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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)

No description available.

No description available.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: myoblasts; dexamethasone; infarction; cryoinjury; desmin; myosin heavy chain; differentiation. Includes bibliographical references (p. 54-59).

Zugl.: Giessen, Univ., Diss., 2009

An alternative gene therapy method, non-autologous somatic-gene therapy, is the use of a genetically modified universal cultured cell line that can be implanted into different allogeneic recipients. When used as recombinant cells in microcapsules, myoblasts possess several advantages over other cell types, namely their ability to terminally differentiate thus preventing overcrowding within the capsular space. However, encapsulated myoblasts demonstrate decreased proliferation and myogenic differentiation when compared to unencapsulated myoblasts due to the unnatural capsule environment. This study aims to improve the microcapsule environment by incorporating basic fibroblast growth factor (bFGF) and insulin-like growth factor-11 (IGF-11) and the extracellular matrix materials, collagen, laminin-1 and merosin (laminin-2) within the microcapsules in an attempt to mimic the natural surrounding required for myoblast growth and differentiation. While bFGF lead to significant increases in encapsulated myoblast proliferation, it did not appear to be an ideal choice for optimizing the microcapsule environment due to its inhibitory effect on differentiation and the relative cost in therapeutic delivery of proteins. Both merosin and the combination of laminin and merosin together provide a better alternative for increasing myoblast growth and survival within microcapsules since they have no apparent inhibitory effect on myogenic differentiation, and produce similar proliferative results seen when using bFGF. In terms of differentiation, the addition of IGF-11 to the microcapsules or the use of a myoblast cell line overexpressing IGF-11, aid in increasing the myogenic differentiation of encapsulated myoblasts, however, differentiation levels still do not approach those seen in unencapsulated myoblasts. The positive results obtained with the growth factors and matrix materials employed in this study are important steps towards the optimization of microcapsules by improving both the proliferation and differentiation of encapsulated myoblasts. However, more study is needed to elucidate possible solutions to the continued problem of decreased differentiation of myoblasts within APA microcapsules in order to achieve myogenic differentiation that is comparable to what is seen in unencapsulated myoblasts. / Thesis / Master of Science (MS)

Cancer cachexia is a wasting syndrome causing involuntary weight loss and muscle atrophy. PARP1 is a nicotinamide dinucleotide-dependent enzyme that modifies target proteins by PARylation. The reversal process, dePARylation, is mediated by the PARG enzyme. PARP1 inhibitors are potent cancer agents, while PARG inhibitors are in clinical trials for similar cancers. Here we examine the role of PARylation on muscle homeostasis in cancer cachexia. We employed mouse models with inducible muscle specific knockouts of Parp1 (Parp1-IMKO) or Parg (Parg-IMKO) to investigate their implications on skeletal muscle in a cancer cachexia model. We assessed muscle loss, grip strength, and gene expression. Results show that Parp1- IMKO mice had increased muscle wasting, while Parg-IMKO had degradation rates similar to wild-type mice during cancer cachexia. This suggests reduced PARylation might worsen cancer cachexia, while an increase does not. This supports PARG inhibitor development as anticancer alternatives. Our study highlights challenges with PARP1 inhibitors and the need to study PARylation and dePARylation in muscle health during cancer cachexia, impacting clinical strategies using PARP1 or PARG inhibitors.

Doctor of Philosophy / Department of Animal Sciences and Industry / John M. Gonzalez / This series of studies focuses on the impact of intra-litter variation on fetal myogenesis, and the ability of porcine progenitor cells to respond to growth promoting stimuli. In study 1, the smallest (SM), median (ME), and largest (LG) male fetuses from each litter were selected for muscle morphometric analysis from gilts at d-60 ± 2 and 95 ± 2 of gestation. On d-60 and 95 of gestation LG fetuses had greater whole muscle cross-sectional area (CSA) than ME and SM fetuses, and ME fetuses had greater whole muscle CSA than SM fetuses. Indicating that SM and ME fetuses are on a delayed trajectory for myogenesis compared to LG fetuses. At d-60 the advanced trajectory of LG compared to ME fetuses was due to increased development of secondary muscle fibers; whereas, the advanced myogenic development of LG and ME fetuses compared to SM fetuses was due to the presence of fewer primary and secondary muscle fibers. At d-95 of gestation the advanced myogenic development of LG and ME was due to increased hypertrophy of secondary muscle fibers. For study 2, porcine fetal myoblasts (PFM) were isolated from SM, ME, and LG fetuses from d-60 ± 2 of gestation fetuses and for study 3, porcine satellite cells (PSC) were isolated from the piglet nearest the average body weight of the litter. Both myogenic cell types were utilized to evaluate effects of porcine plasma on proliferation, differentiation, and indications of protein synthesis. For the proliferation assay, cells were exposed to one of three treatments: high serum which consisted high-glucose Dulbecco's Modified Eagle Medium supplemented with 10% (vol/vol) fetal bovine serum, 2% (vol/vol) porcine serum, 100 U penicillan/mL, 100 µg of strepmycin/mL, and 20 µg of gentamicin/mL (HS), low serum which consisted of HS without 10% FBS (LS), and LS supplemented with 10% (wt/vol) porcine plasma (PP). Treatments for the differentiation and protein synthesis assays consisted of either HS or LS media that either contained porcine plasma at 10% (wt/vol; PPP) or 0% (wt/vol; PPN). The HS-PFM had a greater proliferation rate compared to the LS and PP-PFM, and PP-PFM had a greater proliferation rate compared to LS-PFM. The LG fetuses’ PFM had a reduced proliferation rate compared to SM and ME fetuses’ PFM, which were similar. The PPP-PFM had a decreased myotube diameter compared to PPN-PFM. Small fetuses’ PFM had a greater myotube diameter compared to ME and LG fetuses’ PFM, and ME fetuses’ PFM had a greater myotube diameter compared to LG fetuses’ PFM. The proliferation rate of PP-PSC was decreased compared to the HS- and LS-PSC, and HS-PSC had a greater proliferation rate compared to LS-PSC. The PPP-PSC had greater differentiation capacity and myotube diameter than PPN-PSC. In conjunction these results indicate divergent myogenic development among different fetal sizes within a litter and suggest that porcine plasma supplementation stimulates myogenic progenitor cell activity in an age specific manner.

Background: Large-scale expansion of myogenic progenitors is necessary to support the development of high-throughput cellular assays in vitro and to advance genetic engineering approaches necessary to develop cellular therapies for rare muscle diseases. However, optimization has not been performed in order to maintain the differentiation capacity of myogenic cells undergoing long-term cell culture. Multiple extracellular matrices have been utilized for myogenic cell studies, but it remains unclear how different matrices influence long-term myogenic activity in culture. To address this challenge, we have evaluated multiple extracellular matrices in myogenic studies over long-term expansion. Methods: We evaluated the consequence of propagating mouse and human myogenic stem cell progenitors on various extracellular matrices to determine if they could enhance long-term myogenic potential. For the first time reported, we comprehensively examine the effect of physiologically relevant laminins, laminin 211 and laminin 521, compared to traditionally utilized ECMs (e.g., laminin 111, gelatin, and Matrigel) to assess their capacity to preserve myogenic differentiation potential. Results: Laminin 521 supported increased proliferation in early phases of expansion and was the only substrate facilitating high-level fusion following eight passages in mouse myoblast cell cultures. In human myoblast cell cultures, laminin 521 supported increased proliferation during expansion and superior differentiation with myotube hypertrophy. Counterintuitively however, laminin 211, the native laminin isoform in resting skeletal muscle, resulted in low proliferation and poor differentiation in mouse and human cultures. Matrigel performed excellent in short-term mouse studies but showed high amounts of variability following long-term expansion. Conclusions: These results demonstrate laminin 521 is a superior substrate for both short-term and long-term myogenic cell culture applications compared to other commonly utilized substrates. Since Matrigel cannot be used for clinical applications, we propose that laminin 521 could possibly be employed in the future to provide myoblasts for cellular therapy directed clinical studies.