Дисертації з теми "Muscle cells"

Improving stem cell therapy is a major goal for the treatment of muscle diseases, where physiological muscle regeneration is progressively exhausted. Mesoangioblasts are vessel-associated stem cells that appear to be the most promising cell type for the cell therapy for muscular dystrophies because of their significant contribution to restoration of muscle structure and function in different muscular dystrophy model. Here we report that MAGE protein Necdin enhances muscle differentiation and regeneration by mesoangioblasts. Indeed, when Necdin is constitutively overexpressed, it accelerates their differentiation and fusion in vitro and it increases their efficacy to restore dystrophic phenotype of α-sarcoglycan mutant mouse. Moreover, Necdin confers a enhanced survival ability when mesoangioblasts are exposed to cytotoxic stimuli that mimic inflammatory dystrophic environment. Taken together, these data demonstrate the pivotal role of Necdin in muscle reconstitution from which we could take advantage to boost therapeutic applications of mesoangioblasts.

Vascular smooth muscle cells (VSMC) in the healthy adult arterial wall are a highlydifferentiated cell type with low levels of proliferation. However, when activated these cells can undergo a phenotypic change to become proliferative, migratory and excrete higher levels of extra-cellular matrix. While this cellular change is an essential element of the adaptable vasculature, excessive proliferation of VSMC underpins the development of a number of disease states, including atherosclerosis and restenosis after balloon angioplasty. The activation of VSMC is dependent on intracellular signalling pathways broadly altering gene expression. A key feature of this process is the initial potent regulation of transcription factors such as Egr-1, c-Jun and Ets-1, which then drive further transcriptional changes resulting in phenotypic change. The aim of this thesis was to discover novel genes, particularly transcription factors, regulated early upon stimulation and to characterise their contribution to the activation of VSMC. A key stimulus for activation of VSMC is the release of fibroblast growth factor 2 (FGF-2). A microarray used to explore the effects of FGF-2 exposure demonstrated the extensive nature of transcriptional modulation. In addition, it highlighted a number of transcription factors that were not previously described in VSMC: p8, ATF-4 and SHARP-2. In particular, SHARP-2 was potently upregulated and was reconfirmed in animal models of vascular injury. The subsequent contribution these factors make to VSMC activation was also demonstrated. p8 strongly induced VSMC proliferation, while ATF-4 contributed to cytokine production and SHARP-2 potently downregulated VSMC differentiation markers. A second area that was explored related to a gene known as YRDC, which was found to be upregulated upon stimulation of VSMC. YRDC is highly conserved across almost all cellular life, however its function remains unknown. A number of novel splice variants of YRDC were discovered and demonstrated to be differentially regulated in VSMC upon stimulation. Further work to commence characterising its function showed that it interacts with key ribosomal proteins and most likely plays a role in regulating translation. The discovery of the relevance of these genes to vascular biology in addition to their transcriptional regulation makes an important contribution to increasing our understanding of the molecular mechanisms behind vascular remodelling.

Mitochondrial myopathies are a group of progressive muscle disorders caused by mutations in the mitochondrial genome (mtDNA) for which there is no effective treatment. Culturing of myoblasts from patients with sporadically occurring mitochondrial diseases has suggested that mtDNA mutations may be lower or absent in muscle stem cells (satellite cells). The activation of muscle satellite cells and subsequent repair of muscle fibres may favourably shift the balance of delete to wild-type (WT) mtDNA, thereby decreasing mtDNA mutation load in affected muscle. This research has investigated muscle precursor cells from patients with mitochondrial myopathy due to sporadically occurring mtDNA deletions. This was to determine if they will benefit from attempts to “gene shift” the balance of WT and mutated mtDNA in their muscles using high intensity resistance training. Fluorescently Activated Cell Sorting (FACS) on the basis of CD56 (NCam) was used to isolate satellite cells and real time PCR to analyse them. In all eight patients investigated mtDNA deletions were detected in satellite cells at levels similar to mature muscle. In most of these patients the mtDNA deletions were lost during the culturing of their myoblasts. In some patients, however, the mutation was maintained, although there was a gradual decline in mutation load as the myoblasts headed towards differentiation. It was hypothesised that this difference between patients in the maintenance or loss of mutations in their myoblasts was attributable to an mtDNA bottleneck effect at the point of satellite cell activation. A second selection point occurred during the process of myoblast proliferation, possibly mediated by segregation of WT and delete mtDNA after cell division. Daughter cells that inherit large amounts of delete mtDNA will be unable to continue to proliferate. If efforts to “gene shift” in these patients will involve the activation of satellite cells to repair damaged muscle, it is paramount that this process does not exhaust the muscle stem cell pool. Satellite cell numbers have been determined in patients harbouring sporadically occurring mtDNA deletions, who will be considered potential beneficiaries of exercise based interventions. No significant difference was observed in satellite cell numbers when patients were compared to controls. In addition, a single patient was examined for satellite cell numbers over eleven years and no reduction in numbers was found. Given that the large majority of single deletion patients will lose their mtDNA mutation during the process of muscle regeneration and that they will not suffer from an exhaustion of the satellite cell pool, “gene shifting” remains a viable therapy in these patients. However, the mechanisms behind the process are somewhat different to what was originally hypothesised.

Cellular differentiation is accompanied by the modulation of gene expression. I have compared the expression of various genes, using the slot-blot technique, in two different systems. First, the level of expression of a wide variety of genes was analyzed in the hypertrophied heart of transgenic mice expressing the polyomavirus large T-antigen gene, and compared to normal control heart. I have shown that most changes in gene expression occurred mainly during early stages of heart hypertrophy. These genes code for proteins known to play a role in signal transduction, and transcriptional and growth control. The latter stages of cardiac hypertrophy are accompanied by changes in the expression of genes that are mostly involved in stress responses. Second, we analyzed the expression of various genes in three mouse myogenic cell lines undergoing differentiation in several culture conditions. The adult (C2C12) and fetal-derived (G7 and G8) myoblast cell lines were exposed to either retinoic acid, dimethyl sulfoxide, or transforming growth factor $ beta$. These three molecules are known to have profound effects on cellular growth and differentiation. I have shown that these treatments result in significant changes in expression of a wide variety of genes. Interestingly, all three cell lines differed considerably in their pattern of gene expression. Results from the analysis of these two systems demonstrate that differentially induced morphological changes of muscle cells, result in cell type specific changes in expression of a variety of genes.

GPCRs are the largest family of proteins in the human genome and a target for huge numbers of therapeutic drugs. However, the role of skeletal muscle in the action of these drugs is unclear. Given the unique importance of GPCR signalling in terms of glucose and fatty acid turnover in other tissues, it would be anticipated that GPCR identified to influence metabolism in these tissues might well be expressed in skeletal muscle. This study investigated the expression of genes encoding GPCRs in skeletal muscle and in cultured preparations thereof. In particular, this study focussed on the expression and signalling of adenosine receptors, a2-adrenoceptor, P2Y receptors and CBI cannabinoid receptors and the impact of CBI receptor modulation upon insulin signalling in rat primary skeletal muscle cells. All experiments in this work looked at GPCR expression and their signalling; with either tissues or cultured cells from rats. These experiments included: 1. Transcriptional profiling of skeletal muscle tissue in Wistar rats for GPCRs and proteins in associated signalling pathways. 2. Signalling of GPCRs (adenosine, a2A-adrenoceptor, P2y) in rat primary skeletal muscle cells. 3. Cannabinoid signalling pathways and cross-talk with insulin signalling. 4. CBI cannabinoid receptor antagonist/inverse agonist/agonist treatment of rat primary skeletal muscle cells. Expression of example members of the three major G protein coupling GPCR families was observed in rat skeletal muscle tissue. mRNA encoding Gs- (A2Aadenosine receptor, P2-adrenoceptor), Gi- (AI adenosine receptor, (l2A-adrenoceptor), and Gq-coupled (P2Y 1. P2Y2 and P2Y6 receptors) receptors were detected using gene microarray (Agilent, all ranked <10220 out of 41090). QRT-PCR (Taqman) identified (l2A-adrenoceptor and CBI cannabinoid receptor mRNA expression at low level similar across myoblasts, myotubes and skeletal muscle tissue. Functional responses to example members of the three major G protein coupling families of GPCR were also observed in rat primary skeletal muscle preparations. First, treatment of myotubes with the non-selective adenosine receptor agonist NECA elicited increases in cAMP, which were inhibited in the presence of the A2Badenosine receptorselective antagonist, PSB603. In contrast, the A2A-selective agonist, CGS21680 failed to evoke a significant cAMP elevation in myotubes. Second, neither basal nor forskolinevoked elevation of cAMP was altered in the presence of the Ar-selective agonist, SENBA. Third, the (l2-adrenoceptor agonist UK14304 inhibited forskolin-evoked cAMP levels, however, rauwolscine did not prevent this effect. Treatment with UK14304 also increased phosphorylation of ERK1/2; these responses, however, were inhibited by rauwolscine. In addition, rauwolscine in the absence of other ligands also inhibited ERK phosphorylation. Fourth, ATP and UTP, P2Y receptor agonists, elevated intracellular calcium ion levels in myoblasts. Although expression of mRNA for CBI cannabinoid receptors was detected in myoblasts, myotubes and skeletal muscle tissue, forskolin-evoked elevation of cAMP was unaltered in the presence of the CBI receptor-selective agonist ACEA or the antagonist/inverse agonist rimonabant in cultured myotubes. AICAR-stimulated AMPactivated protein kinase activity was also unaltered by ACEA. However, treatment with ACEA increased activation ofERK1I2 and p38 mitogen-activated protein kinases; these responses were significantly inhibited by rimonabant. Insulin treatment of myotubes increased the activation (phosphorylation) of AKT/protein kinase B, glycogen synthase kinase 3(1 and ~, ERK1I2 and p38 MAP kinases; however, pre-treatment with ACEA for 24 hours failed to alter these responses. In conclusion, these studies indicate expression and functional responses to select members of the three major G protein coupling families of GPCR in rat skeletal muscle preparations. These findings also provided evidence for expression of functionally active CB) cannabinoid receptors in skeletal muscle. However, they fail to support previous reports suggesting an interaction between insulin and CB) receptor signalling in these cells. The impact of CB) receptor function in skeletal muscle should be the subject of further investigation.

The primary cilium has recently been recognised as an essential regulator of the Sonic hedgehog (Shh) signalling pathway. Mutations that disrupt cilia function in humans can cause conditions known as ciliopathies. A wide range of phenotypes is observed in chick and mouse ciliopathy models,including polydactyly, craniofacial defects and polycystic kidneys. The Shh pathway and therefore primary cilia are vital for many developmental processes, including embryonic muscle development, with recent evidence suggesting they may also play a role in adult muscle regeneration. Our studies focus on the Talpid3 gene, which encodes a centrosomal protein required for primary cilia formation and Shh signalling. The Talpid3 loss-of-function mutant has perturbed ciliogenesis and displays many of the phenotypes that are typically associated with developmental Shh mutants and with ciliopathies. Talpid3 mutants have defects in Shh signalling, and processing of Gli transcription factors is affected in structures such as the developing limb buds and the neural tube. However, the role of Talpid3 in muscle development and regeneration remains unknown. The role of Talpid3 in adult muscle regeneration was investigated using a tamoxifen inducible, satellite cell specific knock-out of Talpid3 in mice. This mouse model was generated by crossing Talpid3 floxed mice to a mouse carrying an inducible Pax7-CreERT2 allele. To determine whether loss of Talpid3 affects muscle regeneration a cardiotoxin injury model was used. This showed that loss of Talpid3 in satellite cells results in a regeneration defect as fibres were smaller after 5, 10, 15 and 25 days of regeneration compared to control mice. This defect may be due to a reduced ability of Talpid3 mutant satellite cells to differentiate. We also show that Talpid3 plays a role in satellite cell self-renewal as we observe a complete loss of regeneration in some areas of the muscle following repeat injuries. We provide the first evidence that Talpid3 is critical for the regeneration of skeletal muscle following injury.

Satellite cells are defined by their position beneath the basal lamina of myofibres, and are a source of new myonuclei in adult skeletal muscles. However, other phenotypes also contribute to muscle regeneration, and the relative importance of satellite cells is not known. This work aimed to analyse the stem cell potential of satellite cells by formally investigating their contribution to muscle regeneration. Myofibres isolated from extensor digitorum longus, soleus, and tibialis anterior muscles were found to have respective means of 7,22 and 10 associated satellite cells. When a single myofibre was grafted into an irradiated dystrophic mouse muscle, the associated satellite cells underwent extensive, stem cell-like proliferation, generating progeny which sometimes gave rise to a cluster of more than 100 new myofibres. Cluster size varied according to the muscle group from which the graft was derived, but was not proportional to satellite cell number. Primary myoblasts derived from equivalent muscle groups did not undergo such extensive proliferation, or show inter-muscle variability, suggesting that stem cell activity is critically dependent on a component of the satellite cell niche. Single myofibres isolated from irradiated muscles were non-myogenic after grafting. Satellite cells associated with single myofibres were found to generate new satellite cells in engrafted muscles, demonstrating that satellite cell compartment is maintained by self-renewal. When single myofibre-engrafted muscles were damaged with myotoxin, graft-derived cells underwent rapid clonal expansion to regenerate compact clusters of donor-derived myofibres. The percentage of engrafted muscles containing identifiable donor-derived nuclei was increased after damage, showing that previously inactive cells had been recruited into an active myogenic program. Without experimentally-induced damage, frequency of muscle formation and cluster size were spontaneously augmented over time. These findings demonstrate that satellite cells have several stem cell-like qualities, and thus constitute a self-sufficient and sustainable source of regeneration in adult muscles.

Chez les patients souffrant d'insuffisance rénale chronique, les calcifications vasculaires représentent la première cause de mortalité. Elles résultent de la trans-différenciation des cellules musculaires lisses (CMLs) en cellules de type ostéoblastique et/ou chondrocytaire, en réponse à des cytokines inflammatoires ou à une hyperphosphatémie. Les CMLs forment alors des cristaux par l'activité de la phosphatase alcaline non-spécifique du tissu (TNAP). A la lumière de résultats récents, nous avons émis l'hypothèse que la TNAP module la trans différenciation des CMLs. Nos objectifs étaient donc de déterminer l'effet de la TNAP dans la trans-différenciation des CMLs, et d'étudier les mécanismes impliqués dans son induction, avec un intérêt particulier pour les microRNAs. Nous avons observé que l'ajout de phosphatase alcaline purifiée ou la surexpression de TNAP stimule l'expression de marqueurs chondrocytaires en culture de CMLs et de cellules souches mésenchymateuses. De plus, l'inhibition de la TNAP bloque la maturation de chondrocytes primaires. Nous excluons un rôle des cristaux formés par la TNAP, puisque l'ajout de cristaux seuls ou associés à une matrice collagénique n'a pas reproduit les effets de la TNAP. Nous suspectons que la TNAP agit en hydrolysant le pyrophosphate inorganique (PPi). En effet, c'est la TNAP qui hydrolyse le PPi en culture de CMLs et de chondrocytes, et le PPi mime les effets de l'inhibition de TNAP en culture de chondrocytes. Enfin, nous rapportons le profil de microRNA des artères cultivées en conditions hyperphosphatémiques. Ces résultats pourraient être particulièrement importants dans le développement de nouvelles approches thérapeutiques
In patients with chronic kidney disease (CKD), vascular calcification represents the main cause of mortality. Vascular calcification results from the trans-differentiation of vascular smooth muscle cells (VSMCs) into cells similar to osteoblasts and/or chondrocytes, in response to inflammatory cytokines or hyperphosphatemia. Calcifying VSMCs form calcium phosphate crystals through the activity of tissue nonspecific alkaline phosphatase (TNAP). In light of recent findings, we hypothesized that TNAP also modulates VSMC trans-differentiation. Our objectives were therefore to determine the effect of TNAP activity on VSMC trans-differentiation, and secondly to investigate the molecular mechanisms involved in TNAP expression in aortas, with a particular interest in microRNAs. We first observed that addition of purified alkaline phosphatase or TNAP over-expression stimulates the expression of chondrocyte markers in culture of the mouse and rat VSMC lines, and of mesenchymal stem cells. Moreover, TNAP inhibition blocks the maturation of mouse primary chondrocytes and reduces mineralization. We exclude a role for crystals in TNAP effects, since addition of crystals alone or associated to a collagenous matrix fails to mimic TNAP effects. We rather suspect that TNAP acts through the hydrolysis of inorganic pyrophosphate (PPi). Indeed, PPi is hydrolyzed by TNAP in VSMCs and chondrocytes and addition of PPi mimics the effects of TNAP inhibition on chondrocyte maturation. Finally, we report microRNA signature of aortic explants treated under hyperphosphatemic conditions that induce vascular calcification. These results could be of particular importance in patients with CKD

Thesis (Ph. D.)--University of Missouri-Columbia, 2009.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2009" Includes bibliographical references.

In spite of its post mitotic nature, skeletal muscle maintains remarkable plasticity. Muscle fibres (myofibres) are capable of large alterations in their size as well as an enormous ability to regenerate following injury – thanks to a potent population of resident stem cells (satellite cells). Deciphering the molecular signalling networks responsible for skeletal muscle growth and regeneration is of key scientific interest – not least because of the therapeutic potential these pathways may hold for the treatment of diseases such as muscular dystrophy. In this thesis, the transcriptional co-factor Yes-Associated protein (Yap), the downstream effector of the Hippo Pathway, was investigated in skeletal muscle. Using gain and loss of function approaches within in vitro, ex vivo and in vivo models, the contribution of Yap in regulating both satellite cell behaviour and myofibre growth was investigated. Yap expression and activity are dynamically regulated during satellite cell activation, proliferation and differentiation ex vivo. Overexpression of Yap increased satellite cell proliferation and maintained cells in a ‘naive’, ‘activated’ state by inhibiting myogenic commitment. Knock-down of Yap impaired satellite cell expansion, but did not influence myogenic differentiation. Yap interacts with Tead transcription factors in myoblasts to upregulate genes such as CyclinD1 and Myf5. Forced expression of Yap eventually led to the oncogenic transformation of myoblasts in vitro. Contrary to predictions, constitutive expression of Yap under an inducible muscle-specific promoter in adult mice failed to induce growth and instead led to muscle wasting, atrophy and degeneration – providing evidence against the notion that Yap represents a universal regulator of tissue growth. These data provide the first insight into the function of Yap in skeletal muscle. Results highlight a novel role for Yap in regulating myogenic progression in satellite cells, as well as its propensity to induce oncogenic transformation. The precise function of Yap in adult myofibres remains unclear however, data presented here demonstrates clear cell-type specific roles for Yap compared to observations made in other tissues.

Cardiovascular disease, which involves the cardiac, cerebrovascular and peripheral vascular system, is the major cause of morbidity and mortality in the western world. Changes in the vascular microenvironment trigger cascades of molecular events involving altered signaling, transcription and translation of a gene. The aim of this thesis was to increase our understanding on the molecular regulation of activated vascular smooth muscle cells. The first study looking at PDGF-D expression provides new insights into the regulatory mechanisms controlling the phosphorylation of Sp1. Studies performed identified three amino acids in Sp1 (Thr668, Ser670 and Thr681) that is phosphorylated by PKC-zeta activated by AngII. In the second study, the translational regulatory role of a novel gene YrdC induced by injury was investigated. Current knowledge of translational regulators controlling altered gene expression is little and studies in this thesis shows a splice variant of YrdC playing an important role in controlling mRNA translation and thus protein synthesis in the context of injury. The final study investigated in this study was the increased expression of the apoptotic FasL by the activation of GATA6. Although FasL has been extensively studied over the years, this is the first study linking a GATA factor with FasL in any cell type and provides key insights into the transcriptional events underpinning FasL-dependent SMC apoptosis following exposure to AngII.

Numerous macroscale models of arteries have been developed, comprised of populations of discrete coupled Endothelial Cells (EC) and Smooth Muscle Cells (SMC) cells, an example of which is the model of Shaikh et al. (2012), which simulates the complex biochemical processes responsible for the observed propagating waves of Ca2+ observed in experiments. In a 'homogenised' model however, the length scale of each cell is assumed infinitely small while the population of cells are assumed infinitely large, so that the microscopic spatial dynamics of individual cells are unaccounted for. We wish to show in our study, our hypothesis that the homogenised modelling approach for a particular system can be used to replicate observations of the discrete modelling approach for the same system. We may do this by deriving a homogenised model based on Goldbeter et al. (1990), the simplest possible physiological system, and comparing its results with those of the discrete Shaikh et al. (2012), which have already been validated with experimental findings. We will then analyse the mathematical dynamics of our homogenised model to gain a better understanding of how its system parameters influence the behaviour of its solutions. All our homogenised models are essentially formulated as partial differential equations (PDE), specifically they are of type reaction diffusion PDEs. Therefore before we begin developing the homogenised Goldbeter et al. (1990), we will first analyse the Brusselator PDE with the goal that it will help us to understand reaction diffusion systems better. The Brusselator is a suitable preliminary study as it shares two common properties with reaction diffusion equations: oscillatory solutions and a diffusion term.

The generation of induced pluripotent stem (iPS) cells is a useful tool for regenerative medicine. However, the risk of tumor development of the aforementioned cells should be addressed before they can be used for clinical applications. During the reprogramming process a number of signal pathways are activated, which may lead to direct differentiation of specific cell lineages prior to the cells reaching the pluripotent state. In order to test this hypothesis we designed a combined protocol of reprogramming and differentiation in an attempt to achieve direct differentiation of fibroblasts to specific cell lineages. Human fibroblasts were shortly reprogrammed by overexpression of four reprogramming factors (OCT4, SOX2, KLF4 and c-MYC) and maintained in reprogramming media on a gelatin substrate for four days. These cells were defined as partially induced pluripotent stem (PiPS) cells. PiPS cells did not form tumours in vivo and differentiated into smooth muscle cells (SMCs) when seeded on a Collagen IV substrate and maintained in differentiation media (DM). The PiPS-SMCs expressed a panel of SMC markers such as SMA, SM22 and Calponin at mRNA and protein levels. Immunofluorescent staining of PiPS-SMCs showed positive staining for the above markers, demonstrating a typical SMC morphology. These cells displayed a greater potential to differentiate into SMCs than iPS cells. In order to elucidate the mechanism of PiPS cell differentiation into SMCs, data from a series of experiments indicated that the gene DKK3 was involved in SMC differentiation of PiPS cells. DKK3 was expressed in parallel with SMC markers, while its overexpression or stimulation induced SMC marker expression. Furthermore, DKK3 silencing resulted in downregulation of SMC markers on both the mRNA and protein levels. Finally, additional experiments revealed that the upregulation of SMC markers by DKK3 is mediated by activation of Wnt signalling through interaction of DKK3 with the transmembrane receptor Kremen 1. Therefore, we developed a protocol to generate SMCs from PiPS cells through a DKK3 signal pathway. These findings provide a new insight into the mechanisms of SMC differentiation with therapeutic potential to vascular disease.

Epigenetic mechanisms are of fundamental importance for resolving and maintaining cellular identity. The mechanisms regulating muscle stem and progenitor cell identity have ramifications for understanding all aspects of myogenesis. The epigenetic mechanisms regulating muscle stem cells are therefore important aspects for understanding the regulation of muscle regeneration and maintenance. Important roles for the trithorax H3K4 histone methyltransferase (HMT) MLL1 have been established for early embryogenesis, and for hematopoietic and neural identity. Here, using a conditional Mll1 knockout (KO), we find that in vivo, MLL1 is necessary for efficient muscle regeneration, and for maintenance and proliferation of muscle stem and progenitor cells. Loss of Mll1 in cultured myoblasts reveals an essential role for expression of the myogenic specification gene Pax7. Mll1 KO results in a minor decrease in Pax7 mRNA and a strong decrease of Pax7 protein. While MLL1 was found to bind the Pax7 promoter, Mll1 KO results in a minor decrease of H3K4me3 at Pax7, supporting a recognized non-HMT role for Mll1 at Pax7. Microarray analysis of mRNA expression in Mll1 KO myoblasts finds that Myf5 is the most strongly downregulated of all genes, unexpectedly, mRNA expression of previously identified MLL1 targets are unaffected by loss of MLL1 in myoblasts. Pax7 activates Myf5 expression through recruitment of a H3K4 HMT, and in Mll1 KO myoblasts expression of, and H3K4me3 at Myf5 is lost. Exogenous Pax7 rescues Myf5 expression and H3K4me3 at Myf5 in the absence of MLL1, indicating that Myf5 expression is conditional on Pax7, but not MLL1. We also show that Myf5 DNA is methylated in non-myogenic cells, and in satellite stem cells that have never expressed Myf5, but is not methylated in satellite cells that are committed to the myogenic lineage, indicating that demethylation of Myf5 may be a fundamental step in myogenic commitment. Intriguingly, Myf5 promoter DNA becomes remethylated in Mll1 KO myoblasts. This work finds that Pax7 expression and myogenic identity is partly dependent on MLL1 expression. Further, evidence is uncovered that myogenic commitment is initiated by demethylation of Myf5. These findings add to the understanding of the epigenetic mechanisms that regulate and define muscle stem cells.

Improving treatment and prevention of pelvic organ prolapse, a disorder affecting up to half of parous women, requires thorough mechanical analysis of the vagina and other endopelvic structures at the cellular level. In this study, we tested single vaginal smooth muscle cells (SMCs) to quantify their elastic moduli. Cells were enzymatically isolated from vaginal walls of freshly sacrificed, virgin Long Evans rats and cultured using well-established methods. A custom-built experimental setup was used to perform tensile tests. Micropipettes were fabricated to serve as cantilever-type load cells, which were coated in cellular adhesive. Two pipettes applied tension to SMCs until adhesion between the cell and a pipette failed. During mechanical testing, images of SMCs were collected and translated into strain and stress. Specifically, force/stress data were calculated using Euler-Bernoulli Beam Theory and by making simplifying geometric assumptions. The average initial and total elastic moduli (mean ± SEM) for single vaginal SMCs were 6.06 ± 0.26 kPa and 5.4 ± 0.24 kPa, respectively, which is within the range reported for other types of SMCs, mainly airway and vascular, of various species. This protocol can and will be applied to further investigate mechanics of single cells from the pelvic region with independent variables such as parity, age, body mass index, and various stages of POP. Results of these experiments will provide critical information for improving current treatments like drug therapies, surgical procedures, medical grafts and implants, and preventative practices like stretching and exercise techniques.
Master of Science

Les crises cardiaques et les accidents vasculaires cérébraux sont la principale cause de mortalité dans le monde. La resténose et la thrombose sont parmi les complications les plus fréquentes du traitement de l'athérosclérose. Une étude montre que l'étendue des dommages induits par les dispositifs endovasculaires est sensiblement plus élevée pour une paroi artérielle rigide que pour une paroi souple. Les études sur les cellules musculaires lisses montrent que lorsqu'elles atteignent une densité critique, ces cellules peuvent passer spontanément de feuilles cellulaires plates à des amas tridimensionnels de type sphéroïde. La description des mécanismes physiques régissant l'émergence spontanée de ces intrigants amas tridimensionnels permet de mieux comprendre les troubles liés aux cellules musculaires lisses
Heart attacks and strokes are the leading cause of mortality worldwide. Restenosis and thrombosis are among the most common complications of atherosclerosis treatment. Study shows that the extent of damage induced by endovascular devices is significantly higher for a rigid arterial wall than for a soft wall. The smooth muscle cell investigations demonstrate that upon reaching a critical density, these cells can spontaneously transition from flat cell sheets to three-dimensional spheroid-like clusters. The description of the physical mechanisms governing the spontaneous emergence of these intriguing 3-D clusters offers insight into smooth muscle cell-related disorders

Muscle aging is associated with a decrease in the number of satellite cells and their progeny, muscle progenitor cells (MPCs) that are available for muscle repair and regeneration. However, there is an increase in non-immuno-hematopoietic cells (CD45 negative) in regenerating muscle from aged mice characterized by high stem cell antigen -1(Sca-1) expression. In aged regenerating muscle, 14.2% of cells are CD45neg Sca-1pos while 7.2% of cells are CD45neg Sca-1pos in young adult muscle. In vitro, CD45neg Sca-1pos cells over express genes associated with fibrosis, potentially controlled by Wnt2. These cells are proliferative, non-myogenic and non-adipogenic, and arise in clonally-derived MPCs cultures from aged mice. Both in vitro and in vivo studies suggest that CD45neg Sca-1pos cells from aged muscle are more susceptible to apoptosis than their MPCs, which may contribute to depletion of the satellite cell pool. Therefore, with age, a subset of MPCs takes on an altered phenotype, which is marked by high Sca-1 expression. This altered phenotype prevents these cells from participating in muscle regeneration or replenishing the satellite cell pool, and instead may contribute to fibrosis in aged muscle.

Abdominal aortic aneurysms (AAA) are characterised by a chronic inflammatory infiltrate within the abdominal aortic wall and aortic smooth muscle cell (AoSMC) apoptosis. It is postulated that the inflammatory infiltrate causes AoSMC apoptosis, with resultant aortic wall weakening and aneurysmal degeneration. This putative immune-mediated reaction against aortic wall component suggests that AAA may have features of an auto immune disease. It has been previously demonstrated that natural killer (NK) cells are elevated in the peripheral blood (PB) of AAA patients and display increased cytotoxicity against AoSMC. This study aimed to identify the molecular basis of the increased NK cell cytotoxicity and why an immune-mediated reaction occurs against AoSMC. Using multi-parametric flow cytometry (FC), expression of the activatory receptors NKp30, NKp44, NKp46 and NKG2D were analysed on PB NK cells from AAA patients and age-sex-matched healthy controls. No difference in activatory receptor expression or cell surface density (ΔMFI) existed between the two groups. Region specific (intra-luminal blood and AAA tissue) activatory receptor phenotypes were also investigated in AAA patients. The significant finding was a reduction in the ΔMFI of NKG2D on tissue NK cells, suggesting an interaction between this receptor and potential cognate ligands within the aortic wall. Characterised AoSMC explanted from AAA tissue were subjected to analysis using qRT-PCR and FC to identify the expression of death receptors (Fas, TRAIL-RI and TRAIL R2) and NKG2D ligands (MICA, MICB, ULBPI-3). AoSMC expressed mRNA for all NKG2D ligands. FC confirmed the cell-surface expression of NKG2D ligands and the death receptors. A significantly greater percentage of NK cells from AAA patients were CD107a+ when co-cultured with AAA AoSMC, thus accounting for the increased cytotoxicity in this group. Despite using anti-NKG2D it was not possible to inhibit NK cell degranulation in response to the NKG2D ligands on AoSMC. This work has demonstrated that AoSMC from AAA express death receptors and NKG2D ligands, potentially accounting for the NK cell molecular mechanism that leads to AoSMC apoptosis. The expression of NKG2D ligands, which have been demonstrated in other auto immune diseases, favours the hypothesis that AAA are an immune-mediated process directed against the abdominal aortic wall.

Embryonic stem (ES) cells can differentiate into many different cell lines, including vascular smooth muscle cells (SMCs). The aim of this project is to characterize protein changes during this differentiation process. Mouse ES cells are pre-differentiated by withdrawal of the leukemia inhibitory factor-1 from the culture medium. Subsequently, stem cell antigen-1 positive (Sca-1) cells are sorted by magnetic labelling cell sorting with anti-Sca-1 microbeads and cultured in differentiation medium with or without platelet-derived growth factor (PDGF). Protein extracts of ES cells and Sca-1+ cells are separated by two-dimensional electrophoresis. About 300 protein species of each cell lines are analyzed by mass spectrometry. Proteome maps are available online (http:/ /vwvw.v ascular-proteomicsc. om). After stimulation with PDGF for 5 passages, Sca-1+ cells differentiate into SMCs (esSMCs) with 95% staining positive for SMC markers such as smooth muscle a-actin, calponin, and smooth muscle myosin heavy chain. Protein profiles of esSMCs and mouse aortic SMCs are compared using the difference gel electrophoresis approach. esSMCs display decreased expression of myofilaments but increased oxidation of redox-sensitive proteins due to higher levels of reactive oxgen species (ROS). While immunoblotting reveals an upregulation of numerous antioxidants in esSMCs, enzymatic assays demonstrate lower glutathione concentrations compared to aortic SMCs despite a 3-fold increase in glutathione reductase activity. Mitochondrial superoxide measurement revealed the mitochondria-derived superoxide is the main source of ROS in esSMCs and inhibition of electron transport chain complex III by antimycin A showed remarkable increase of ROS in esSMCs. Moreover, depletion of glutathione by diethyl maleate or inhibition of glutathione reductase by carmustine (BCNU) results in a remarkable loss of cell viability in esSMCs compared to aortic SMCs while adding 2-mercaptoethanol increased esSMCs survival. These results indicate that esSMCs require additional antioxidant protection for survival and consequently, treatment with anti-oxidants could be beneficial for tissue repair from ES cells.

Muscular dystrophies are a category of diseases in which the muscle fibres degrade over time. At present there is no known cure, however a great deal of promise exists in cell replacement therapy, which has been successful in alleviating animal models of muscular dystrophy. Unfortunately, attempts to use stem cell therapy to cure or treat muscular dystrophies in humans have been unsuccessful, despite many different approaches to isolating and transplanting potentially myogenic cells. While skeletal muscle differentiation of embryonic stem cells has previously been reported, a simple and efficient method for the isolation of myogenic precursors from human ES cells has not been established. Recently, advances in induced pluripotent stem cell technology have brought the possibility of patient-specific pluripotent cell lines within reach, though a great deal of work needs to be done to understand the reprogramming process and the differentiation potential of these cells. This technology provides another avenue for cell therapy treatment of muscular dystrophies. Aims: The primary goals of the work described in this thesis were to develop a novel method of differentiating human embryonic stem cells to muscle satellite cells or comparable myogenic precursors and to isolate them using fluorescence activated cell sorting based on the expression of satellite cell-specific genes or surface proteins. Results: Myoblast conditioned medium was used as the primary means of driving myogenic differentiation of hES cells, measured by flow cytometry analysis of surface marker expression and quantitative PCR analysis of myogenic gene expression. During ES cell differentiation, isolation of a pure, differentiated population of cells can be difficult. A variety of satellite cell surface markers were examined in human adult and foetal myoblast lines to test potential targets for FACS isolation. In addition, a reporter construct was developed with the intent of having the PAX7 promoter drive expression of GFP and a line of hES cells containing this construct was established. The differentiation strategy developed for hES cells was also tested on a line of iPS cells and a new line of iPS cells were generated from a patient with Duchenne muscular dystrophy. Conclusions: Several viable candidates for surface marker selection of satellite cells were identified including CD56, CD106, and M-cadherin. However, despite trying a number of different approaches of differentiating hES cells, none resulted in a highly efficient method for generating myogenic precursors. The small number of myogenic cells produced was confirmed by flow cytometry, qPCR, and immunostaining analysis.

Thesis (M.S.)--Boston University
Cardiac and vascular disease syndromes and abnormalities have long been the leading causes of death in the United States, but the cause of congenital defects remain unclear due to the lack of appropriate model systems for scientific investigation. Moreover, the predominance of cardiovascular disease has stimulated scientists to focus on developing tissue-engineered blood vessels (TEBV) for vascular reconstruction and replacement. Major challenges remain in generating large amounts of epithelial cells (EC) and vascular smooth muscle cells (VSMC) for vascular reconstruction and in reducing the immunoresponse in patients after replacement. To address both issues of disease model generation and vascular tissue engineering, we can use induced pluripotent stem (iPS) cells discovered by Shinya Yamanaka in 2006: iPS cells generated from adult tissue and organs through the forced expression of two to four transcription factors are phenotypically indistinguishable from embryonic stem (ES) cells. First, by creating iPS from cardiovascular patients, we can generate patient-specific cell lines for scientific research investigation (i.e. elucidate molecular mechanisms and potential drug targets). Second, EC and VSMC derived from patient-specific iPS cell lines can be used for vascular reconstruction with cells of the patient's own genetic background, which should overcome many of the immunological limitations that currently impede vascular replacement (i.e. provide therapeutic interventions). The goal of this project is to study the differentiation capacity of iPS cells into smooth muscle cells (SMC). This project aims to develop a protocol that can maximize the derivation of purified smooth muscle cells from mouse induced pluripotent stem (iPS) cells through three linear developmental stages: induction of a posterior primitive- streak (PS) like population, formation of Flk1+ mesoderm, and induction of smooth muscle cells. Low dosage of Activin A and Wnt was used to differentiate iPS into a PS-like population, while the administration of BMP4 differentiates the cells to mesoderm via a posterior PS intermediate. Smooth muscle cells were induced from mesoderm by the addition of platelet-derived growth factor (PDGF-BB) and transforming growth factor b (TGF-β). The linear developmental progression from PS formation through mesoderm induction to smooth muscle cells were tracked by RT-qPCR and FACS for the expression of genes indicative of each individual stage, Flk1, and SMαA respectively. The results of this project can be used as a basis for in vitro derivation of purified mammalian smooth muscle cells from a mouse model system that can be further modified. Moreover, the differentiated SMCs can be further used in cell sheet construction as vascular patches for drug testing.

Skeletal muscles have a remarkable capacity to repair and regenerate in response to injury by virtue of their unique population of resident muscle stem cells (MuSCs). Recently, several studies have reported that mitochondria are important regulators of fate and function in various types of stem cells including MuSCs. Furthermore, emerging evidence has shown that accumulation of dysfunctional mitochondria leads to stem cell aging, premature commitment and impaired self-renewal. Preliminary evidence from publicly available transcriptomics datasets processed by our lab showed that Phosphatase and tensin homolog (PTEN)-induced putative kinase 1(PINK1) and Parkin/PARK2 genes, two key regulators of mitophagy are expressed in quiescent MuSCs and are transiently down-regulated as MuSCs activate. This led us to hypothesize that maintenance of an optimally functioning population of mitochondria through mitophagy would be important for self-renewal and muscle repair. In vitro single myofiber cultures isolated from mitophagy reporter mice (mito-QC mice), show that mitophagy is active in quiescent MuSCs and is transiently decreased upon MuSCs activation. We also show that mitophagy is re-activated in differentiating and self-renewing MuSCs. To further study muscle regeneration, we used a cardiotoxin (CTX) injury model of the Tibialis anterior (TA) muscle in mouse models harboring a knockout (KO) of PINK1 and Parkin. We show that loss of PINK1 in vivo promotes commitment of MuSCs in response to acute injury and ultimately leads to depletion of the MuSC pool and impaired muscle regeneration compared to wild type (WT) mice following repetitive injuries. Similarly, loss of Parkin in MuSCs in vivo impaired their self-renewal capacity. Consistent with these results, in vitro single myofiber cultures isolated from PINK1-deficient mice showed increased MuSCs commitment and impaired self-renewal. In vitro preliminary results from MuSCs-specific KO of Parkin revealed altered lineage progression, differentiation and self-renewal of MuSCs. Together, these findings suggest that PINK1/Parkin-dependent mitophagy acts as an important mitochondrial quality control mechanism which could be required for regulating MuSCs fate and function during muscle regeneration.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on January 5, 2010). Thesis advisor: William P. Fay. Includes bibliographical references

Le muscle squelettique adulte est un tissu avec une grande plasticité étant donné qu’il adapte sa taille suite à la surcharge fonctionnelle et il régénère suite à une lésion. La base de cette plasticité est la myofibre et les cellules souches associées, les cellules satellites (CS). Suite aux stimuli, les CS sortent de la quiescence, elles s’activent, proliférent, s’engagent dans la voie myogénique et fusionnent entre elles ou bien avec la fibre pre-éxistante. Une partie des CS retourne à la quiescence afin de maintenir le « pool » de progéniteurs. Ce projet a pour objectif de mieux caractériser des voies de signalisation responsables des adaptations des CS au cours de la régénération et le l’hypertrophie compensatoire. Srf est un facteur de transcription, particulièrement exprimé dans les muscles. Les gènes cibles de Srf sont des gènes qui participent à la régulation de la prolifération cellulaire et des gènes codant des protéines sarcomériques du muscle ou bien des gènes ayant un rôle dans l’adhésion cellulaire, la migration et l’organisation du cytosquelette. Il a été montré que la perte de fonction de Srf dans la lignée de cellules musculaire C2C12 inhibe leur prolifération et leur différenciation et que Srf contrôle l’expression de MyoD qui est un gène de détermination myogénique. Aucune donnée n’est disponible à ce jour concernant la fonction de Srf dans les CS in vivo. Nous avons généré des souris dépourvues de Srf spécifiquement dans les CS adultes. Les CS ont été recruitées par l’hypertrophie et la régénération musculaire. En parallèle des études ex vivo ont été menées afin de préciser si les phénotypes observés sont cellule-autonomes et afin de disséquer les mécanismes sous-jacents. Nous montrons que la perte de Srf dans les CS affecte fortement les processus de régénération et d’hypertrophie suggérant un rôle de Srf dans le contrôle du destin cellulaire de CS. Nos études montrent que la perte le Srf dans les SC n’affecte pas leur prolifération et leur engagement dans la différenciation myogénique. Par contre, leur motilité et leur capacité de fusion sont fortement réduites. Afin d’identifier les effecteurs de Srf impliqués dans la motilité et le défaut de fusion des CS mutantes, nous avons réalisé des études transcriptomiques et identifié le set de gènes dont l’expression est altérée par la perte de Srf dans des conditions de prolifération et de différenciation. L’analyse des fonctions altérées nous a indiqué que la voie de signalisation du cytosquelette d’actine était perturbée. En effet les CS dépourvues de Srf expriment moins d’actine et présentent une organisation du cytosquelette d’actine perturbée. Des expériences de sauvetage utilisant un modèle de souris permettant la surexpression inductible d’actine alpha dans les CS dépourvues de Srf ont montré que la surexpression d’actine chez les mutants Srf était suffisante pour rétablir partiellement l’organisation du cytosquelette et améliorer les capacités de fusion des CS. De manière intéressante, seule la fusion hétérotypique (entre une cellule contrôle et une cellule mutante), et pas la fusion homotypique (entre deux cellules mutantes), est rétablie par l’expression de l’actine. In vivo, le rétablissement de la fusion hétérotypique restaure la croissance hypertrophique des muscles alors que l’altération de la régénération chez les mutants Srf n’est que faiblement améliorée par la surexpression de l’actine. Cette étude nous a permis d’avoir une vision d’ensemble et mécanistique de la contribution du facteur de transcription Srf dans la biologie des CS et de mettre en évidence l’importance structurale du maintien du cytosquelette d’actine pour la fusion des cellules musculaires
The adult skeletal muscle is a high plastic tissue as it adapts its size upon overload and it is capable of regeneration upon muscle lesion. The skeletal muscle is composed of a specialized syncytium, the myofiber, which is the functional unit of the muscle and a small population of myogenic progenitors, residing adjacent to the myofibers, termed as satellite cells (SCs). SCs are the muscle-specific stem cells which endow the skeletal muscle with its remarkable capacity to repair and to maintain homeostasis during muscle turnover. In resting adult muscles, SCs are quiescent but they activate upon exposure to stimuli. The activated SCs (myoblasts) proliferate extensively and subsequently differentiate and fuse between them or pre-existing myofibers, a series of cellular events called myogenesis. In parallel to the myogenesis, a reserve population of SCs escapes the myogenic program and self-renews to replenish the SC pool. The current project aims to further characterize the signalling pathways involved in SC functions during muscle regeneration and compensatory hypertrophy (CH). Srf is a muscle-enriched transcription factor with Srf-target genes implicated in cell proliferation, differentiation (sarcomeric proteins), adhesion, migration and cellular cytoskeleton. Studies in C2C12 mouse myogenic cell line showed that Srf loss prevent the myoblast proliferation and differentiation by down-regulating the expression of the myogenic determinant MyoD gene. We used a genetic murine model for adult SC-specific Srf-loss in order to conduct in vivo and ex vivo studies for the Srf role in SCs. Compensatory hypertrophy and regeneration are the two means by which SCs were recruited. We show that loss of Srf in SCs affects the regeneration process and the CH suggesting the Srf role in the SC fate. Srf-depleted SCs display probably no defect in their proliferation and differentiation but reduced capacity in motility and fusion. Transcriptomic analysis revealed altered actin cytoskeleton and signalling. Srf-depleted SCs show reduced actin expression and altered actin cytoskeleton. Rescue of actin expression in Srf-depleted SCs partially restored the cytoskeleton organization and the fusion process. Interestingly by actin overexpression only the heterotypic/asymmetric fusion was established but not the homotypic/symmetric fusion. Therefore actin overexpression restored the hypertrophic growth in the CH (in vivo model of heterotypic fusion) but failed to do so in the regeneration (in vivo model of homotypic fusion). This study contributed to the in vivo investigation of the Srf mechanistic role in adult SCs and underlined the importance of actin cytoskeleton maintenance in the fusion of myogenic cells

Introduction: Stem cell biology has received much interest because of its potential in both therapeutic application and in vitro modeling of diseases. In particular embryonic stem cells have good proliferative and differentiative abilities, but their use is still associated to ethical concerns and problems related to their teratogenic potential. Adult stem cells have also been described to be pluripotent both in vitro and in vivo. However, their use is limited because they are difficult to isolate and expand, particularly in a clinical setting. In this scenario, it would be advantageous to obtain a cell population with high selfrenewal and differentiation capacities, without ethical problems. In 2007 our group described that amniotic fluid stem (AFS) cells could be derived selecting amniocytes using c-Kit antibody. AFS cells have clonogenic capability and can be directed into a wide range of cell types representing the three primary embryonic lineages. Aim: This work aiming at characterize the myogenic potential of mouse AFS cells using a mouse model of spinal muscular atrophy and in particular at analyzing their ability to differentiate into satellite cells and colonize the muscle stem cell niche. Materials and Methods: Mouse AFS cells were obtained by amniocentesis and selected for the marker c-Kit with immunomagnetic beads. Freshly isolated AFS cells were analyzed for the expression of different markers (CD90, CD45, CD44, CD34, CD31, Flk1, SCA1, CD105) by flow cytometry and the expression of Oct4, Sox2, c-Myc, Klf4 and Sca-1 by qRT-PCR at different embryonic stages. For the treatment of HSA-Cre, SmnF7/F7 mutant mice, GFP+ AFS cells were injected via the tail vein and animals were sacrificed one and fifteen months after transplantation. Clinical aspects were observed and analyzed after transplantation to evaluate AFS cells’ effects. Several muscles were stained with hematoxylin and eosin, Masson’s trichrome and analyzed by immunofluorescence with anti-GFP and anti-dystrophin antibodies. To demonstrate the ability of AFS cells to replenish the muscle niche, staining for satellite cell markers and secondary transplantation were performed. The myogenic potential of AFS cells was also evaluated with transplantation after in vitro expansion. Results: Mouse AFS cell number changes during the course of gestation. At E12.5 these cells express hematopoietic markers (CD45, CD34, SCA1), mesenchymal markers (CD90, CD105) together with Flk1, CD31 and CD44. Gene expression analysis showed that mouse AFS cells express at low levels Oct4 and Sox2 and at high levels c-Myc and Klf4, whereas they are negative for the expression of myogenic genes. Mild muscular mutant HSA-Cre, SmnF7/F7 mice die at the age of 10 months and show evident clinical complications such as kyphosis and muscle shrinkage. After transplantation with GFP+ AFS or bone marrow (BM) cells mice survival rate increased by 75% and 50% respectively. Animals treated with AFS cells recovered more than 75% of force compared to the untreated. One month after transplantation, muscles obtained from AFS-treated mice displayed 37% of GFP+ fibers, with very low number of regenerating myofibers (<1%) and normal dystrophin expression. Fifteen months after transplantation BM-treated mice displayed a high number of central nucleated fibers and consistent infiltration of interstitial tissue and no GFP+ myofibers, while AFS-treated mice had a normalized phenotype, close to the same age WT mice, and 58% of the myofibers were GFP+. Similar results were obtained with transplantation of mouse AFS cells expanded in culture. Discussion: Mouse AFS cells are a heterogeneous population, and their phenotype changes during the course of gestation. At E12.5 they express mesenchymal, hematopoietic and endothelial markers, but most importantly don not express myogenic factors, indicating that no myogenic progenitor cells are present in this stem cell population. When injected in a muscular mutant mouse model, AFS cells showed a myogenic potential, even after long-term transplantation, suggesting an interesting therapeutic potential. They indeed were able to differentiate into satellite cells localizing in the muscle stem cell niche and expressing Pax7, a7integrin and SM/c-2.6, exclusively markers of satellite cell population. Moreover, AFS cells could contribute to the formation of new myofibers even after in vitro expansion.
Introduzione: Negli ultimi anni lo studio delle cellule staminali ha suscitato molto interesse, sia per il grande potenziale di queste cellule nelle terapie e applicazioni cliniche, sia come modello di studio in vitro per diversi tipi di malattie. In particolare, le cellule staminali embrionali hanno una elevata capacità proliferativa e di differenziazione, ma il loro utilizzo è ancora associato a problematiche etiche. Anche le cellule staminali adulte possiedono grandi potenzialità differenziative sia in vitro che in vivo, tuttavia il loro utilizzo è limitato in quanto difficili da isolare ed espandere, soprattutto in ambito clinico. In questo scenario sarebbe vantaggioso poter ottenere una popolazione di cellule con elevata capacità di proliferazione e differenziazione, senza dover affrontare però problemi di tipo etico. Nel 2007 il nostro gruppo ha isolato una popolazione di cellule staminali dal liquido amniotico (cellule AFS), utilizzando come marcatore il recettore c-Kit. Queste cellule hanno capacità clonogenica e possono essere dirette a differenziare in una vasta gamma di tipi cellulari appartenenti a tutti e tre i foglietti germinativi. Obiettivo: Questo lavoro mira a caratterizzare il potenziale miogenico delle cellule staminali del liquido amniotico di topo utilizzando un modello murino di atrofia spinale muscolare. In particolare è volto ad analizzare la capacità delle cellule AFS di dare origine a cellule staminali muscolari e colonizzare la nicchia staminale del muscolo scheletrico. Materiali e Metodi: Le cellule AFS sono state ottenute mediante amniocentesi e selezionate per la positività al marcatore c-kit con metodo immmunomagnetico. Appena isolate le cellule AFS sono state analizzate per l'espressione di diversi marcatori (CD90, CD45, CD44, CD34, CD31, Flk1, SCA1, CD105) tramite citometria a flusso; inoltre, attraverso qRT-PCR è stata analizzata l'espressione di Oct4, Sox2, c-Myc, Klf4 e Sca-1 delle cellule AFS isolate a diversi stadi embrionali. Per la terapia di topi transgenici HSA-Cre, SmnF7/F7, le cellule AFS GFP+ sono state iniettate per via sistemica attraverso la vena caudale; gli animali sono stati poi sacrificati a uno e a quindici mesi dopo il trapianto. Sono stati osservati e analizzati alcuni parametri clinici per valutare l’effetto del trapianto cellulare. Diversi muscoli sono stati raccolti ed analizzati con ematossilina e eosina, tricromica di Masson e mediante immunofluorescenza con anticorpi anti-GFP e anti-distrofina. Per dimostrare la capacità delle cellule AFS di colonizzare la nicchia staminale del muscolo, sono state eseguite delle immunofluorescenze per i marcatori specifici delle cellule satelliti e sono stati eseguiti dei trapianti secondari. Il potenziale miogenico delle cellule AFS è stato valutato anche con trapianto dopo espansione in vitro. Risultati: Il numero medio di cellule AFS presenti nel liquido amniotico varia nel corso della gestazione murina; all’età di 12.5 giorni queste cellule sono circa l’1% del totale ed esprimono marcatori ematopoietici (CD45, CD34, SCA1), marcatori mesenchimali (CD90, CD105) unitamente a Flk1, CD31 e CD44. L’analisi di espressione genica ha dimostrato che le cellule AFS esprimono a bassi livelli Oct4 e Sox2 e alti livelli di c-Myc e Klf4, mentre, nonostante la composizione mista di questa popolazione, non è stata rilevata espressione di marcatori o fattori di trascrizione tipici dei precursori muscolari. I topi HSA-Cre, SmnF7/F7 mediamente muoiono all'età di 10 mesi e durante il corso della loro vita mostrano evidenti complicazioni cliniche come una pronunciata cifosi e atrofia a livello muscolare. Dopo il trapianto con cellule AFS GFP+ o con cellule del midollo osseo, il tasso di sopravvivenza di questi animali aumenta rispettivamente del 75% e 50%. Gli animali trattati con cellule AFS hanno recuperato più del 75% della forza rispetto agli animali non trattati. Un mese dopo il trapianto, i muscoli di topi trattati con cellule AFS presentano il 37% di fibre GFP+, un numero molto basso di miofibre rigeneranti (< 1%) ed una normale espressione di distrofina. Quindici mesi dopo il trapianto, gli animali trattati con cellule del midollo osseo mostrano un elevato numero di fibre centro nucleate, un’importante infiltrazione di tessuto interstiziale e nessuna miofibra GFP+, mentre i topi trattati con cellule AFS hanno un fenotipo molto simile a quello di topi sani della stessa età, e il 58% delle miofibre è GFP+. Risultati simili sono stati ottenuti trattando lo stesso modello animale con cellule AFS dopo espansione in cultura. Discussione: Le cellule AFS isolate dal liquido amniotico di topo sono una popolazione eterogenea; queste cellule esprimono marcatori mesenchimali, ematopoietici e marcatori endoteliali. Va evidenziato che, nonostante la composizione mista di questa popolazione staminale, non esistono precursori muscolari al suo interno, e quindi qualunque differenziamento in senso muscolare di queste cellule è dovuto ad una differenziazione delle cellule AFS e non ad una maturazione di cellule già pre-commited. Quando vengono iniettate in un modello di atrofia muscolare, le cellule AFS mostrano un grande potenziale miogenico, anche a lungo termine, dimostrandosi una interessante fonte cellulare per scopi terapeutici. Queste cellule infatti sono state in grado di differenziare in cellule satelliti localizzandosi nella nicchia delle cellule staminali muscolari ed esprimendo Pax7, a7integrina e SM/c-2.6, tutti marcatori esclusivi delle cellule satelliti. Inoltre, le cellule AFS possono contribuire alla formazione di nuove miofibre anche dopo espansione in cultura, aumentando così lo spettro di possibili applicazioni terapeutiche.

Abstract Skeletal muscles are composed of long, multinucleated cells called myofibers, which are highly differentiated cells and therefore unique in structure. In the present study the organization of the endocytic and exocytic pathways in isolated rat skeletal myofibers was defined with confocal and electron microscopic methods. In isolated myofibers the I band areas were shown to be active in endocytosis. The sorting endosomes were distributed in a cross-striated fashion while the recycling and late endosomal compartments were located to perinuclear areas and interfibrillar spaces, where they followed the course of microtubules. Protein trafficking in the different stages of muscle cell differentation was also analyzed. The studies with L6 myoblasts and myotubes showed that during myogenesis varying fractions of different viral glycoproteins were sorted from the endoplasmic reticulum (ER) into a specific compartment that did not recycle with the Golgi apparatus. This compartment is suggested to be the sarcoplasmic reticulum (SR). The studies with living muscle cells showed further changes in vesicle trafficking taking place during myogenesis. With GFP-tagged tsO45G protein, transport containers were detected in 20% of the infected myofibers, while all infected L6 myoblasts or myotubes showed intense movement of corresponding structures. We also detected significant differences between the pre-and post-Golgi traffickings in myofibers. When the distribution of the ER in adult myofibers was studied, the confocal microscopic data showed that the labeling patterns of the rough endoplasmic reticulum (RER) and the SR markers were different. Blocking of different cargo proteins in the RER revealed two discrete distribution patterns, neither of them identical with the SR. The collected electron microscopic data supported the idea that in mature myofibers there are two separate RER compartments. We suggest that the RER compartment capable of export function located around the myonuclei and on the Z lines, while the non-exporting RER compartment localized to terminal cisternae and probably took care of the synthesis of the SR proteins.