Muscle Stem Cells Regulate the Bioenergetic Function of Myofibers in Mitochondrial Myopathies

Skeletal muscle tissue exhibits a high degree of plasticity due to their muscle stem cells, which are indispensable for muscle fiber repair, and the unique architecture of their mitochondria, which provide the energy for muscle fiber function, maintenance, and regeneration. In response to injury, quiescent muscle stem cells (MuSCs) undergo myogenesis to activate, differentiate, and fuse into the muscle fiber as new myonuclei that regulate myofiber repair. To meet the high energy demands of muscle regeneration, MuSCs increase in mitochondrial content through the various phases of myogenesis. However, when muscle mitochondria become dysfunctional, such as in peripheral artery disease (PAD) and Duchenne muscular dystrophy (DMD), MuSCs are debilitated and myofiber mitochondria remain defective, resulting in mitochondrial myopathy. Despite the characterization of mitochondrial dysfunction in these two disease models, the relationship between MuSC mitochondria and the bioenergetic function of the myofiber has not been investigated. To address this, the overarching objective of this thesis was to explore the role of MuSCs in remodeling the mitochondrial network and function of the myofiber in mitochondrial myopathies. We first correlated the MuSC response with the stages of mitochondrial network remodeling following a murine hindlimb ischemia (HLI) model of PAD and discovered that MuSC-derived myonuclei drive mitochondrial biogenesis. As direct evidence of MuSC-mediated remodeling of mitochondria, we then revealed that mitochondrial dysfunction in the MuSC yields deficient bioenergetic function of the dystrophic myofiber, which can be rescued by transplantation of MuSCs with healthy mitochondria. We tested this further in ischemic muscle after aging, which exacerbated the mitochondrial dysfunction, and again observed significant improvements in bioenergetic function following transplantation of healthy MuSCs. Overall, this thesis established that MuSC mitochondria play a consequential role on myofiber bioenergetic function, identified a source of mitochondrial dysfunction in dystrophic muscle, developed a model of age-associated PAD, and provided a conceptual framework for MuSC transplantation as a therapeutic approach for mitochondrial myopathies.Ph.D

Prolotherapy Induces an Inflammatory Response in Human Tenocytes In Vitro

Proliferative therapy, or prolotherapy, is a controversial treatment method for many connective tissue injuries and disorders. It involves the injection of a proliferant, or irritant solution, into the site of injury, which causes small-scale cell death. This therapeutic trauma is theorized to initiate the body's wound-healing cascade, perhaps leading to tissue repair. The immediate effects of many of these proliferants are poorly characterized, as are the cellular responses to them; here, we sought to evaluate the immediate effects of two common proliferants (dextrose and P2G, a combination of phenol, glucose, and glycerin) on the cellular response of human tenocytes, and begin to explicate the mechanisms with which each proliferant functions. We asked: What are the effects of treating cultured tenocytes with proliferative treatment agents on their (1) cellular metabolic activity, (2) RNA expression, (3) protein secretion, and (4) cell migration? Using human hamstring and Achilles tendon cells, we attempted to answer our research questions. We used a colorimetric metabolic assay to assess the effect of dextrose and P2G proliferant treatment on cell mitochondrial activity compared with nontreated tenocytes. Next, using quantitative PCR, ELISA, and a reporter cell line, we assessed the expression of several key markers involved in tendon development and inflammation. In addition, we used a scratch wound-healing assay to evaluate the effect of proliferant treatment on tenocyte migration. Results showed that exposure to both solutions led to decreased metabolic activity of tenocytes, with P2G having the more pronounced effect (75% +/- 7% versus 95% +/- 7% of untreated control cell metabolic levels) (ANOVA; p < 0.01; mean difference, 0.202; 95% CI, 0.052-0.35). Next, gene expression analysis confirmed that treatment led to the upregulation of key proinflammatory markers including interleukin-8 and cyclooxygenase-2 and downregulation of the matrix marker collagen type I. Furthermore, using a reporter cell line for transforming growth factor-beta (TGF-beta), a prominent antiinflammatory marker, we showed that treatments led to decreased TGF-beta bioactivity. Analysis of soluble proteins using ELISA revealed elevated levels of soluble prostaglandin E2 (PGE2), a prominent inducer of inflammation. Finally, both solutions led to decreased cellular migration in the tenocytes. Taken together, these results suggest that prolotherapy, more so with P2G, may work by decreasing cellular function and eliciting an inflammatory response in tenocytes. Additional studies are needed to confirm the cellular signaling mechanisms involved and the resulting immediate response in vivo. If these preliminary in vitro findings can be confirmed in an in vivo model, they may provide clues for a possible cellular mechanism of a common alternative treatment method currently used for certain soft tissue injuries

Cross-talk between human tenocytes and bone marrow stromal cells potentiates extracellular matrix remodeling in vitro

\u3cp\u3eTendon and ligament (T/L) pathologies account for a significant portion of musculoskeletal injuries and disorders. Tissue engineering has emerged as a promising solution in the regeneration of both tissues. Specifically, the use of multipotent human mesenchymal stromal cells (hMSC) has shown great promise to serve as both a suitable cell source for tenogenic regeneration and a source of trophic factors to induce tenogenesis. Using four donor sets, we investigated the bidirectional paracrine tenogenic response between human hamstring tenocytes (hHT) and bone marrow-derived hMSC. Cell metabolic assays showed that only one hHT donor experienced sustained notable increases in cell metabolic activity during co-culture. Histological staining confirmed that co-culture induced elevated collagen protein levels in both cell types at varying time-points in two of four donor sets assessed. Gene expression analysis using qPCR showed the varied up-regulation of anabolic and catabolic markers involved in extracellular matrix maintenance for hMSC and hHT. Furthermore, analysis of hMSC/hHT co-culture secretome using a reporter cell line for TGF-β, a potent inducer of tenogenesis, revealed a trend of higher TGF-β bioactivity in hMSC secretome compared to hHT. Finally, hHT cytoskeletal immunostaining confirmed that both cell types released soluble factors capable of inducing favorable tenogenic morphology, comparable to control levels of soluble TGF-β1. These results suggest a potential for TGF-β-mediated signaling mechanism that is involved during the paracrine interplay between the two cell types that is reminiscent of T/L matrix remodeling/turnover. These findings have significant implications in the clinical use of hMSC for common T/L pathologies. J. Cell. Biochem. 117: 684-693, 2016.\u3c/p\u3

Cross-Talk between Human Tenocytes and Bone Marrow Stromal Cells Potentiates Extracellular Matrix Remodeling in Vitro

Tendon and ligament (T/L) pathologies account for a significant portion of musculoskeletal injuries and disorders. Tissue engineering has emerged as a promising solution in the regeneration of both tissues. Specifically, the use of multipotent human mesenchymal stromal cells (hMSC) has shown great promise to serve as both a suitable cell source for tenogenic regeneration and a source of trophic factors to induce tenogenesis. Using four donor sets, we investigated the bidirectional paracrine tenogenic response between human hamstring tenocytes (hHT) and bone marrow-derived hMSC. Cell metabolic assays showed that only one hHT donor experienced sustained notable increases in cell metabolic activity during co-culture. Histological staining confirmed that co-culture induced elevated collagen protein levels in both cell types at varying time-points in two of four donor sets assessed. Gene expression analysis using qPCR showed the varied up-regulation of anabolic and catabolic markers involved in extracellular matrix maintenance for hMSC and hHT. Furthermore, analysis of hMSC/hHT co-culture secretome using a reporter cell line for TGF-β, a potent inducer of tenogenesis, revealed a trend of higher TGF-β bioactivity in hMSC secretome compared to hHT. Finally, hHT cytoskeletal immunostaining confirmed that both cell types released soluble factors capable of inducing favorable tenogenic morphology, comparable to control levels of soluble TGF-β1. These results suggest a potential for TGF-β-mediated signaling mechanism that is involved during the paracrine interplay between the two cell types that is reminiscent of T/L matrix remodeling/turnover. These findings have significant implications in the clinical use of hMSC for common T/L pathologies

Cross-Talk between Human Tenocytes and Bone Marrow Stromal Cells Potentiates Extracellular Matrix Remodeling in Vitro

Tendon and ligament (T/L) pathologies account for a significant portion of musculoskeletal injuries and disorders. Tissue engineering has emerged as a promising solution in the regeneration of both tissues. Specifically, the use of multipotent human mesenchymal stromal cells (hMSC) has shown great promise to serve as both a suitable cell source for tenogenic regeneration and a source of trophic factors to induce tenogenesis. Using four donor sets, we investigated the bidirectional paracrine tenogenic response between human hamstring tenocytes (hHT) and bone marrow-derived hMSC. Cell metabolic assays showed that only one hHT donor experienced sustained notable increases in cell metabolic activity during co-culture. Histological staining confirmed that co-culture induced elevated collagen protein levels in both cell types at varying time-points in two of four donor sets assessed. Gene expression analysis using qPCR showed the varied up-regulation of anabolic and catabolic markers involved in extracellular matrix maintenance for hMSC and hHT. Furthermore, analysis of hMSC/hHT co-culture secretome using a reporter cell line for TGF-β, a potent inducer of tenogenesis, revealed a trend of higher TGF-β bioactivity in hMSC secretome compared to hHT. Finally, hHT cytoskeletal immunostaining confirmed that both cell types released soluble factors capable of inducing favorable tenogenic morphology, comparable to control levels of soluble TGF-β1. These results suggest a potential for TGF-β-mediated signaling mechanism that is involved during the paracrine interplay between the two cell types that is reminiscent of T/L matrix remodeling/turnover. These findings have significant implications in the clinical use of hMSC for common T/L pathologies