the encapsulated myoblasts may be optimized to provide a stable vehicle for gene delivery. / Thesis / Master of Science (MS)

Identiferoai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23221
Date06 1900
CreatorsBowie, Kelly
ContributorsChang, P. L., Biology
Source SetsMcMaster University
LanguageEnglish
Detected LanguageEnglish
TypeThesis

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