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Development of strategies to enhance the contribution of hematopoietic cells to skeletal muscle repair Long, Michael Anthony Aug 31, 2010

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DEVELOPMENT OF STRATEGIES TO ENHANCE THE CONTRIBUTION OF HEMATOPOIETIC ELS TO SKELETAL MUSCLE REPAIR by MICHAEL ANTHONY LONG  B.Sc., McMaster University, 201  A THESIS SUBMITED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGRE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Medical Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2010 © Michael Anthony Long, 2010       ii ABSTRACT The ability of bone marow derived cels to contribute to skeletal muscle repair may represent a novel means of cel therapy for myopathies. However, this phenomenon takes place at exceedingly low frequencies and has failed to yield any measurable functional improvement in disease models. In an efort to increase the eficiency of this process, we designed Cre/loxP-based tracing strategies to identify the lineages and mechanisms involved. However, these experiments were complicated by a previously unknown limitation of comon Cre-reporter strains.  We have studied the Z/AP, Z/EG and R26R-EYFP reporter strains and have demonstrated that although each reporter can be reliably activated by Cre during early development, exposure to Cre in adult hematopoietic cels results in a much lower frequency of reporter-positive cels in the Z/AP or Z/EG strains than in the R26R-EYFP strain. In reporter-negative cels derived from the Z/AP and Z/EG strains, the transgenic promoter is methylated and Cre-mediated recombination of the locus is inhibited. These findings sugest that the Z/AP and Z/EG strains may not be suitable for the investigation of developmental plasticity in adult models. As bone marow derived cels are now believed to contribute to skeletal muscle repair primarily via fusion, we also constructed a chimeric measles hemaglutinin, Hα7, which eficiently mediates the fusion of diverse cel types with skeletal muscle. When compared directly to polyethylene glycol in vitro, Hα7 consistently generated a ten to fiften fold increase in heterokaryon yield and induced insignificant levels of toxicity. More importantly, Hα7 was also capable of increasing the contribution of mouse and human bone marow derived cels to skeletal muscle repair in vivo.   iii TABLE OF CONTENTS ABSTRACT................................................................................................................ii TABLE OF CONTENTS............................................................................................iii LIST OF FIGURES....................................................................................................vi CO-AUTHORSHIP STATEMENT............................................................................vi CHAPTER 1. INTRODUCTION..................................................................................1 1.1 SKELETAL MUSCLE DEVELOPMENT AND REGENERATION...............1 1.2 MUSCULAR DYSTROPHY AND EXISTING THERAPIES........................4 1.3 SKELETAL MUSCLE REPAIR BY BONE MAROW DERIVED CELS...8 1.3.1 Hematopoiesis.............................................................................8 1.3.2 Discovery of a circulating myogenic progenitor..........................11 1.3.3 Phenotype of the circulating myogenic progenitor......................13 1.3.4 Recruitment of the circulating myogenic progenitor...................17 1.3.5 Fusion versus transdiferentiation..............................................20 1.3.6 Nuclear reprograming.............................................................23 1.3.7 Prospects for therapy.................................................................26 1.4 SPECIFIC AIMS......................................................................................28 1.4.1 Identification of hematopoietic lineages responsible for the generation of bone marow derived muscle.....................................28 1.4.2 Identification of the mechanism responsible for the generation of bone marow derived muscle...........................................................30 1.4.3 Increase the contribution of bone marow derived cels to skeletal muscle repair...................................................................................33 1.5 REFERENCES........................................................................................34 CHAPTER 2. SILENCING INHIBITS CRE-MEDIATED RECOMBINATION OF THE Z/AP AND Z/EG REPORTERS IN ADULT CELS..............................................44 2.1 INTRODUCTION.....................................................................................44 2.2 RESULTS................................................................................................48 2.2.1 Ineficient activation of the Cre reporter in hematopoietic stem cels derived from adult Z/EG mice..................................................48   iv 2.2.2 Ineficient activation of the Cre reporter in myeloid cels derived from adult Z/AP and Z/EG mice.......................................................51 2.2.3 The transgenic locus is resistant to Cre-mediated recombination in a subset of granulocytes derived from Z/EG mice.......................54 2.2.4 The transgenic locus is methylated in reporter-negative granulocytes derived from LysM-Cre/Z/EG and LysM-Cre/Z/AP mice.........................................................................................................56 2.2.5 Expression of the pre-excision reporter is also variegated in peripheral blod leukocytes derived from Z/AP and Z/EG mice.......59 2.3 DISCUSION..........................................................................................61 2.4 MATERIALS AND METHODS.................................................................64 2.4.1 Ethics statement.........................................................................64 2.4.2 Transgenic mice.........................................................................64 2.4.3 TIE2-tTA/Tet-O-Cre/Z/EG mice..................................................64 2.4.4 Flow cytometry...........................................................................65 2.4.5 Excision analysis........................................................................65 2.4.6 Bisulfite sequencing...................................................................66 2.5 REFERENCES........................................................................................67 CHAPTER 3. TARGETED CEL FUSION ENHANCES THE CONTRIBUTION OF HEMATOPOIETIC ELS TO SKELETAL MUSCLE FIBERS............................70 3.1 INTRODUCTION.....................................................................................70 3.2 RESULTS................................................................................................73 3.2.1 Design, construction and characterization of Hα7......................73 3.2.2 Verification of bona fide heterokaryons......................................75 3.2.3 Comparison of Hα7 and PEG induced fusion.............................77 3.2.4 Nuclear reprograming folowing Hα7 mediated fusion.............80 3.2.5 Hα7 mediated fusion in vivo.......................................................82 3.3 DISCUSION..........................................................................................85 3.4 METHODS...............................................................................................88 3.4.1 Construction of Hα7...................................................................88 3.4.2 In vitro fusion assays..................................................................89  v 3.4.3 Imunofluorescence and FISH..................................................90 3.4.4 Quantitative real-time gene expression analysis........................91 3.4.5 Lentiviral vectors........................................................................92 3.4.6 In vivo fusion assays..................................................................93 3.5 REFERENCES........................................................................................95 CHAPTER 4. CONCLUSION...................................................................................98 4.1 CONCLUSION.........................................................................................98 4.2 REFERENCES......................................................................................107 APENDIX A. ANIMAL CARE CERTIFICATES...................................................10   vi LIST OF FIGURES Figure 1.1 Tentative model of hematopoiesis…………………………………………..9  Figure 1.2 Phenotype of the circulating myogenic progenitor………………………..14  Figure 1.3 Signals afecting recruitment & diferentiation of myogenic progenitors..18  Figure 1.4 Strategy for the identification of hematopoietic lineages capable of contributing to skeletal muscle repair…………………………………………………...29  Figure 1.5 Strategy to distinguish fusion from transdiferentiation in the generation of bone marow derived muscle…………………………….……………………………...31  Figure 2.1 Genomic organization of Cre-reporter transgenes……………………….45  Figure 2.2 Activation of the EGFP reporter is ineficient in the blod of adult TIE2-tTA/Tet-O-Cre/ZEG mice…………………………………………………………….…..49  Figure 2.3 Activation of the Cre-reporter gene is less eficient in Z/AP and Z/EG mice than in R26R-EYFP mice…………………………………………………………..52  Figure 2.4 The transgenic locus is resistant to Cre-mediated recombination in a subset of granulocytes derived from the Z/EG mice…………………………………..5  Figure 2.5 The transgenic locus is methylated in reporter-negative granulocytes derived from LysM-Cre/Z/EG and LysM-Cre/Z/AP mice………………………………57  Figure 2.6 Expression of the pre-excision reporter is variegated in peripheral blod leukocytes derived from Z/AP and Z/EG mice………………………………………….60  Figure 3.1 Design, construction and characterization of Hα7……………………….74  Figure 3.2 Formation of bona fide heterokaryons……………………………………..76  Figure 3.3 Comparison of Hα7 and PEG-mediated fusion eficiencies……………..78  Figure 3.4 Nuclear reprograming folowing Hα7-mediated fusion…………………81  Figure 3.5 Hα7-mediated fusion in vivo………………………………………………..83  Figure 4.1 Model ilustrating the proposed methylation status of the Z/EG locus during early embryogenesis………………………………………………………….…10  vii CO-AUTHORSHIP STATEMENT Chapter 2 M. Long designed experiments, performed experiments, analyzed data and wrote the manuscript. F. Rossi designed experiments and edited the manuscript. Chapter 3 M. Long designed experiments, performed experiments, analyzed data and wrote the manuscript. J. Brind’Amour performed FISH analysis. F. Rossi designed experiments and edited the manuscript.  1 CHAPTER 1.  INTRODUCTION 1.1  SKELETAL MUSCLE DEVELOPMENT AND REGENERATION In vertebrate organisms, cels that are specialized for contraction may be broadly divided into four categories: skeletal muscle fibers, cardiomyocytes, smoth muscle cels and myoepithelial cels[1]. These cel types serve unique, non-overlaping functions and regulate a multitude of processes. Skeletal muscle, as the name implies, is conected by tendons to the bones of the skeleton and generates the forces required for virtualy al voluntary movements. Conversely, cardiac muscle, smoth muscle and myoepithelial cels are responsible for involuntary movements such as the beating of the heart, peristalsis and dilation of the iris respectively. Although the ability to generate contractile forces is conserved among these cel types, they are otherwise dissimilar in many respects. Skeletal muscle fibers in particular, exhibit a number of characteristics that are the direct result of a unique mode of development and regeneration. During embryonic development, skeletal muscle formation is initiated by the migration of somite-derived progenitor cels into the developing limb buds as wel as into the epaxial myotome, which eventualy forms the muscles of the main body axis[2]. In these locations, a number of signals including Wnts stimulate the proliferation of skeletal muscle progenitor cels and induce expression of the myogenic determination factors Myf5 and MyoD[3]. These basic helix-lop-helix (bHLH) transcription factors are extremely powerful regulators of gene expression and initiate a hierarchical cascade that promotes myogenesis. In fact, expression of MyoD alone is suficient to induce myogenesis in a number of diferentiated adult  2 somatic cel types including fibroblasts and chondroblasts[4, 5]. Folowing the proliferative phase, the expression of Myf5 and MyoD is repressed, alowing other transcription factors including Myogenin and Mef2 family members to activate a diferentiation program[6, 7]. However, unlike the vast majority of somatic cels, the diferentiation of skeletal muscle progenitor cels ultimately culminates in large-scale cel fusion. As a result, diferentiated skeletal muscle fibers, also known as myofibers or myotubes, are long cylindrical cels containing several hundred nuclei and can reach a length of 2-3cm in adult humans[1]. Folowing fusion, the nuclei contained within myofibers withdraw from the cel cycle and their transcriptional activity is altered to serve the function of adult skeletal muscle. This involves expression of al components of the contractile aparatus as wel as secretion of a specialized extracelular matrix known as the basal lamina, which completely surounds each myofiber, providing tensile strength and stability[8].  Importantly, during embryonic development not al skeletal muscle progenitor cels undergo the process of fusion. Folowing the initial wave of myogenesis described above, a second wave of progenitor cels, whose developmental origins remain uncertain, migrate into sites of developing muscle and take up residence betwen the plasma membrane and basal lamina of each myofiber[9]. These cels, known as satelite cels, persist during development and remain in the sublaminar position throughout adulthod[10]. Under homeostatic conditions in adult skeletal muscle, satelite cels are quiescent and are relatively rare, representing roughly 2-5% of the total nuclei present[1]. However, in response to injury, these cels proliferate and contribute to myofiber repair in a maner that recapitulates several aspects of  3 embryonic myogenesis. Although some of the factors involved in the repair of adult skeletal muscle tend to be dictated by the nature of the injury, the basic mechanisms underlying the regenerative process remain the same. In general, damaged muscle tissue is rapidly infiltrated by neutrophils and later by macrophages, which serve to phagocytose and eliminate fragments of necrotic myofibers[12]. These inflamatory cels, as wel as damaged myofibers themselves also secrete a batery of growth factors including fibroblast growth factors (FGFs), which serve to activate satelite cels within 2-6hr of injury[13]. Reminiscent of embryonic myogenesis, this activation process is characterized by entry into the cel cycle and induced expression of MyoD[14]. At this stage, activated satelite cels are often refered to as myoblasts and undergo several rounds of division within 72hr of injury[15]. Unlike embryonic myogenesis however, the subsequent specification of cel fate results in two distinct outcomes. Most myoblasts diferentiate in a myogenin dependent maner and fuse to form new myofibers within 7 days of damage. A subset of myoblasts however, prematurely downregulate MyoD, withdraw from the cel cycle and return to quiescence, thereby replenishing the satelite cel pol[16]. Although the signals governing these mutualy exclusive outcomes are curently unclear, asymmetric divisions induced by Notch signaling are suspected to play a role[17]. Formation of nascent myotubes is finaly folowed by a maturation period during which inervation occurs, such that normal tissue architecture is restored within two weks of injury[12].    4 1.2  MUSCULAR DYSTROPHY AND EXISTING THERAPIES By virtue of their central role in locomotion, skeletal muscle fibers are subjected to significant mechanical forces. However, these forces are not borne entirely by the myofibers themselves. As described above, each skeletal muscle fiber is surounded by structure known as the basal lamina, which is composed largely of a latice network of colagen IV and lamini[8]. Although the tensile strength of the latice network itself provides a level of suport to skeletal muscle fibers, the basal lamina must be linked across the plasma membrane to the cytoskeleton of each myofiber in order to provide mechanical strength and stability. In order to serve this purpose, diferentiated skeletal muscle fibers also express the laminin binding receptors, alpha7 integrin and dystroglycan[18, 19]. These transmembrane receptors are bound to beta1 integrin and sarcoglycans respectively at the cel surface as wel as to intracelular linker proteins including dystrophin, which in turn are bound to the actin cytoskeleton[20]. Although this system generaly serves to maintain myofiber integrity during contraction, mutations in a number of the components have ben described, which lead to various forms of a condition known colectively as muscular dystrophy. For example, the most comon form of this condition, Duchene muscular dystrophy, is known to be caused by mutations in the dystrophin gene[21]. Aditionaly, mutations in laminin and a number of sarcoglycans have ben described, which result in congenital muscular dystrophy and limb-girdle muscular dystrophy respectively[2-26]. In each of these conditions, myofibers are not adequately stabilized by the basal lamina and as a result, the plasma membrane is damaged and eventualy destroyed by the forces involved in simple voluntary  5 movements[27].  As described earlier, adult skeletal muscle retains a remarkable capacity for regeneration. However, this ability is not without limits. In al forms of muscular dystrophy, ongoing muscle damage eventualy exceeds the capabilities of the satelite cel-mediated repair pathway. Although the clinical severity of this outcome can be relatively mild and involve generalized muscle weakness, patients sufering from Duchene muscular dystrophy typicaly lose the ability to walk by the age of 10 and die by the age of 30[28]. At present, these patients are primarily treated with physical therapy and respiratory assistance in adition to the corticosteroid drugs Prednisone and Deflazacort, which are believed to exert their therapeutic benefit by a combination of anabolic and imunosupressive efects[29]. However, long-term use of corticosteroids is wel known to induce serious side efects including imunosupression, osteoporosis and weight gain[30]. Moreover, these treatments merely delay the progression of symptoms and do not adress the underlying cause of the disease. Given the eficiency of satelite cel-mediated skeletal muscle repair, myoblast transplantation has ben an atractive candidate for treatment of muscular dystrophies[31]. In theory, satelite cels may be harvested via muscle biopsy from a healthy donor, expanded in culture and transplanted into patients where they wil participate in muscle regeneration and provide nascent myofibers with functional copies of a defective gene. In reality however, clinical trials of this strategy over the past 20 years have yielded litle success. The failure of these protocols is now generaly atributed to two main factors.  6 I. Myoblasts survive porly folowing transplantation. In fact, several groups have reported that at least 75% of donor myoblasts die within 72hr of transplantation[32-34]. Once again, the precise mechanisms responsible for this phenomenon are unclear, although oxidative stress and fre radicals derived from inflamatory cels are suspected to play a role[35]. Perhaps more importantly, al donor-derived myoblasts are rejected in less than two weks if recipients are not placed on imunosupressive therapy[36]. At present, strategies intended to overcome these limitations are relatively unsophisticated, involving transplantation of larger numbers of cels and sustained imunosupression with FK506[31]. However, as with long-term use of corticosteroids, sustained treatment with FK506 is known to induce a number of serious side efects including nephrotoxicity and diabetes[30].   I. Myoblasts migrate porly folowing transplantation. Due to the fact that myoblasts canot be recruited from the circulation to sites of muscle damage, al clinical trials conducted to date have involved intramuscular transplantation of donor cels[37]. Unfortunately, folowing this mode of delivery, myoblasts do not migrate further than 20µm from the site of injection[38]. Therefore, systemic myoblast-based treatment of muscle tissue in Duchene muscular dystrophy patients wil require an enormous number of intramuscular injections. For example, in recent clinical trials, patients received 10 injections per cm2 to a total of 400[39-41]. Although this protocol has yielded the most encouraging results of any clinical trial to date, in the best case, dystrophin expression was restored in only 35% of myofibers and no therapeutic efect was observed in any patient. Given the severity of the disease and the absence of alternative treatment options, patients sufering from  7 Duchene muscular dystrophy may be wiling to undergo this procedure on a larger scale. However, it is clear that cel therapy of muscular dystrophies would be greatly facilitated by the identification of an alternative myogenic progenitor that is capable of homing to sites of damage via the circulation.  8 1.3  SKELETAL MUSCLE REPAIR BY BONE MAROW DERIVED CELS 1.3.1  Hematopoiesis In adition to the satelite cels of skeletal muscle, adult stem cels have ben identified in numerous tissues including bone marow[42], peripheral nervous system[43], central nervous system[4], myocardium[45], intestine[46], liver[47] and skin[48] where they apear to play a role in homeostatic maintenance and injury repair. Among these, hematopoietic stem cels residing in adult bone marow are the most highly characterized. In adult vertebrates, these cels are responsible for the generation of al mature blod lineages via a process of stepwise comitment, which progressively restricts the diferentiation potential of intermediate progenitors[49] (Figure 1.1).  9   Figure 1.1 Tentative model of hematopoiesis. Self-renewing, long-term reconstituting hematopoietic stem cels (LT-HSC) first give rise to transiently reconstituting, short-term hematopoietic stem cels (ST-HSC). ST-HSC in turn, produce comon myeloid progenitors (CMP) and comon lymphoid progenitors (CLP). CLP are the source of comited progenitors that eventualy give rise to T and B lymphocytes. CMP further give rise to megakaryocyte-erythroid progenitors (MEP) and granulocyte-macrophage progenitors (GMP). MEP are the source of comited progenitors that eventualy give rise to erythrocytes and megakaryocytes whereas GMP are the source of comited progenitors that eventualy give rise to mast-cels, neutrophils, eosinophils and monocytes.        10 While the mechanisms involved in the diferentiation of hematopoietic stem cels are extremely complex and remain the focus of intense study, it apears that both stochastic and instructive factors play a role in this process. According to the so- caled, intrinsic theory, individual hematopoietic stem cels and multipotent progenitors co-express low levels of several lineage-specific transcription factors, with eventual diferentiation being the result of stochastic reinforcement of a particular gene expression cascade and repression of al other alternatives[50]. Under homeostatic conditions, promiscuous gene expression in both hematopoietic stem cels and multipotent progenitors has ben convincingly demonstrated[51, 52]. However, extrinsic signals also clearly play a role in hematopoiesis. For example, diferentiation of T lymphocytes is absolutely dependent on IL-7 stimulation of progenitor cels within the thymus[53]. Importantly, signals derived from a number of pathological conditions may also instructively alter hematopoietic lineage diferentiation. For example, in response to acute bacterial infection, bone marow stromal cels secrete the cytokines, GM-CSF and GCSF, which stimulate multiple levels of granulopoiesis resulting in a selective increase in neutrophil levels[54]. Likewise, in response to anemia or hypoxia, the kidneys produce increased levels of erythropoietin, which stimulates increased production of erythrocytes from bone marow derived progenitors[54]. Thus, adult hematopoietic stem cels and the process of hematopoiesis in general exhibit a remarkable plasticity and are able to respond apropriately to diverse physiolgical demands.   11 1.3.2  Discovery of a circulating myogenic progenitor Originaly, it was believed that the repair of adult tissues was a semi-autonomous process as the multipotency of adult stem cels was restricted to regeneration of their tissue of origin. In 198 however, a groundbreaking study of skeletal muscle repair caled such dogma into question. This report by Ferari et al. sugested that folowing a bone marow transplant, donor derived cels were infrequently able to participate in the regeneration of skeletal muscle which had ben injured with snake venom cardiotoxin[5]. Although bone marow derived cels were found to contribute to muscle far less eficiently than satelite cels, the observation generated an enormous amount of interest in the plastic diferentiation potential of adult stem cels and provided hope for a novel, cel-based therapeutic strategy aimed at the systemic treatment of muscle degenerative diseases. This study was quickly folowed by further demonstrations of the ability of bone marow derived cels to contribute to skeletal muscle repair[56, 57] as wel as to other tissues including the heart[57], central nervous system[58, 59] and liver[60, 61]. Unfortunately, the rapid succession of such reports did not alow for a great deal of standardization of methodology. Therefore it may not be surprising that other groups, including at least two high profile reports, were unable to detect any contribution of bone marow derived cels to the brain[62] or skeletal muscle[63], casting a degre of doubt on the existence of the phenomenon and creating a great deal of controversy. Although such discrepancies have subsequently ben atributed to diferences in experimental protocol[64], a great deal of controversy remained. At the heart of the debate was a simple hypothesis, which was proposed by the earliest reports describing the  12 formation of donor-derived tissues folowing bone marow transplantation. That is, resembling their wel-known role in the repopulation of al blod lineages, hematopoietic stem cels also retain the ability to diferentiate into lineages of several other tissues. However, to date this has not ben conclusively demonstrated. Thus, at the outset of the research described here, any efort to improve upon the exceedingly low frequency of this non-classical repair process for therapeutic purposes would first require a greater understanding of the cel types and mechanisms involved.  13 1.3.3  Phenotype of the circulating myogenic progenitor For at least five years folowing the initial report of Ferari et al., the uncomited nature of hematopoietic stem cels implicated them as the direct precursors of donor derived tissues. However, early studies utilizing whole bone marow involved transplantation of a complex mixture of several lineages including mature hematopoietic cels, adipocytes, osteoblasts, and endothelial cels thereby rendering claims of hematopoietic stem cel plasticity somewhat premature. In subsequent years, few atempts have ben made to identify the phenotype of the plastic bone marow-derived lineage and most remain inconclusive due to technical dificulties. For example, studies involving intramuscular injection of fractionated bone marow have ben hampered by low viability of transplanted cels and have often yielded results difering form those obtained folowing a bone marow transplant[65-67]. Alternatively, transplantation of purified hematopoietic stem cels has ben equaly inefective in the confirmation of hematopoietic stem cel plasticity due to the potential heterogeneity of the ‘purified’ population. In an efort to circumvent these problems, a number of groups have demonstrated the occurence of donor-derived muscle in mice whose hematopoietic system had ben reconstituted with a single hematopoietic stem cel[63, 68-70]. While these experiments prove that the hematopoietic lineage contains a circulating myogenic progenitor, it remains a formal possibility that any daughter lineage of the hematopoietic stem cel is actualy directly responsible for this phenomenon. In fact Camargo et al. have sugested that the hematopoietic cels participating in the repair of skeletal muscle are myelomonocytic in origin (Figure 1.2B)[69].   14     Figure 1.2 Phenotype of the circulating myogenic progenitor. The majority of skeletal muscle repair is performed by satelite cels, which are located betwen the sarcolea and basal lamina of each muscle fiber (A). These cels are activated folowing muscle injury and fuse with multinucleated myofibers to repair damage. Recently it has ben demonstrated that bone marow derived cels also infrequently contribute to skeletal muscle repair and several models have emerged which atempt to define the lineage and mechanism involved. Camargo et al. have proposed that myelomonocytic cels are capable of fusing directly with damaged myofibers (B). Doyonas et al. have demonstrated that c-kit+ myelomonocytic precursors are capable of contributing to skeletal muscle and evidence from the same group sugests that this may occur through a satelite cel intermediate (C). Sherwod et al. have found bone marow derived cels in the satelite cel niche that apear to be non-hematopoietic in origin (D).    15 Unfortunately this study also remains inconclusive due to the fact that the selected marker of myeloid cels, Lysozyme-M, has also ben shown to be expressed in a fraction of hematopoietic stem cels[52]. This caveat may eventualy prove more academic than practical however, as other groups have also implicated myelomonocytic cels in the transfer of donor derived markers to the liver[71, 72] and skeletal muscle[73]. Utilizing fluorescence-activated cel sorting of bone marow derived cels, Doyonas et al. have demonstrated that only fractions containing c-kit+ imature myelomonocytic precursors are capable of contributing to myofibers folowing intramuscular injection (Figure 1.2C)[73]. However, in the absence of data describing the survival or expansion of each fraction folowing intramuscular injection, these results are dificult to interpret. Moreover, this technique has in the past proven to be less eficient than bone marow transplantation in the generation of donor derived muscle[6, 74], sugesting that parameters such as sustained hematopoietic engraftment or recruitment from the circulation may be important in the process. Hence, lineages other than myelomonocytic cels may also possess the ability to contribute to skeletal muscle regeneration in other experimental models.  As an alternative to the entire concept of developmental plasticity, Ratajczak et al. have proposed that circulating myogenic progenitors are simply satelite cels, which express the chemokine receptor CXCR4 and therefore accumulate in the bone marow as a result of local expression of the CXCR4 ligand, stromal-derived factor 1 (SDF-1)[75]. Clearly, this theory does not account for the presence of donor-derived muscle in mice reconstituted with a single hematopoietic stem cel. However, it remains a formal possibility that bone marow may inded contain low numbers of  16 bona fide myoblasts or that other non-hematopoietic bone marow derived lineages may also contribute to muscle repair. Along these lines, Sherwod et al. have recently demonstrated that the only subset of donor-derived cels found within the satelite cel niche which are capable of myogenesis upon co-culture with diferentiating myoblasts do not express the pan-hematopoietic marker, CD45[76]. While it may be reasonable to concede that this marker could be down regulated in a myogenic environment, Sherwod et al. also demonstrate that this population is only generated folowing transplantation of whole bone marow and not folowing transplantation of highly purified hematopoietic stem cels, sugesting a mesenchymal rather than hematopoietic origin (Figure 1.2D). Thus short of once again invoking variations introduced by disparate methodologies, one must postulate the existence of multiple sources of a circulating myogenic progenitor in order to reconcile these various reports.       17 1.3.4  Recruitment of the circulating myogenic progenitor While many of the factors involved in the conversion of bone marow derived cels to muscle remain unknown, it is now quite clear that muscle damage is an important requirement for this phenomenon. For example, intramuscular injection of snake venom toxins such as cardiotoxin or notexin has ben demonstrated to reliably induce regeneration mediated via bone marow derived cels, yet the efect is rarely observed in contralateral, unijured muscles[68, 69]. In a more clinicaly relvant seting, the chronic myofiber degeneration observed in the murine mdx model of Duchene muscular dystrophy apears to be suficient to induce the myogenic conversion of bone marow derived cels[69]. Notably, the recruitment of macrophages to damaged muscle is observed in both of these models and has ben interpreted as further prof of the involvement of myelomonocytic cels in the contribution of bone marow derived cels to skeletal myofibers[29, 69]. The process of inflamation however, involves extremely sophisticated means of recruiting cels from the circulation and the chemokines involved may also specificaly sumon other progenitor cels to damaged muscle. Recently, a role for SDF-1 in such traficking has ben sugested by a number of groups (Figure 1.3A)[7-79].          18          Figure 1.3 Signals afecting recruitment and diferentiation of myogenic progenitors. A number of groups have demonstrated that the recruitment of bone marow derived cels to damaged muscle is enhanced by local expression of stromal derived factor-1 (SDF-1) (A). However, delivery of this chemokine to uninjured muscle is insuficient to promote such recruitment, sugesting a role for unidentified, damage-induced factors in the homing process. Polesskaya et al. have shown that daaged muscle also upregulates various Wnt isoforms, which subsequently act upon resident CD45+/Sca1+ cels to induce expression of myogenic markers (B).         19  In adition to describing the SDF-1 based recruitment of satelite cels to bone marow, Ratajczak et al. have also sugested that these cels are re-mobilized to peripheral blod and return to damaged muscle in response to a gradient of SDF-1 expressed therein folowing injury[7]. Likewise, myocardial infarction has ben shown to transiently induce expression of SDF-1 in injured cardiac tissue, greatly enhancing the recruitment of bone marow derived cels, including a c-kit+ fraction, to the heart[78, 79]. Conversely, expression of viraly delivered or localy injected SDF-1 as wel as G-CSF induced mobilization of bone marow progenitor cels to the peripheral blod has proven insuficient to promote such homing in the absence of injury, sugesting a role for auxiliary damage-induced factors in the recruitment process[78, 79]. In agrement, Musaro et al. have demonstrated that recruitment of bone marow derived cels to damaged skeletal muscle is also enhanced by local expression of insulin-like growth factor 1 (IGF-1)[80]. Interestingly, although this study as wel as those of SDF-1 demonstrate significantly increased recruitment of bone marow derived cels to damaged muscle tissue, none have reported a resultant increase in the frequency of donor-derived skeletal myofibers or cardiomyocytes. These observations imply that the mere recruitment of bone marow derived cels to a site of injury may not be the rate limiting step in the formation of donor derived muscle and shift the focus to localy acting processes which folow homing.     20 1.3.5  Fusion versus transdiferentiation As mentioned earlier, the studies reported by Ferari et al. as wel as those that folowed were imediately and widely atributed to so-caled stem cel plasticity despite a paucity of evidence to suport either the participation of stem cels or plasticity in the process. Therefore yet another caveat to temper this burgeoning field has ben the possibility that the contribution of donor derived markers to non-hematopoietic recipient tissues is a consequence of fusion betwen donor and recipient cels as oposed to spontaneous, plastic diferentiation of stem cels across lineage boundaries or transdiferentiation of mature lineages. This scenario was initialy proposed folowing the observation of spontaneous fusion betwen co-cultured embryonic stem cels and GFP positive bone marow resulting in hyper-diploid cels expressing GFP yet retaining pluripotency[81]. Folowing up on this in vitro data, a number of subsequent reports have inded confirmed the role of cel fusion in the transfer of donor derived markers to liver, heart and central nervous system tissues folowing a bone marow transplant[82-86]. This is not to say that the process of environmentaly induced reprograming has ben completely dismissed. In fact, fusion does not apear to play a role in the contribution of bone marow-derived markers to insulin producing pancreatic islet cels[87] or epithelial cels of the lung, liver and skin[8]. In the case of skeletal muscle, the role of either fusion or transdiferentiation in the formation of myofibers expressing donor derived markers remains contentious for several reasons including the confounding efects of myoblast fusion in the physiological formation and repair of this tissue. This is certainly the case in studies that utilize the co-expression of  21 donor and recipient specific markers within a single myofiber to sugest fusion betwen donor-derived hematopoietic cels and mature host myofibers[69, 74]. Formaly, these experiments do not exclude the possibility that such data may be due to the fusion of endogenous myoblasts with a nascent, autonomous, donor-derived myofiber. Nor do they exclude the possibility that the donor-derived cel had converted to a myogenic phenotype prior to fusion with an existing myofiber. On the other hand, studies implicating transdiferentiation in the formation of myofibers expressing donor-derived markers are not without their own problems. For example, LaBarge and Blau have proposed an enticingly intuitive mechanism in which iradiation induced damage first recruits bone marow-derived cels to the satelite cel niche beneath the basal lamina where local environmental cues induce these cels to convert to a resting myogenic phenotype. Subsequently, exercise-induced damage activates these cels and elicits the contribution of donor-derived markers to regenerating myofibers via the physiological satelite cel mediated repair pathway (Figure 1.2C)[89]. Although subsequent studies have reported similar results[90, 91], others have contradicted both this model and each other, finding either no donor-derived satelite cels[69] or donor-derived cels occupying the satelite cel niche that, while expressing some myogenic markers, were not autonomously myogenic in vitro[76]. The later discrepancy may be reconciled based on the fact that the myoblast isolation employed by LaBarge and Blau to test the myogenic potential of bone marow-derived cels, included a nine-day expansion of mononuclear cels from the muscle of transplant recipients prior to re-plating in clonal conditions. This step therefore, may have recapitulated the co-culture of donor  22 derived and endogenous myofiber associated cels, which Sherwod et al. demonstrate to be efective in inducing a myogenic phenotype in a subset of myofiber-associated bone marow derived cels[76]. Furthermore, both studies agre that the donor-derived cels occupying the satelite cel niche express a variety of myogenic markers and are capable of contributing to the formation of donor derived myofibers in vivo.  As for the local environmental cues involved in the myogenic conversion of bone marow derived cels, Polesskaya et al. have shown that the Wnt isoforms up-regulated within damaged skeletal muscle, activate the canonical Wnt signaling pathway in resident CD45+/Sca1+ cels in vivo and induce expression of early myogenic markers including Pax 7 within the same cels in vitro (Figure 1.3B)[92]. Although expression of the time-tested hematopoietic marker, CD45, strongly sugests that these cels may be the progeny of hematopoietic stem cels, their origin in bone marow has yet to formaly proven. The biological progression from bone marow through satelite cel to myofiber described above provides an atractive model for transdiferentiation. However, none of the relevant reports formaly exclude the possibility that the intermediate myogenic precursors may be themselves the product of fusion. Karyotypic analysis sugesting that these cels are diploid was performed after in vitro expansion, during which reductive divisions may have led to the loss of the extra chromosomes and the reversion to a diploid genotype[89]. Therefore at present, the precise mechanism responsible for the generation of donor derived skeletal myofibers remains unclear and it is entirely possible that both fusion and transdiferentiation play a role.  23 1.3.6  Nuclear reprograming Many reports have described the expression of donor-derived markers in non-hematopoietic recipient tissues folowing a bone marow transplant as ‘plasticity’ or a ‘contribution’ of bone marow derived cels. Yet very few studies have examined the degre to which gene expression is altered in donated nuclei or how significant this contribution is to the function of any given recipient tissue. In many cases, donor-derived cels are marked with a transgene such as LacZ or GFP, which is ubiquitously expressed and therefore provides no evidence of nuclear reprograming. Furthermore, the expression of such genes would hardly be expected to contribute to the function of engrafted tissues. This however, is not to say that the nuclei of mamalian somatic cels are completely refractory to significant and meaningful alterations in gene expression. In fact, the ability of cytoplasmic factors to activate previously silenced genes was demonstrated in heterokaryons over two decades ago[93, 94]. More importantly, the generation of cloned animals via transfer of adult somatic cel nuclei to enucleated ocytes has demonstrated that terminaly diferentiated cels retain the potential to restablish the gene expression profile required to produce functional cels of any tissue[95-97]. Although both of these examples are reminiscent of cel fusion as they involve the exposure of nuclei from one cel type to cytoplasmic factors in another, in vitro and in vivo examples of transdiferentiation are also known to occur. For example, the conversion of cultured myoblasts to adipocytes has ben accomplished via ectopic expression of the transcription factors PARγ and C/EBPα[98] or inhibition of Wnt signaling[9]. In vivo, the diferentiation of pancreatic epithelial progenitor cels into  24 hepatocytes folowing engraftment of the adult rat liver has also ben described[10]. More recently, the transfer of a smal number of transcription factors including Oct4, Sox2, Klf4 and c-myc has ben shown to revert several diferentiated cel types to a state of pluripotency, thereby creating so-caled ‘induced pluripotent stem cels’ or iPS cels[101-104]. These cels possess the capacity for diferentiation into diverse lineages in vitro as wel as the ability to contribute to chimeric animals in vivo[105-108]. As an extension of this phenomenon, the transfer of other smal groups of transcription factors has ben shown to directly convert pancreatic exocrine cels to insulin-producing beta cels as wel as to stimulate the direct conversion of fibroblasts to functional neurons[109, 10]. Thus, nuclear reprograming of adult somatic cels is clearly possible. The issue now confronting the plasticity of bone marow derived lineages regards the completeness of such reprograming.  In a limited number of cases, a degre of reprograming has ben demonstrated by activation of the muscle specific, myosin light chain 3F promoter[5, 69] or by expression of dystrophin[56, 57] folowing incorporation of bone marow derived cels into skeletal myofibers. These experiments however, do not imply complete conversion from a hematopoietic to myogenic gene expression profile within donor-derived nuclei. On the contrary, the generation of endogenous, revertant fibers via exon skiping in mdx mice is wel documented, casting doubt on the reliability of dystrophin expression as a halmark of reprograming[11]. Furthermore, Lapidos et al. have recently reported that in a murine model of cardiomyopathy and muscular dystrophy caused by targeted disruption of the of δ-sarcoglycan gene, the majority of  25 donor derived cels engrafting either skeletal or cardiac muscle fail to activate δ-sarcoglycan expression despite exposure to syncytial myogenic transcription factors[12]. As stated by the authors however, the extent to which these donor-derived nuclei may have ben reprogramed is unknown. Early markers of myogenesis such as MyoD or desmin may have ben activated yet reprograming may not have reached a threshold required to facilitate expression of late markers including δ-sarcoglycan[13]. Therefore, the infrequency with which bone marow derived cels contribute to skeletal muscle also curently prevents systematic investigation of subsequent nuclear reprograming processes.  26 1.3.7  Prospects for therapy To date, the most convincing demonstration of the ability of bone marow derived cels to contribute to the repair of a non-hematopoietic tissue has ben in a murine model of the genetic liver disease, fumarylacetoacetate hydrolase (Fah) deficiency. In this model, a functional copy of the Fah enzyme is provided to Fah-/- host hepatocytes via fusion with wild type myelomonocytic cels folowing a bone marow transplant. As a result, donor derived hepatocytes are given a growth advantage alowing them to expand and regenerate the majority of the liver, curing mice of the disease. Unfortunately, an analogous myopathy model does not exist and with rare exceptions, the expansion of bone marow derived myogenic cels has not ben described[14].  More recently, a number of studies have demonstrated that transplanted bone marow cels are also capable of contributing to functional improvement folowing stroke injury[15]. At present it apears that this efect is mediated by the secretion of cytokines and growth factors as wel as by direct participation of bone marow derived cels in angiogenesis[16, 17]. Interestingly, both fusion and transdiferentiation have ben shown to play a role in the formation of blod vessels folowing stroke and are responsible for generating bone marow derived pericytes and endothelial cels respectively[18, 19]. Although this process, much like the generation of donor derived muscle is known to be enhanced by inflamation, very litle is known about the particular mediators involved, thereby precluding significant enhancement of either phenomenon[18].    27 Aside from discordant reports of treatment success in animal models of muscular dystrophy, most groups agre that the frequency with which bone marow-derived cels contribute to regenerating muscle is exceedingly low, thus precluding any clinical aplication[120-123]. Inded, similarly low frequencies of donor-derived nuclei have ben observed in myofibers of Duchene’s patients who received a bone marow transplant to treat an unrelated pathology[124]. Despite this lack of preclinical success, the desire to transfer findings from the bench to the bedside has led rapidly to clinical trials involving intracoronary transplantation of hematopoietic progenitor cels in an atempt to regenerate cardiac myocytes lost as a result of an infarct[125]. However, recent data demonstrates that under these conditions hematopoietic stem cels make no contribution to the ischemic myocardium, sugesting that this strategy may be il founded[6, 67], or that the mechanism underlying potential beneficial efects is not based on myocyte replacement. These results underscore the fact that clinical advances must await more rigorous characterization of the mechanisms involved in the generation of bone marow derived muscle as wel as the development of strategies to increase its eficiency to therapeutic levels.  28 1.4  SPECIFIC AIMS 1.4.1  Identification of hematopoietic lineages responsible for the generation of bone marow derived muscle. This aim was to be adressed utilizing a Cre-loxP based tracing strategy. In theory, specific hematopoietic lineages would be labeled by crossing Z/EG reporter mice[126] with transgenic strains expressing Cre-recombinase in T cels (Lck-Cre)[127], B cels (CD19-Cre)[128] or myelomonocytic cels (LysM-Cre)[129]. Thus, folowing transplantation of labeled bone marow into wildtype mice, the presence of GFP-positive myofibers in recipients would identify the capability of an individual lineage to contribute to skeletal muscle (Figure 1.4).   29                              Figure 1.4 Strategy for the identification of hematopoietic lineages capable of contributing to skeletal muscle repair. (A) In Z/EG reporter mice, transcription of a β-galactosidase/neomycin phosphotransferase fusion gene (β-geo) is driven by a hybrid CMV/β-actin prooter (pCAGS) and terminated by polyadenylation sites (white boxes). Cre recombines loxP sites (white triangles) to remove the β-geo gene, activating expression of enhanced gren fluorescent protein (EGFP). (B) In the experimental set-up, the Z/EG strain is first crossed with mice expressing Cre-recobinase in a specific hematopoietic lineage. Bone marow from the resulting ofspring is then transplanted into wild-type recipients. In this way, contribution of the labeled lineage to skeletal muscle wil be easily detectable by expression of the donor-derived reporter in recipient muscle fibers (C).                Skeltal Muscle  Bone Marrow Transplant into WT Mice  Fusion or Transdiferentiation  Donor Derived Muscle  Lineage-Specific Cre Strain  Z/EG x Labeled Lineage Labeled-Lineage Mice  A B Myofiber  BM Transplant  GFP+ Myofiber pCAGGS EGFP  β-geo pCAGGS EGFP C Cre  30 1.4.2  Identification of the mechanism responsible for the generation of bone marow derived muscle This aim was to be adressed utilizing a modification of the Cre-loxP based tracing strategy described above in order to distinguish fusion events from transdiferentiation events. PCX-NLS-Cre transgenic mice[130], which ubiquitously express Cre recombinase, were to be transplanted with bone marow from the Z/EG reporter strain. Thus in transplant recipients, the expression of β-galctosidase would identify products of transdiferentiation while EGFP expression would distinguish products of fusion (Figure 1.5)                         31                                  Figure 1.5 Strategy to distinguish fusion from transdiferentiation in the generation of bone marow derived muscle. PCX-NLS-Cre mice are transplanted with bone marow derived from the Z/EG reporter strain. Thus, if hematopoietic cels contribute to skeletal muscle by fusion, Cre expressed by recipient nuclei wil activate EGFP expression in donor nuclei (A). Alternatively, if hematopoietic cels contribute to skeletal muscle by transdiferentiation, donor nuclei wil not be exposed to Cre and continue to express β-galactosidase (B).               PCX-NLS-Cre Skeltal Muscle  Transdiferentiation  Donor Derived Muscle  B                PCX-NLS-Cre Skeltal Muscle  Fusion  Donor Derived Muscle  Z/EG Hematopoietic Cel A Recombination  Z/EG Hematopoietic Cel Myofiber  BM Transplant  GFP+ Myofiber  Myofiber  BM Transplant  LacZ+ Myofiber   32 Clearly these experiments rely on eficient activation of the EGFP reporter in Z/EG mice. Therefore, we first analyzed the labeling eficiency of adult hematopoietic cels derived from this reporter strain. As described in Chapter 2, we discovered that epigenetic silencing inhibits Cre-mediated recombination of the Z/EG reporter in adult cels, thereby precluding our initial specific aims.    33 1.4.3  Increase the contribution of bone marow derived cels to skeletal muscle repair. The contribution of bone marow derived cels to skeletal muscle repair is now largely agred to be due to the fusion of macrophages with damaged myofibers[69, 73]. However, the mechanisms involved in this process remain insuficiently understod to enhance its eficiency for therapeutic purposes. As stated earlier, recruitment of bone marow derived cels to the site of injury does not apear to be the rate limiting step in the formation of donor derived muscle. In fact, in several human skeletal myopathies, muscle tissue is chronicaly inflamed as a result of ongoing fiber degeneration[131]. Therefore, we have designed a system to increase the eficiency with which these inflamatory cels fuse with skeletal muscle fibers. As described in Chapter 3, we have created a chimeric measles hemaglutinin, which specificaly and eficiently mediates the fusion of diverse cel types with skeletal muscle both in vitro and in vivo. We anticipate that this reagent may facilitate the development of novel cel and gene therapies for skeletal myopathies.    34 1.5  REFERENCES 1. 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An Neurol, 1984. 16(2): p. 193-208.            44 CHAPTER 2.  SILENCING INHIBITS CRE-MEDIATED RECOMBINATION OF THE Z/AP AND Z/EG REPORTERS IN ADULT CELLS1 2.1  INTRODUCTION The bacteriophage P1 enzyme, Cre, recognizes and recombines two copies of a specific 34 base-pair sequence known as a loxP sequence located at each end of the phages linear genome. This process converts the genome to a unit-copy circular plasmid ensuring proper replication and partitioning of the prophage [1,2,3]. This process does not require cofactors, therefore loxP sites inserted into the murine genome are also recognized and recombined by Cre, resulting in excision, inversion or translocation of chromosomal sequence depending on the orientation and location of the loxP sites [4]. To date, hundreds of transgenic mouse strains have ben created which express Cre under the control of tissue specific or inducible promoters and in combination with transgenic mice containing loxP-flanked aleles, have revolutionized the study of the genetic factors involved in a multitude of biological processes. In order to facilitate such studies, several so-caled Cre-reporter strains have also ben created [4]. These mice generaly cary the gene for an easily detectable marker, the expression of which is only activated folowing exposure to Cre. For example, in the Z/AP reporter strain (Figure 2.1A), transcription of a β-galactosidase/neomycin phosphotransferase fusion gene (β-geo) is driven by a hybrid CMV enhancer/chicken β-actin promoter (pCAGS) and terminated by a trimer of SV40 polyadenylation sites [5].                                                 1 A version of this chapter has ben published. Long MA and Rosi FMV (209) Silencing Inhibits Cre-Mediated Recombination of the Z/AP and Z/EG Reporters in Adult Cels. PLoS ONE. 4(5):e5435  45          Figure 2.1 Genomic organization of Cre-reporter transgenes. In the Z/AP strain (A) and Z/EG strain (B), transcription of a β-galactosidase/neomycin phosphotransferase fusion gene (β-geo) is driven by a hybrid CMV/β-actin promoter (pCAGS) and terminated by polyadenylation sites (white boxes). Cre recombines loxP sites (white triangles) to remove the β-geo gene, activating expression of human placental alkaline phosphatase (hPLAP) or enhanced gren fluorescent protein (EGFP) in the Z/AP and Z/EG strains respectively. In the R26R-EYFP strain (C), Cre-mediated recombination of loxP sites removes a phosphoglycerate kinase (PGK) promoter and a neomycin phosphotransferase gene (NeoR), alowing the endogenous ROSA26 genoic locus to drive expression of the downstream enhanced yelow fluorescent protein (EYFP) gene.       46 The β-geo gene and polyadenylation sites are flanked by loxP sites oriented in the same direction. Therefore, upon exposure to Cre, the β-geo gene is excised and transcription of the human placental alkaline phosphatase (hPLAP) gene is activated by proximity to the pCAGS promoter. The Z/EG strain (Figure 2.1B) was derived from the Z/AP reporter and contains the cDNA for enhanced gren fluorescent protein (EGFP) in place of hPLAP [6]. Thus, al cels of Z/AP and Z/EG mice are designed to exhibit a binary readout of Cre activity, expressing β-galactosidase by default and activating expression of hPLAP or EGFP respectively upon exposure to Cre. The R26R-EYFP strain (Figure 2.1C) was generated by targeted insertion of a Cre reporter cassete into the ROSA26 genomic locus, which has ben shown to be ubiquitously expressed throughout development and in adult tissues [7,8]. The reporter cassete contains a PGK promoter driving expression of a neomycin phosphotransferase gene, both of which are removed by Cre mediated recombination of flanking loxP sites. Folowing excision, the endogenous ROSA26 promoter drives expression of the downstream enhanced yelow fluorescent protein (EYFP) gene. Cre-reporter strains have ben utilized to validate the expression profile of Cre transgenes [9], to act as a surogate marker for excision of a second alele [10], to ireversibly label cels for lineage tracing experiments [1] and to diferentiate betwen fusion and transdiferentiation in studies of stem cel plasticity [12]. Although it is known that the chromosomal integration site of loxP sequences can afect the eficiency with which they are recombined [13], to date there has not ben a systematic comparison of the labeling eficiencies of some of the most widely used  47 Cre-reporter strains. We have undertaken such a comparison and have demonstrated that the eficiency of reporter activation in adult cels derived from the Z/AP and Z/EG strain is much lower than in adult cels derived from the R26R-EYFP strain. Furthermore, our evidence sugests that the ineficient labeling eficiency observed in the Z/AP and Z/EG strains is due to methylation of the pCAGS promoter which prevents both reporter expression and Cre-mediated recombination of the transgenic locus.   48 2.2  RESULTS 2.2.1  Ineficient activation of the Cre reporter in hematopoietic stem cels derived from adult Z/EG mice The Z/EG reporter strain has become a valuable tol for studying embryonic development [14,15]. However, we were interested in utilizing this strain to study adult hematopoiesis and therefore created the triple transgenic TIE2-tTA/Tet-O-Cre/Z/EG strain. In these mice, expression of the tetracycline-transactivator is driven by the TIE2 promoter, which has ben shown to be active in hematopoietic stem cels [16,17]. Therefore folowing removal of doxycycline from the diet of mice, the tetracycline-transactivator is able to bind to the tet-operator and drive expression of Cre in hematopoietic stem cels, theoreticaly resulting in expression of the EGFP reporter in al hematopoietic lineages. In order to determine the kinetics of reporter activation in this system, we analyzed the blod of triple transgenic mice for expression of EGFP at several intervals folowing removal of doxycycline from the diet (Figure 2.2A-C).  49                                Figure 2.2 Activation of the EGFP reporter is ineficient in the blod of adult TIE2-tTA/Tet-O-Cre/ZEG mice. EGFP expression in the blod of a representative triple transgenic mouse, 3, 6 and 9 months folowing removal of doxycycline from the diet is shown in (A-C) respectively.  EGFP expression in the blod of a representative triple transgenic mouse, bred in the absence of doxycycline is shown in (D). In al triple transgenic mice analyzed, activation of the EGFP reporter was not observed in peripheral blod leukocytes (PBL) earlier than 6 months folowing removal of doxycycline and remained low at nie months post induction (E).   50 Although EGFP positive cels were eventualy detected in the blod of al triple transgenic mice, in the best case less than 10 percent of peripheral blod leukocytes were labeled folowing nine months of induction (Figure 2.2E). Conversely, the blod of triple transgenic mice that developed in the absence of doxycycline was labeled quite eficiently, validating that the system does function properly during development (Figure 2.2D). While this sugests that the Z/EG reporter may not function as wel in adult hematopoietic cels as it is during development, TIE2 expression has ben proposed to maintain hematopoietic stem cels in a quiescent state, thus it remains possible that in triple transgenic mice, TIE2 positive hematopoietic stem cels are labeled quite eficiently and simply do not contribute significantly to the peripheral blod [18]. Furthermore, although unlikely, it is possible that folowing removal of doxycycline from the diet of triple transgenic mice, the levels of the drug remaining in vivo are suficient to supress expression of Cre.  51 2.2.2  Ineficient activation of the Cre reporter in myeloid cels derived from adult Z/AP and Z/EG mice In an efort to eliminate pharmacological complications and to test the hypothesis that the Z/EG reporter does not function as eficiently in adult hematopoietic cels as it does during development, we crossed Z/EG mice to the LysM-Cre strain, which expresses Cre in al myelomonocytic cels and to the general deleter strain pCX-NLS-Cre [19,20]. As a basis for comparison, LysM-Cre and pCX-NLS-Cre mice were also bred to the Z/AP reporter strain as wel as to the R26R-EYFP reporter strain and peripheral blod leukocytes were analyzed for expression of the apropriate post-excision reporter. As expected, the R26R-EYFP reporter was activated in al peripheral blod leukocytes of pCX-NLS-Cre/R26R-EYFP mice (Figure 2.3A) and in 85 percent of granulocytes of LysM-Cre/R26R-EYFP mice (Figure 2.3B).  52                              Figure 2.3 Activation of the Cre-reporter gene is les eficient in Z/AP and Z/EG mice than in R26R-EYFP mice. (A) Al reporter strains were crossed to the general deleter strain, pCX-NLS-Cre, and activation of each reporter gene was assessed by flow cytometry of peripheral blod leukocytes at 12 weks of age. (B) Al reporter strains were also crossed to the myeloid-specific LysM-Cre strain, and activation of each reporter gene was assessed by flow cytometry of peripheral blod granulocytes at 12 weks of age. Representative histogras demonstrating reporter expression in peripheral blod granulocytes are shown in (C-E) for LysM-Cre/R26R-EYFP, LysM-Cre/ZAP and LysM-Cre/Z/EG mice respectively (solid lines). Reporter expression in BL/6 mice is shown in doted lines.   53 A similar labeling eficiency of LysM-Cre/R26R-EYFP granulocytes has previously ben reported and most likely aproaches the maximum labeling eficiency of these short-lived cels utilizing a Cre transgene expressed from a promoter which is activated during their life-cycle [21]. In contrast to these results, the average labeling eficiency of granulocytes in the LysM-Cre/Z/AP and LysM-Cre/Z/EG strains was only 57 percent and 36 percent respectively (Figure 2.3B). A similar labeling eficiency has ben reported for the Z/EG strain folowing a cross to a separate myeloid specific-Cre strain [9]. These data, taken together with the observation that it is possible to activate the hPLAP and EGFP reporter in virtualy al blod cels of most pCX-NLS-Cre/Z/AP and pCX-NLS-Cre/Z/EG mice (Figure 2.3A), further suports the hypothesis that the Z/AP and Z/EG reporters do not function as eficiently in adult hematopoietic cels as they do during development. The presence of a significant percentage of hPLAP negative and EGFP negative cels in a subset of pCX-NLS-Cre/Z/AP and pCX-NLS-Cre/Z/EG mice (Figure 2.3A) has also ben reported by others and demonstrates that even under conditions of embryonic exposure to high levels of Cre recombinase, the Z/AP and Z/EG reporters are not completely reliable [6,12,2].  54 2.2.3  The transgenic locus is resistant to Cre-mediated recombination in a subset of granulocytes derived from Z/EG mice The diferential labeling eficiencies observed in LysM-Cre/R26R-EYFP, LysM-Cre/Z/AP and LysM-Cre/Z/EG mice are unlikely to be due to variable expression of functional Cre recombinase as al mice contain the same LysM-Cre transgene and were maintained on the same genetic background. Therefore, we reasoned that the ineficient labeling of Z/EG and Z/AP granulocytes may be due to impaired recombination and/or ineficient expression of the transgenic loci. In order to diferentiate betwen these two scenarios, we designed a PCR-based strategy to examine the eficiency of Cre-mediated excision of the β-geo gene. Granulocytes from LysM-Cre/Z/EG mice were first sorted into reporter-negative and reporter-positive populations (Figure 2.4A). Genomic DNA from these groups was then subjected to PCR reactions containing primers designed to generate an amplicon only in the presence of a recombined transgenic locus. As seen in Figure 2.4B, reporter positive cels contain a recombined locus as expected. In the reporter-negative population however, the recombined locus was not detected, sugesting that Cre may be unable to eficiently access the loxP sites in the genomic DNA of cels derived from the LysM-Cre/Z/EG strain.  55                      Figure 2.4 The transgenic locus is resistant to Cre-mediated recombination in a subset of granulocytes derived from the Z/EG mice. (A) Gr-1 positive cels from a LysM-Cre/Z/EG mouse were sorted into reporter negative (-) and reporter positive (+) populations. (B) Genomic DNA from these populations was then analyzed by PCR utilizing primers binding within the pCAGS promoter and EGFP cDNA. This combination of priers (Exc) generates a 240bp fragent in the presence of the recombined locus whereas the distance across the intact locus is to large to facilitate exponential amplification under the conditions used. Primers recognizing the IL-2 inducible T-cel kinase (ITK) gene were also utilized as a positive control in reporter negative (-) and reporter positive (+) reactions as wel as in a no-template (NT) control.  56 2.2.4  The transgenic locus is methylated in reporter-negative granulocytes derived from LysM-Cre/Z/EG and LysM-Cre/Z/AP mice As DNA methylation is known to be one of the primary mechanisms by which eukaryotic cels silence foreign DNA, we hypothesized that the Z/EG transgene is methylated and incorporated into heterochromatin in adult hematopoietic cels, thereby reducing the accessibility of the loxP sites for Cre mediated recombination [23,24]. We therefore subjected a 20 base-pair segment of the pCAGS promoter containing 36 CpG dinucleotides to bisulfite sequencing in order to examine its methylation status in both reporter-negative and reporter-positive granulocytes from the LysM-Cre/Z/EG strain. As seen in Figures 2.5A,C, this segment of the pCAGS promoter is inded methylated to a greater extent in reporter negative cels than it is in reporter positive cels.  57    Figure 2.5 The transgenic locus is methylated in reporter-negative granulocytes derived from LysM-Cre/Z/EG and LysM-Cre/Z/AP mice. Genomic DNA from reporter negative and reporter positive populations was subjected to bisulfite sequencing in order to determine the methylation status of a 20 bp region of the pCAGS promoter. This region contains 36 CpG dinucleotides, significantly more of which were methylated (filed circles) in clones derived from reporter negative cels than in those derived from reporter positive cels (A,B). The degre of methylation at the pCAGS and ROSA promoters in both reporter negative (-) and reporter positive (+) populations is shown in (C) and (D) for cels derived from LysM-Cre/ZEG and LysM-Cre/Z/AP mice respectively. The degre of methylation at the ROSA promoter in cels derived from the LysM-Cre/R26R-EYFP strain is shown in (E).   58 In order to demonstrate that hypermethylation is not a global phenomenon in reporter negative cels, we also determined the methylation status of a 253 base-pair segment of the endogenous ROSA26 promoter containing 28 CpG dinucleotides in both reporter-negative and reporter positive cels. As seen in Figure 2.5C, very litle methylation was observed at this locus in al clones examined, regardless of reporter expression. A similar corelation betwen hypermethylation of the pCAGS promoter and ineficient expression of the Cre-reporter gene was also observed in granulocytes obtained from LysM-Cre/Z/AP mice (Figures 2.5B,D). A corolary to our hypothesis that DNA methylation inhibits Cre-mediated activation of the reporter gene in the Z/AP and Z/EG strains is that the ROSA26 promoter should be unmethylated in granulocytes derived form the LysM-Cre/R26R-EYFP strain. As seen in Figure 2.5E, this is inded the case.  59 2.2.5  Expresion of the pre-excision reporter is also variegated in peripheral blod leukocytes derived from Z/AP and Z/EG mice As a final confirmation of the fact that the Z/AP and Z/EG loci are silenced in adult hematopoietic cels, we also quantified expression of the pre-excision reporter, β-galactosidase, via fluorescein-di-beta-D-galctopyranoside (FDG) staining of peripheral blod leukocytes taken from these mice. As seen in Figure 2.6, the pre-excision reporter is also ineficiently expressed in adult hematopoietic cels of Z/AP and Z/EG mice presumably due to epigenetic silencing of the transgenic locus.    60             Figure 2.6 Expresion of the pre-excision reporter is variegated in peripheral blod leukocytes derived from Z/AP and Z/EG mice. Silencing of the Z/AP and Z/EG transgenic loci was demonstrated via quantification of the percentage of peripheral blod leukocytes expressing β-galctosidase by FDG staining. Data are shown as mean ± s.d. (n=3).  61 2.3  DISCUSION We have demonstrated a diminished sensitivity to Cre-mediated recombination in adult hematopoietic cels derived from Z/AP and Z/EG mice. These reporter cassetes were randomly inserted into the mouse genome and as such are more likely to be subjected to position efect variegation than the R26R-EYFP reporter, which was inserted into the ubiquitously expressed ROSA26 genomic locus. In accordance with this hypothesis we have also demonstrated that expression of the pre-excision reporter is variegated in adult hematopoietic cels derived from Z/AP and Z/EG mice. The diference in the degre of silencing observed betwen Z/AP and Z/EG cels is unlikely to be due to diferences in the location or extent to which the pCAGS promoter is methylated as these parameters apear to be quite similar betwen cels derived from the two strains (Figure 2.5A-D). Thus, diferences in the chromatin conformation of the loci into which the transgenes have ben inserted likely influence the frequency and not the extent to which the pCAGS promoter is subjected to methylation, underlying the observed diferences in silencing. Interestingly, the percentage of cels expressing the pre-excision reporter (Figure 2.6) in both Z/AP and Z/EG mice is remarkably similar to the percentage of cels expressing the post-excision reporter (Figure 2.3B) in LysM-Cre/Z/AP and LysM-Cre/Z/EG mice respectively. This observation sugests that the population of cels that express the pre-excision reporter may be the only cels capable of undergoing Cre-mediated activation of the post-excision reporter in adult granulocytes.   62 In order to explain these findings, we propose a model wherein the Z/AP and Z/EG loci are demethylated after fertilization as a result of the genome wide demethylation that is known to occur in pre-implantation embryos [25]. Therefore, if these transgenes are exposed to Cre recombinase shortly after fertilization, such as by crossing to the pCX-NLS-Cre strain, which ubiquitously expresses Cre, the locus is accessible, the β-geo gene is eficiently excised and the downstream reporter is expressed. As embryonic development progresses however, the Z/AP and Z/EG transgenes become methylated, resulting in the eventual incorporation of the transgenic loci into heterochromatin, which inhibits access of transcription factors and Cre recombinase. Therefore, if these transgenes are exposed to Cre recombinase in adult cels, such as by crossing to the LysM-Cre strain, the β-geo gene is ineficiently excised and the locus is ineficiently expressed. The presence of the β-galactosidase (LacZ) sequence within the β-geo gene may be a significant contributor to this efect as the CpG-rich LacZ cDNA is known to induce silencing of some genes to which it is fused [26]. In accordance with this notion, we have demonstrated that excision of the β-geo gene shortly after fertilization significantly reduces silencing of the reporter loci in adult cels (Figure 2.3A). Although we have restricted our analysis to the labeling eficiency of hematopoietic cels, other groups have also reported low labeling eficiency utilizing the Z/EG strain in the adult kidney, liver, testis, adrenal glands, fat tissue, lung, pituitary gland, splen and retina [27,28]. Furthermore, Rotolo et al. have recently developed a method for the analysis of neuronal morphology, which is based in part on the ineficiency with which the Z/AP locus is recombined in the adult brain [29]. Our  63 findings highlight the potential shortcomings of utilizing these particular Cre-reporters as surogate markers of excision or in lineage tracing experiments. Therefore, the R26R-EYFP reporter may be the strain of choice for researchers interested in tracing the expression of Cre beyond early development.  64 2.4  MATERIALS AND METHODS  2.4.1  Ethics statement Al experiments were performed in accordance with the rules of the Animal Care Comite at the University of British Columbia. 2.4.2  Transgenic mice The Z/AP, Z/EG, pCX-NLS-Cre, TIE2-tTA and Tet-O-Cre strains were generously provided by Dr. Corine Lobe and the LysM-Cre and R26R-EYFP mice were generously provided by Dr. Thomas Graf. Mice were housed in a specific pathogen fre facility and each strain was maintained by backcrossing to the C57BL/6 strain. 2.4.3  TIE2-tTA/Tet-O-Cre/Z/EG mice Double transgenic TIE2-tTA/Tet-O-Cre mice were crossed with Z/EG mice and breders were fed Dox-Diet (Bio-Serv) containing 20mg/kg doxycycline. After weaning, triple transgenic pups were maintained on Dox-Diet until 8 weks of age at which time doxycycline was removed from the diet. Peripheral blod then was taken daily for a wek, wekly for a month and at 2, 3, 6 and 9 months post induction. Peripheral blod leukocytes were analyzed by flow cytometry for the expression of the Cre-reporter transgene, EGFP.      65 2.4.4  Flow cytometry Peripheral blod samples were taken from the tail vein of each mouse and erythrocytes were lysed in a hypotonic solution. For experiments requiring identification of granulocytes or hPLAP, cels were stained with a PE-conjugated anti-Gr-1 antibody (eBioscience) or an anti-human hPLAP antibody (Serotec) respectively. For FDG staining, blod cels were incubated in a hypotonic solution containing 1 mM fluorescein-di-beta-D-galactopyranoside for 1 minute at 37oC. The mixture was then diluted 10-fold in PBS and incubated for 1 hour on ice. Al data was colected with a Becton-Dickinson FACSCalibur and analyzed with FlowJo software. 2.4.5  Excision analysis Peripheral blod samples were prepared and stained with a PE-conjugated anti-Gr-1 antibody as described above. Reporter negative and reporter positive granulocytes were sorted utilizing a Becton-Dickinson FACSVantage and genomic DNA was prepared from roughly 3x104 sorted cels from each population (DNeasy Blod and Tissue Kit, QIAGEN). PCR primers designed to amplify a segment of the ITK gene were as folows: ITK-F: 5’-GCGTAATGACAGTGTG-3’ and ITK-R: 5’-TGCTCAGACTGTGAGAGTCG-3’. Primers designed to identify a recombined Z/EG locus were pCAGS-F: 5’-GGCACGTGCTGTGT-3’ and EGFP-R: 5’-CAGCTCGACAGATG-3’.     66 2.4.6  Bisulfite sequencing Genomic DNA from reporter negative and reporter positive populations was converted utilizing the EpiTect Bisulfite Kit (QIAGEN). For analysis of the pCAGS promoter, DNA was subjected to a semi-nested PCR reaction utilizing primers BABF6: 5’-GAGAGTGYGYGTAGTATAGAG-3’ and BABR5d: 5’-AACCTCAACTTCACRCACACA-3’ for the first round folowed by BABF6 and BABR4c: 5’-TCATAACAACRCTATACACC-3’ for the second round. Analysis of the ROSA26 promoter utilized the primers ROSAF2: 5’- GAAYGTATGATYGTAYGGAT-3’ and ROSAR3: 5′-ACTATCTCACAACRACTCACAC-3′. PCR products were cloned into the pCR2.1 vector (Invitrogen) and sequenced from the T7 priming site utilizing Aplied Biosystems BigDye v3.1 Terminator Chemistry at the NAPS Unit, UBC.  67 2.5  REFERENCES 1. Sternberg, N. and D. Hamilton, Bacteriophage P1 site-specific recombination. I. Recombination betwen loxP sites. J Mol Biol, 1981. 150(4): p. 467-86. 2. Austin, S., M. Ziese, and N. Sternberg, A novel role for site-specific recombination in maintenance of bacterial replicons. Cel, 1981. 25(3): p. 729-36. 3. Sternberg, N., et al., Bacteriophage P1 cre gene and its regulatory region. Evidence for multiple promoters and for regulation by DNA methylation. J Mol Biol, 1986. 187(2): p. 197-212. 4. Branda, C.S. and S.M. Dymecki, Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev Cel, 204. 6(1): p. 7-28. 5. Lobe, C.G., et al., Z/AP, a double reporter for cre-mediated recombination. Dev Biol, 199. 208(2): p. 281-92. 6. Novak, A., et al., Z/EG, a double reporter mouse line that expresses enhanced gren fluorescent protein upon Cre-mediated excision. Genesis, 200. 28(3-4): p. 147-55. 7. Srinivas, S., et al., Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol, 201. 1: p. 4. 8. Zambrowicz, B.P., et al., Disruption of overlaping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cels. Proc Natl Acad Sci U S A, 197. 94(8): p. 3789-94. 9. Feron, M. and J. Vacher, Targeted expression of Cre recombinase in macrophages and osteoclasts in transgenic mice. Genesis, 205. 41(3): p. 138-45. 10. Muncan, V., et al., Rapid loss of intestinal crypts upon conditional deletion of the Wnt/Tcf-4 target gene c-Myc. Mol Cel Biol, 206. 26(2): p. 8418-26. 1. Jiang, X., et al., Fate of the mamalian cardiac neural crest. Development, 200. 127(8): p. 1607-16. 12. Haris, R.G., et al., Lack of a fusion requirement for development of bone marow-derived epithelia. Science, 204. 305(5680): p. 90-3. 13. Voijs, M., J. Jonkers, and A. Berns, A highly eficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent. EMBO Rep, 201. 2(4): p. 292-7.  68 14. Zhu, X., D.E. Bergles, and A. Nishiyama, NG2 cels generate both oligodendrocytes and gray mater astrocytes. Development, 208. 135(1): p. 145-57. 15. Kider, B.L., et al., Embryonic stem cels contribute to mouse chimeras in the absence of detectable cel fusion. Cloning Stem Cels, 208. 10(2): p. 231-48. 16. Iwama, A., et al., Molecular cloning and characterization of mouse TIE and TEK receptor tyrosine kinase genes and their expression in hematopoietic stem cels. Biochem Biophys Res Comun, 193. 195(1): p. 301-9. 17. Yano, M., et al., Expression and function of murine receptor tyrosine kinases, TIE and TEK, in hematopoietic stem cels. Blod, 197. 89(12): p. 4317-26. 18. Arai, F., et al., Tie2/angiopoietin-1 signaling regulates hematopoietic stem cel quiescence in the bone marow niche. Cel, 204. 18(2): p. 149-61. 19. Clausen, B.E., et al., Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res, 199. 8(4): p. 265-7. 20. Nagy, A., Cre recombinase: the universal reagent for genome tailoring. Genesis, 200. 26(2): p. 9-109. 21. Ye, M., et al., Hematopoietic stem cels expressing the myeloid lysozyme gene retain long-term, multilineage repopulation potential. Imunity, 203. 19(5): p. 689-99. 22. Guo, C., W. Yang, and C.G. Lobe, A Cre recombinase transgene with mosaic, widespread tamoxifen-inducible action. Genesis, 202. 32(1): p. 8-18. 23. Walsh, C.P., J.R. Chailet, and T.H. Bestor, Transcription of IAP endogenous retroviruses is constrained by cytosine methylation. Nat Genet, 198. 20(2): p. 16-7. 24. Jahner, D., et al., De novo methylation and expression of retroviral genomes during mouse embryogenesis. Nature, 1982. 298(5875): p. 623-8. 25. Rougier, N., et al., Chromosome methylation paterns during mamalian preimplantation development. Genes Dev, 198. 12(14): p. 2108-13. 26. Cohen-Tanoudji, M., C. Babinet, and D. Morelo, lacZ and ubiquitously expressed genes: should divorce be pronounced? Transgenic Res, 200. 9(3): p. 23-5. 27. Julien, N., et al., Conditional transgenesis using Dimerizable Cre (DiCre). PLoS ONE, 207. 2(12): p. e135.  69 28. Zhang, X.M., et al., Transgenic mice expressing Cre-recombinase specificaly in retinal rod bipolar neurons. Invest Ophthalmol Vis Sci, 205. 46(10): p. 3515-20. 29. Rotolo, T., et al., Geneticaly-directed, cel type-specific sparse labeling for the analysis of neuronal morphology. PLoS ONE, 208. 3(12): p. e409.   70 CHAPTER 3.  TARGETED CEL FUSION ENHANCES THE CONTRIBUTION OF HEMATOPOIETIC ELS TO SKELETAL MUSCLE FIBERS1 3.1  INTRODUCTION The induced fusion of cels in vitro has ben an essential technique for research in a number of fields including the study of nuclear reprograming[1], the production of monoclonal antibodies[2] and the generation of dendritic-cel hybrids for cancer imunotherapy[3]. However, advances in these and other areas are curently inhibited by the limitations of traditional fusogenic agents. The most comonly utilized techniques for inducing cel fusion in vitro, namely polyethylene glycol (PEG)[4] and electrofusion[5] were first described roughly thirty years ago and although incremental refinements have gradualy increased their eficacy, each of these methods remain notoriously ineficient. As a result of mechanisms that rely on random agregation and membrane damage in order to achieve cel fusion, PEG and electrofusion protocols generaly produce heterokaryons with low eficiency and high toxicity. Methods that employ micromanipulation[6], afinity crosslinking[7] or microfluidic devices[8] to properly pair two cel types are capable of increasing the eficiency of fusion. However, these systems continue to rely on PEG or electric pulses to initiate membrane fusion.                                                    1 A version of this chapter wil be submited for publication. Long MA and Rosi FMV (2010) Targeted Cel Fusion Enhances the Contributiuon of Hematopoietic Cels to Skeletal Muscle Fibers.  71 In vivo, the ability of bone marow derived cels to contribute to the repair of several organs is largely thought to be due to the fusion of circulating cels with damaged tissues[9]. This discovery has raised the prospect that cel fusion may represent a viable therapeutic strategy for several genetic and degenerative diseases. However, the ineficiency with which this phenomenon occurs has also precluded its therapeutic utility. Atempts to increase the eficiency of this process, including injection of snake venom toxins, apear to function by simply damaging tissue which in turn recruits inflamatory cels to the site of injury where they infrequently fuse to regenerating tissue[10]. Clearly these reagents, like PEG and electrofusion are unlikely to be suitable for clinical aplication. Members of the Paramyxoviridae family of viruses, including measles and Sendai virus have long ben known to induce cel fusion in vivo and in vitro[1, 12]. In the case of measles virus, infection is initiated via recognition of human CD46 or CD150 on the surface of cels by the viral hemaglutinin (H) protein[13, 14]. This interaction is believed to induce a conformational change in the associated viral fusion (F) protein, exposing a hydrophobic peptide, which inserts into the target plasma membrane and mediates fusion of the virus with the cel[15]. Subsequent display of measles H and F on the surface of infected cels then initiates fusion betwen neighbouring cels, ultimately resulting in large multinucleated syncitia, which die primarily as a result apoptosis[16]. This cytopathic efect has motivated the development of oncolytic measles viruses, capable of specificaly recognizing and infecting tumour cels. A number of groups have accomplished this by the adition of peptides[17], growth factors[18], single chain antibodies (scFv)[19] or cytokines[20]  72 to the carboxyl-terminus of the hemaglutinin protein, efectively retargeting the tropism of the measles virus, resulting in fusion and death of cels expressing the cognate receptor or antigen. To date however, chimeric hemaglutinin glycoproteins have not ben developed for the generation of stable heterokaryons in vitro or for cel therapy in vivo.  Here we present a system based on a chimeric measles virus hemaglutinin glycoprotein, which is capable of generating stable heterokaryons with high eficiency both in vitro and in vivo. This modified measles virus hemaglutinin, Hα7, was produced by adition of a scFv that recognizes the muscle specific integrin, α7, to the carboxyl-terminus of a mutant hemaglutinin. Co-transfection of plasmids encoding Hα7 and measles F, induced fusion of al cel types tested with cultured skeletal muscle fibers. Moreover, the eficiency of Hα7-mediated fusion was clearly superior to PEG mediated fusion and demonstrated insignificant levels of toxicity. Interspecific heterokaryons generated via Hα7 mediated fusion retained morphology characteristic of diferentiated myotubes and activated transcription of human myogenic genes. Expression of Hα7 on both mouse and human inflamatory cels also increased the contribution of these cels to muscle fiber repair in vivo.    73 3.2  RESULTS 3.2.1  Design, construction and characterization of Hα7 We generated a muscle-specific fusion reagent, Hα7, by ading an anti-alpha7-integrin scFv to the carboxyl terminus of a mutated measles hemaglutinin, H481A,53A, which lacks the ability to bind either measles receptor[21] (Figure 3.1A). The scFv was constructed from the wel-characterized CA5.5 monoclonal antibody, which has ben employed extensively in the purification and characterization of myoblasts[2]. As seen in Figures 3.1B-E, the scFv retains the specificity and afinity of the parental monoclonal antibody, as demonstrated by its ability to stain C2C12 myoblasts but not NIH/3T3 fibroblasts. We then tested the ability of cels expressing our chimeric hemaglutinin to fuse with diferentiated skeletal myotubes. In order to accomplish this, 293T cels were co-transfected with plasmids encoding Hα7, F and GFP. The folowing day, these cels were mixed with cultures of diferentiated C2C12 myotubes and twenty-four hours after mixing, the percentage of GFP-positive myotubes was determined. As seen in Figure 3.1F, transfected 293T cels can be made to fuse with nearly every myotube in the culture, with an eficiency proportional to the amount of input plasmid. Importantly, insignificant numbers of GFP-positive myotubes were observed in the same assay when H481A,53A was used in place of Hα7 or when Hα7 or F were omited from the transfection (Figure 3.1F). Moreover, myotubes remained viable folowing fusion and exhibited a morphology characteristic of diferentiated C2C12 cels (Figure 3.1G).   74                               Figure 3.1 Design, construction and characterization of Hα7. (A) Schematic representation of Hα7, aproximating the locations each blade (β1-β6) in the β-propelor fold [23, 24] as wel as the location of mutations that abrogate CD46 binding (Y481A) and CD150 binding (R53A). The anti-α7 integrin scFv is displayed as a carboxy-terminal extension of the type I transmembrane glycoprotein. Standard one-leter abreviations are used to denote amino acid residues. N: Amino-terminal cytoplasmic tail. TM: Transmembrane domain. (B-E) Evaluation of the anti-α7 integrin scFv by flow cytometry. The scFv (B, solid line) retains the ability of the parental monoclonal antibody (D, solid line) to stain C2C12 myoblasts, whereas neither antibody stains NIH/3T3 fibroblasts (C and F, solid line). In al plots, the staining level of cels incubated with secondary antibody alone is shown in doted lines. (F) Hα7 mediates fusion of transfected 293T cels and diferentiated C2C12 myotubes with an eficiency that is proportional to the amount of transfected plasmid and is absolutely dependent on the presence of the anti-α7 integrin scFv and the measles F protein. Data are shown as mean ± s.d. (n=3). (G) Morphology of yotubes folowing fusion.  75 3.2.2  Verification of bona fide heterokaryons In order to eliminate the possibility that the multinucleated, GFP-positive cels observed in co-cultures were exclusively derived from the homotypic fusion of transfected cels, we first cultured 293T cels in myogenic diferentiation medium folowing co-transfection with Hα7, F and GFP. This treatment did not result in the formation of syncitia (Figure 3.2A), sugesting that transfected 293T cels are unable to autonomously initiate the fusion process and demonstrating the inability of Hα7 to facilitate fusion betwen cels that do not express alpha7 integrin. In co-cultures however, multinucleated, GFP-positive cels were found to express murine myosin heavy chain (Figure 3.2B-D), confirming the presence of proteins derived from both 293T and C2C12 cels within these syncitia. Furthermore, in order to confirm that these cels were heterokaryons by definition, we identified the presence of both human and murine nuclei within syncitia by diferential DAPI staining (Figure 3.2E, F) as wel as by fluorescent-in situ-hybridization (FISH) stainig of human and murine satelite repeat DNA (Figure 3.2G). In the FISH assay, double positive nuclei were never observed, confirming that the Hα7-mediated fusion of 293T cels with diferentiated C2C12 myotubes results in the generation of true heterokaryons.  76                                 Figure 3.2 Formation of bona fide heterokaryons. (A) 293T cels co-transfected with Hα7, F and GFP do not fuse with one another. (B-D) Folowing co-culture of transfected human 293T cels with diferentiated mouse C2C12 myotubes, elongated GFP positive cels (B) express mouse myosin heavy-chain (C) and contain multiple nuclei (D, merged). (E,F) Diferential DAPI staining demonstrates the presence of both human and mouse nuclei within heterokaryons. Mouse nuclei contain dense chromocenters while human nuclei (arows) stain difusely and exhibit dark nucleoli. (G) Fluorescent in situ hybridization of human α-satelite DNA (red) and mouse γ-satelite DNA (gren) further confirms the presence of both human and mouse nuclei within heterokaryons.  77 3.2.3  Comparison of Hα7 and PEG induced fusion PEG remains the most widely used fusogenic agent for the production of heterokaryons. Therefore, we sought to compare the eficiency Hα7-mediated fusion with that of a standard PEG-mediated fusion protocol. In this case, 293T cels were either co-transfected with plasmids encoding Hα7, F and GFP or transfected with a plasmid encoding GFP alone. The folowing day, equal numbers of 293THα7,F,GFP or 293TGFP cels were mixed with cultures of diferentiating C2C12 cels and wels containing 293TGFP were treated with PEG to induce fusion. The number of GFP-positive myotubes as wel as the total number of myotubes per low-power field was determined daily thereafter for each condition. As seen in Figure 3.3, the number of GFP-positive myotubes generated by Hα7-mediated fusion was roughly ten to fiften-fold greater than the number generated by standard PEG-mediated fusion. This finding is unlikely to be due to improper use of PEG, as previous studies employing this method have reported similar fusion eficiencies[25]. A slight increase in the percentage of GFP positive myotubes was observed in wels subjected to Hα7 mediated fusion betwen day-one and day-two post fusion (Figure 3.3A versus Figure 3.3B). This observation is most likely due to that fact that diferentiation of C2C12 myoblasts continues and terminates over this time frame. Thus, in the first 24 hours of co-culture, nascent myotubes are forming at a rate that simply outpaces the ability of cels expressing Hα7 to encounter and fuse with them.  78               Figure 3.3 Comparison of Hα7 and PEG-mediated fusion eficiencies. (A-C) The total number of myotubes (white bars) as wel as the number of GFP-positive myotubes (gren bars) was determined by visual inspection of randomly selected, low power (5x) fields at (A) 1 day (B) 2 days and (C) 3 days post fusion. ***: P<0.01 (unpaired t-test). Data are shown as mean ± s.d. (n=6). Values above each bar represent the average fusion eficiency.   79 At al timepoints, the total number of myotubes surviving in the Hα7 treatment group was nearly twice as great as the number surviving PEG treatment. In fact, the total number of myotubes present folowing Hα7-mediated fusion was not significantly diferent from controls lacking any fusogen, demonstrating the greatly reduced toxicity of this method with respect to PEG. A decrease in the total number of myotubes was observed on day thre post-fusion as diferentiated muscle cels began to contract and detach from the dish. However, this phenomenon uniformly afected the total number of myotubes across al treatment groups and did not preferentialy afect GFP-positive myotubes within any group.  80 3.2.4  Nuclear reprograming folowing Hα7 mediated fusion To investigate the potential utility of Hα7 mediated fusion for nuclear reprograming studies, we analyzed the expression of human muscle transcripts in heterokaryons comprised of MRC-5 human lung fibroblasts and diferentiating C2C12 myotubes. Similar experiments performed with PEG have demonstrated that folowing the fusion of several human cel types with C2C12 myotubes, muscle genes are activated in human nuclei with kinetics that resemble myogenic diferentiation[26, 27]. Therefore, we chose to examine the expression of MyoD and myogenin, as markers of early and late myogenic diferentiation respectively. As seen in Figure 3.4, isolated MRC-5 cels do not express either of these transcription factors. However, MyoD expression was upregulated within 72 hours of Hα7 mediated fusion and reached a peak roughly 48 hours later (Figure 3.4A). On the other hand, the levels of myogenin transcript remained relatively low at early time points while steadily increasing over time (Figure 3.4B). These data are in agrement with previous reports[26, 27], confirming that heterokaryons generated via Hα7 mediated fusion retain the capacity to undergo nuclear reprograming.  81              Figure 3.4 Nuclear reprograming folowing Hα7-mediated fusion. (A,B) Quantitative RT-PCR analysis of (A) human MyoD and (B) human myogenin transcript levels at daily intervals folowing Hα7-mediated fusion of MRC-5 cels and diferentiating C2C12 myotubes. Al values were normalized to β-actin transcript levels and subsequently to the mean expression level on Day 1. Data are shown as mean ± s.d. (n=3). (C) Endpoint RT-PCR reactions demonstrating the specificity of huan MyoD and myogenin primers.  82 3.2.5  Hα7 mediated fusion in vivo Finaly, to evaluate the potential utility of targeted cel fusion for regenerative medicine, we investigated the ability of Hα7 to increase the eficiency of fusion betwen inflamatory cels and muscle fibers in vivo. Bone marow derived from the Z/AP transgenic strain[28], which expresses β-galactosidase in the majority of hematopoietic cels was infected with the lentiviral vectors LV-HIG and LV-FIY, which encode Hα7-IRES-GFP and F-IRES-YFP respectively. Infected bone marow was then transplanted into congenic C57BL/6 recipients and four weks later, muscle damage was induced in the tibialis anterior muscle by notexin injection. Al recipients remained healthy throughout the treatment and at eight weks post-transplant, mice were sacrificed and hind limbs were examined for the presence of β-galactosidase-positive muscle fibers. Unfortunately, the eficiency of lentiviral transduction in al recipients was low (Figure 3.5A,B). However, significantly more donor-derived muscle fibers were observed in a subset of recipients that were transplanted with infected bone marow as compared to control recipients that received uninfected bone marow transplants (Figure 3.5C).  83      Figure 3.5 Hα7-mediated fusion in vivo. (A,B) Expression of lentiviral transgenes in the peripheral blod of mice four weks after transplantation with (A) infected or (B) uninfected bone marow. (C) The maximum number of donor derived muscle fibers per section at eight weks post transplant in recipients of infected and uninfected bone marow. (D,E) Expression of lentiviral transgenes in U937 cels folowing infection with (D) LV-HIG and LV-FIY or (E) LV-FIY alone. (F) Donor derived, GFP positive muscle fibers in NOD/SCID recipients one wek folowing transplantation of U937Hα7,F. (G) Same image displayed in (F) with the gren chanel removed to emphasize intact basal lamina surounding donor derived muscle fibers.   84 As a model of human inflamatory cels, the monocyte cel line U937, was also infected with LV-HIG and LV-FIY (Figure 3.5D). Doubly infected U937Hα7,F cels or single infected U937F control cels (Figure 3.5E) were then injected into the tibialis anterior muscle of NOD/SCID recipients. One wek after transplantation, mice were sacrificed and hind limbs were examined for the presence of GFP positive muscle fibers. As seen in Figures 3.5F and G, donor derived fibers exhibiting normal morpholgy and surounded by basal lamina were regularly observed in recipients of U937Hα7,F cels. In al tissues obtained from control animals however, only a single GFP-positive muscle fiber was ever observed.  85 3.3  DISCUSION We have created a cel fusion reagent, Hα7, which overcomes the low eficiency, high toxicity and lack of specificity exhibited by existing chemical and physical fusogens. As oposed to PEG and electrofusion, our system is based on a specific ligand-receptor interaction, which simultaneously promotes the proper pairing and eficient fusion of cels. This feature maximizes the generation of heterokaryons and virtualy eliminates the non-productive formation of homokaryons. In vitro, Hα7 mediated fusion eficiencies routinely exceeded ninety percent and consistently generated ten to fiften fold more heterokaryons than a standard PEG mediated protocol. A similar increase in fusion eficiency has recently ben described utilizing a microfluidic device to control cel pairing[8]. While this represents a significant improvement over existing techniques, the microfluidic device is limited to the manipulation of a maximum of six thousand cel pairs per run. Our method on the other hand, has no such limitations and is therefore capable of producing far more of heterokaryons per experiment. The ability of Hα7 to increase fusion eficiency was not gained at the expense of cel viability. At al time points analyzed, the total number of myotubes present folowing Hα7-mediated fusion was not significantly diferent from controls lacking any fusogen. Moreover, heterokaryons generated via Hα7 treatment exhibited normal healthy morphology, characteristic of diferentiated C2C12 myotubes. In contrast, PEG mediated fusion resulted in the death of roughly fifty percent of myotubes. This diference is likely due to the fact that unlike PEG, the measles fusion glycoprotein complex is capable of initiating and stabilizing the fusion process  86 without relying on the induction of membrane damage. Folowing Hα7 mediated fusion of MRC-5 human lung fibroblasts and diferentiating C2C12 myotubes, expression of the myogenic transcription factors MyoD and myogeni was induced in human nuclei. The kinetics of this reprograming process was nearly identical to previous reports based on PEG mediated fusion[26, 27]. While it remains possible that the expression of Hα7 and F may alter gene expression in heterokaryons, it is unlikely that this efect wil be greater than the perturbations caused by the toxic efects of PEG. Therefore, we anticipate that the increased yield and quality of heterokaryons generated via Hα7 mediated fusion wil facilitate the systematic identification of the factors and mechanisms involved in the process of nuclear reprograming. Skeletal muscle is naturaly repaired by satelite cels, which proliferate and fuse to multinucleated myofibers[29]. However, in several human skeletal myopathies, ongoing cycles of fiber degeneration overwhelm this regenerative process and muscle tissue becomes chronicaly inflamed as a result[30]. Therefore, we examined the ability of Hα7 to increase the eficiency of fusion betwen inflamatory cels and multinucleated myofibers in vivo. In two distinct models, Hα7 expression increased the contribution of mouse and human inflamatory cels to skeletal muscle repair. Unfortunately, in the mouse bone-marow transplantation model, the magnitude of this efect was limited by the low eficiency of lentiviral transduction. Despite this fact, these results demonstrate that Hα7 is capable of enhancing fusion of inflamatory cels and skeletal muscle fibers in an imunocompetent host. In agrement, Iankov et al, have demonstrated that cels infected with measles virus are capable of  87 undergoing fusion in vivo even in the presence of pre-existing humoral imunity[31]. Therefore we further anticipate that the creation of circulating myogenic progenitors via expression of Hα7 on inflamatory cels wil facilitate the development of novel cel and gene therapies for skeletal myopathies.   88 3.4  METHODS 3.4.1  Construction of Hα7 RNA was prepared from the CA5.5 hybridoma (RNeasy, Qiagen) and cDNA was produced utilizing Superscript I (Invitrogen) and an oligo-dT primer. The variable region of the imunoglobulin heavy chain was then amplified utilizing Platinum Pfx DNA Polymerase (Invitrogen) and the degenerate primers VH: 5’-TGA GT GCA GCT GA GA GTC-3’ and Cγ: 5’-AGA CG ATG GG CTG TG TT TG C-3’. The variable region of the imunoglobulin light chain was amplified utilizing the degenerate primers Vκ: 5’-GAC AT CTG ATG AC CAG TCT-3’ and Cκ: 5’-TG ATA CAG TG GTG CAG CAT CAG C-3’. PCR products were cloned into pCR-BluntI-TOPO (Invitrogen) and sequenced from the T7 priming site. (Aplied Biosystems BigDye v3.1 Terminator Chemistry, NAPS Unit, UBC). These sequences were utilized to identify the particular variable region genes expressed in CA5.5 cels and gene specific primers were designed. The variable region of the imunoglobulin heavy chain was then reamplified from CA5.5 cDNA utilizing the primers CA5.5H-F: 5’-AA GA TCT GC CA GC GC CA GT GCA GCT GA GA GTC-3’and CA5.5H-R: 5’-GAC GT GAC CAT GAC TC TG G-3’. The variable region of the imunoglobulin light chain was reamplified utilizing the primers CA5.5L-F: 5’-AA GAG CTC GCT GAC CA GTC TC TGC TT G-3’ and CA5.5L-R: 5’-AA CTC GAG CG CG CC GT TCA AT CA GCT TG TGC-3’. A complete scFv was then assembled by cloning the heavy chain fragment upstream and the light chain fragment downstream of a glycine-serine (G4S1)3 linker contained in pASK85-9E10 utilizing BglI, BstEI and SacI, XhoI sites respectively, thereby creating pCA5.5scFv.  89 The CA5.5scFv was subsequently fused to a human light chain constant region by subcloning into pLC-huCκ with BglI and NotI. This plasmid, pCA5.5scFv- huCκ, was transiently transfected into 293T cels via standard calcium phosphate precipitation and 48 hours later, neat supernatant containig the CA5.5scFv-huCκ fusion protein was utilized to stain C2C12 myoblasts and NIH/3T3 fibroblasts in paralel with a 0.5µg/mL dilution of the CA5.5 monoclonal antibody. The goat anti-human-kapa-PE (Southern Biotech) and the goat anti-rat-PE secondary (Southern Biotech) antibodies were used to detect the CA5.5scFv-huCκ and CA5.5 staining respectively. Al flow cytometry data was colected with a Becton-Dickinson FACSCalibur and analyzed with FlowJo software. Folowing confirmation of specificity, the CA5.5scFv was fused to the carboxyl-terminus of a mutant measles hemaglutinin contained in pTNH6-Ha using SfiI and NotI, thereby creating pHα7. 3.4.2  In vitro fusion asays 293T and C2C12 cels were maintained in DMEM (Gibco) suplemented with 10% and 20% fetal bovine serum (Gibco) respectively. To induce diferentiation, C2C12 cels were plated in DMEM suplemented with 2% horse serum (Invitrogen) on colagen-coated dishes (Sigma, Becton Dickinson) at a density of 4x104 cels/cm2. Twenty-four hours later, cytosine β-D-arabinofuranoside (Ara-C) (Sigma) was aded to a concentration of 1x10-5M in order to eliminate proliferating myoblasts. 293T cels were transfected with calcium phosphate twenty-four hours prior to co-culture and were plated onto C2C12 cels at a density of 4x104 cels/cm2. Co-cultures were initiated folowing two or five days of C2C12 diferentiation and are refered to as  90 diferentiating or diferentiated cultures respectively. PEG mediated fusion of cels was caried out as described previously. Briefly, 293T cels were mixed with diferentiating C2C12 myoblasts and alowed to setle and adhere for four to six hours. Medium was then completely aspirated and replaced with prewarmed 50% PEG 150 (Roche) for sixty seconds. PEG was then removed and cels were washed thre times in prewarmed DMEM. Cultures were subsequently maintained in DMEM suplemented with 2% horse serum, 1x10-5M Ara-C and 1x10-5M ouabain (Sigma) to eliminate unfused human cels. Fusion eficiency was quantified at selected intervals by enumerating the total number of myotubes as wel as the number of GFP-positive myotubes present in at least six randomly selected low power (5x) fields. 3.4.3  Imunofluorescence and FISH To detect mouse myosin-heavy chain expression, heterokaryons were first fixed in 4% paraformaldehyde (PFA) for 5 minutes at rom temperature, washed in PBS and permeabilized in 0.5% Triton X-10 for 5 minutes at rom temperature. Cels were then stained with mouse anti-mouse myosin-heavy chain (Developmental Studies Hybridoma Bank) overnight at 4oC, folowed by a 1 hour incubation with goat anti-mouse Alexa 568 (Molecular Probes) at rom temperature. Nuclei were counterstained with 4´,6-diamidino-2-phenylindole (DAPI) (1µg/mL). For FISH, cels were post-fixed with 4% formaldehyde, treated with 1 mg/ml pepsin, dehydrated in increasing series of ethanol and air-dried. Cels were denatured for 3 minutes at 80°C in hybridization mixture (70% formamide, 0.5 µg/ml  91 of Cy-3–conjugated PNA probe specific to human α-satelite sequences (CTCAATATCACTGC), 0.5 µg/ml of Cy-5–conjugated PNA probe specific to mouse major satelite (GAGACTGATATG) and 0.25% (w/v) blocking reagent (DuPont) in 10 mM Tris (pH 7). Hybridization was performed at rom temperature for 1 hour and slides were then washed with 70% formamide/10 mM Tris (pH 7.2; twice for 15 min each) and with 0.05 M Tris/0.15 M NaCl (pH 7.2) containing 0.05% Twen-20 (thre times for 5 min each). Slides were dehydrated, air dried and counterstained with 0.2 µg of DAPI/ml, and mounted in antifading solution (DABCO). The images were acquired with the DeltaVision RT imaging system (Aplied Precision) on an inverted microscope (IX70 Olympus) equiped with a Colsnap HQ digital camera. Images stacks were acquired for each wavelength in 12 bit grey scale through a 60/1.4 oil imersion lens. Deconvolution was performed on the Deltavision RT imaging system and single plane projection of individual images was done using SoftWoRx software (Aplied Precision). 3.4.4  Quantitative real-time gene expresion analysis Heterokaryons were generated in 24-wel plates as described above. Folowing fusion, RNA was harvested daily (RNeasy, Qiagen) from a single wel for each treatment condition for a total of eight days. Purified RNA was treated with DNAse (Fermentas) and cDNA was then produced utilizing Superscript I (Invitrogen) and random hexamer primers (Invitrogen). qPCR reactions were set up with Maxima SYBR Gren/ROX qPCR Master Mix (Fermentas) and the folowing primers pairs hMyoDF: 5’-CAC TC GT CC AA TGT AG-3’ and hMyoDR: 5’-GT ATA AC GTA CA AT CC TGT A-3’. hMyogeninF: 5’-CAG CGA ATG CAG CTC TCA C-3’  92 and hMyogeninR: 5’-CAG AG TAG TG CAT CTG TG-3’. β-actinF: 5’-TT GAG AC TC AC AC CA GC-3’ and β-actinR: 5’-AT GTC ACG CAC GAT TC CG C-3’. Gene expression was quantified using a 790HT Fast Real-Time PCR System and the 700 SDS relative quantification software (Aplied Biosystems). 3.4.5  Lentiviral vectors The transfer vector, pLV-HIG, was constructed by inserting the Hα7 cDNA contained in pHα7, downstream of the EF1α promoter in the third generation lentiviral vector, pCL.sin.cPPT.EF1α.SET7.IRES.GFP.WPRE utilizing BamH1. The transfer vector, pLV-FIY, was constructed by first inserting the measles fusion protein cDNA contained in pCGF, downstream of the EF1α promoter in same the third generation lentiviral vector described above, utilizing Xma1 and Xba1. The EYFP cDNA contained in pEYFP (Clontech) was then cloned downstream of the IRES utilizing Nco1 and BsrG1. Lentiviruses were produced by cotransfecting 293T cels with the apropriate transfer vector as wel as with the packaging plasmids, pMDL, pRev and pVSVG utilizing calcium phosphate. Supernatant was harvested 36 to 60 hours later, filtered through a 0.45µm filter (Pal) and concentrated by ultracentrifugation at 19,40rpm for 2hr at 20oC in a Beckman SW28 rotor. Viral pelets were resuspended in 10µL of HBS and stored in 20µL aliquots at -80oC. In order to determine the biolgical titer of each lentivirus preparation, 293T cels were infected with serial dilutions of stock solutions and infection eficiency was quantified 48hr later via flow cytometry (BD FACScan and FlowJo software).  93 3.4.6  In vivo fusion asays In the mouse bone marow transplantation model, 1.5x105 Sca-postitive bone marow cels derived from a Z/AP transgenic mouse were plated in StemSpan SFEM (StemCel Technologies) suplemented with rhFlt-3 ligand, rhSCF, rhTPO (C10, StemCel Technologies), al at a concentration of 10ng/mL. Folowing a 2 hour prestimulation, cels were incubated for 4 hours with 1.5x106 infectious units of each lentivirus, vHα7-IRES-GFP and vF-IRES-YFP, in the presence of 5µg/mL polybrene. 7.5x104 cels were then transplanted via tail vein injection into C57BL/6 recipients that had ben lethaly iradiated (1Gy) in two doses, nine hours and five hours prior to transplantation. Four weks after transplantation, the tibialis anterior muscle of each recipient was injured via intramuscular injection of 10ng of notexin. Muscle tissue was then alowed to heal for four weks prior to analysis. In the human monocyte transplantation model, U937 cels maintained in RPMI medium suplemented with 10% fetal bovine serum were infected with vHα7-IRES-GFP and vF-IRES-YFP. Infected cels were sorted (Becton-Dickinson FACSVantage) and 1x105 cels were injected into the tibialis anterior muscle of NOD/SCID recipients. Muscle tissue was then alowed to heal for one wek prior to analysis. For analysis, mice were first terminaly anesthetized with avertin, then perfused with PBS containing 10mM EDTA and finaly perfused with 4% PFA in PBS. Al lower leg muscles were then removed from recipients and post-fixed in 4% PFA overnight prior to overnight cryoprotection in 20% sucrose. Muscle tissue was then embeded (OCT, Sakura) and cut into 20µm sections (Leica CM3050 S). In the  94 mouse bone marow transplantation model, sections were stained overnight with 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) (Sigma). In the human monocyte transplantation model, sections were stained with rabit anti-mouse laminin (Abcam) overnight at 4oC, folowed by a 1 hour incubation with goat anti-rabit Alexa 568 (Molecular Probes) at rom temperature. In both cases the presence of donor-derived fibers was detected by examination using a Zeiss Axioplan2 microscope.  95 3.5  REFERENCES 1. Blau, H.M., C.P. Chiu, and C. Webster, Cytoplasmic activation of human nuclear genes in stable heterocaryons. Cel, 1983. 32(4): p. 171-80. 2. Kohler, G. and C. Milstein, Continuous cultures of fused cels secreting antibody of predefined specificity. Nature, 1975. 256(517): p. 495-7. 3. Gong, J., et al., Induction of antitumor activity by imunization with fusions of dendritic and carcinoma cels. Nat Med, 197. 3(5): p. 58-61. 4. Pontecorvo, G., Production of mamalian somatic cel hybrids by means of polyethylene glycol treatment. Soatic Cel Genet, 1975. 1(4): p. 397-400. 5. Zimerman, U. and J. Vienken, Electric field-induced cel-to-cel fusion. J Mebr Biol, 1982. 67(3): p. 165-82. 6. Stromberg, A., et al., Manipulating the genetic identity and biochemical surface properties of individual cels with electric-field-induced fusion. Proc Natl Acad Sci U S A, 200. 97(1): p. 7-1. 7. Bakker Schut, T.C., et al., Selective electrofusion of conjugated cels in flow. Biophys J, 193. 65(2): p. 568-72. 8. Skeley, A.M., et al., Microfluidic control of cel pairing and fusion. Nat Methods, 209. 6(2): p. 147-52. 9. Alvarez-Dolado, M., et al., Fusion of bone-marow-derived cels with Purkinje neurons, cardiomyocytes and hepatocytes. Nature, 203. 425(6961): p. 968-73. 10. Camargo, F.D., et al., Single hematopoietic stem cels generate skeletal muscle through myeloid intermediates. Nat Med, 203. 9(12): p. 1520-7. 1. Warthin, A.S., Occurence of numerous large giant cels in the tonsils and pharyngeal mucosa in the prodroal stage of measles - Report of four cases. Archives of Pathology, 1931. 11(6): p. 864-874. 12. Okada, Y., Analysis of giant polynuclear cel formation caused by HVJ virus from Ehrlich's ascites tumor cels. I. Microscopic observation of giant polynuclear cel formation. Exp Cel Res, 1962. 26: p. 98-107. 13. Dorig, R.E., et al., The human CD46 molecule is a receptor for measles virus (Edmonston strain). Cel, 193. 75(2): p. 295-305. 14. Tatsuo, H., et al., SLAM (CDw150) is a celular receptor for measles virus. Nature, 200. 406(6798): p. 893-7.  96 15. Yin, H.S., et al., Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation. Nature, 206. 439(7072): p. 38-4. 16. Esolen, L.M., et al., Apoptosis as a cause of death in measles virus-infected cels. J Virol, 195. 69(6): p. 395-8. 17. Halak, L.K., et al., Targeted measles virus vector displaying echistatin infects endothelial cels via alpha(v)beta3 and leads to tumor regression. Cancer Res, 205. 65(12): p. 5292-300. 18. Schneider, U., et al., Recombinant measles viruses eficiently entering cels through targeted receptors. J Virol, 200. 74(21): p. 928-36. 19. Peng, K.W., et al., Oncolytic measles viruses displaying a single-chain antibody against CD38, a myeloma cel marker. Blod, 203. 101(7): p. 257-62. 20. Alen, C., et al., Interleukin-13 displaying retargeted oncolytic measles virus strains have significant activity against gliomas with improved specificity. Mol Ther, 208. 16(9): p. 156-64. 21. Nakamura, T., et al., Antibody-targeted cel fusion. Nat Biotechnol, 204. 22(3): p. 31-6. 22. Blanco-Bose, W.E., et al., Purification of mouse primary myoblasts based on alpha 7 integrin expression. Exp Cel Res, 201. 265(2): p. 212-20. 23. Hashiguchi, T., et al., Crystal structure of measles virus hemaglutinin provides insight into efective vaccines. Proc Natl Acad Sci U S A, 207. 104(49): p. 19535-40. 24. Colf, L.A., Z.S. Juo, and K.C. Garcia, Structure of the measles virus hemaglutinin. Nat Struct Mol Biol, 207. 14(12): p. 127-8. 25. Palermo, A., et al., Nuclear reprograming in heterokaryons is rapid, extensive, and bidirectional. FASEB J, 209. 23(5): p. 1431-40. 26. Teranova, R., et al., Acquisition and extinction of gene expression programs are separable events in heterokaryon reprograming. J Cel Sci, 206. 19(Pt 10): p. 2065-72. 27. Pomerantz, J.H., et al., Reprograming to a muscle fate by fusion recapitulates diferentiation. J Cel Sci, 209. 122(Pt 7): p. 1045-53. 28. Long, M.A. and F.M. Rossi, Silencing inhibits Cre-mediated recombination of the Z/AP and Z/EG reporters in adult cels. PLoS One, 209. 4(5): p. e5435.   97 29. Lipton, B.H. and E. Schultz, Developmental fate of skeletal muscle satelite cels. Science, 1979. 205(412): p. 1292-4. 30. Arahata, K. and A.G. Engel, Monoclonal antibody analysis of mononuclear cels in myopathies. I: Quantitation of subsets according to diagnosis and sites of accumulation and demonstration and counts of muscle fibers invaded by T cels. An Neurol, 1984. 16(2): p. 193-208. 31. Iankov, I.D., et al., Infected cel cariers: a new strategy for systemic delivery of oncolytic measles viruses in cancer virotherapy. Mol Ther, 207. 15(1): p. 14-22.     98 CHAPTER 4.  CONCLUSION 4.1  CONCLUSION Existing models of cel therapy for the treatment of skeletal myopathies are mainly based on the transplantation of satelite cels[1]. Unfortunately, these cels exhibit several properties which limit their clinical utility, namely imunogenicity[2], relative rarity[3], and inability to migrate from the circulation to muscle tissue[4]. Several groups have atempted to overcome these limitations via imunosupression[5], expansion in culture[6] and local injection[7], respectively. However, recent evidence demonstrates that expansion in culture significantly reduces the regenerative capacity of myoblasts[8]. Moreover, the large number of local injections required to systemicaly treat heritable myopathies remains a major limitation of these protocols and certainly hampers regeneration of porly accessible sites, such as the diaphragm. Clearly, the treatment of skeletal myopathies would be greatly facilitated by an alternative, abundant source of cels capable of delivery via the circulation. The ability of bone marow derived cels to participate in the repair of skeletal muscle initialy generated hope that this phenomenon may represent an alternative means of cel therapy[9]. Unfortunately, this process has ben shown to occur at exceedingly low frequencies, thereby precluding clinical aplication. At the outset of the work described here, the factors involved in the generation of bone marow derived muscle were almost completely uncharacterized. As a result, early atempts to increase the eficiency of the process were il founded and met with failure due to a lack of knowledge regarding the cel types and mechanisms involved[10, 1]. Therefore we designed a Cre/loxP based tracing strategy to identify the  99 hematopoietic lineages responsible for the generation of bone marow derived muscle as wel as a strategy to identify the role of fusion or transdiferentiation in the process (Figures 1.4 and 1.5). However, these strategies were not pursued upon discovery of ineficient labeling in our chosen Cre-reporter strain. We analyzed the labeling eficiency of hematopoietic cels in thre Cre-reporter strains, Z/AP, Z/EG and R26R-EYFP[12-14]. Each of these reporter strains was capable of eficient hematopoietic labeling when exposed to Cre during early embryonic development. However, when Cre was expressed in adult hematopoietic cels, the labeling eficiency of the Z/AP and Z/EG reporter was much lower than the R26R-EYFP reporter. We subsequently demonstrated that in unlabeled adult hematopoietic cels derived from Z/AP and Z/EG mice, the transgenic promoter was methylated and Cre-mediated recombination of the locus was inhibited. In order to explain these data, we propose the folowing model (Figure 4.1).  100  Figure 4.1 Model ilustrating the proposed methylation status of the Z/EG locus during early embryogenesis. (A) The Z/EG locus is methylated (white circles) and refractory to Cre-mediated recombination in a subset of adult cels. (B) Folowing fertilization, this epigenetic modification is removed during a genome wide demethylation process, which is known to peak at 3.5 days post-coitum (DPC). The deethylated locus may be recombined by Cre-recombinase present during this time. (C) Although we do not know the precise kinetics of the process, we hypothesize that if recombination does not occur within this temporal window, the β-geo transgene nucleates methylation of the locus during a genome wide methylation process, which is known to peak at 7.5 DPC. (D) This epigenetic modification then spreads to the promoter, silencing expression of β-geo and inhibiting Cre-mediated recombination. Thus, Cre transgenes expressed folowing this window, such as LysM-Cre, are unable to eficiently activate expression of the EGFP reporter. DPC 3.5 7.5 0 Global DNA Methylation Cre Expresion PCX-NLS Cre LysM Cre A B C D  101 After fertilization, the Z/AP and Z/EG loci are demethylated as a result of the genome wide demethylation that is known to occur in pre-implantation embryos[15]. This process provides a window wherein Cre recombinase may eficiently access the reporter locus and excise the β-geo transgene. However, if Cre-recombinase is not present during this developmental time window, the β-geo transgene remains intact and nucleates methylation of the locus as development proceeds. This prediction is based in part on the fact that removal of the β-geo transgene during early embryogenesis prevents silencing of the locus in adult cels (Figure 2.3A). We propose that methylation then spreads outward and inhibits both expression of β-geo transgene and Cre-mediated recombination of loxP sites. These efects may be mediated by direct inhibitory methylation of transcription factor binding sites[16] or loxP sites[17]. However DNA methylation is also known to be involved in the establishment and maintenance of a repressive chromatin state[18]. Therefore these efects may also be due to incorporation of the transgenic loci into heterochromatin. In suport of this model, it should be noted that the percentage of cels expressing β-geo in both Z/AP and Z/EG mice (Figure 2.6) is remarkably similar to the percentage of cels expressing the post-excision reporter (Figure 2.3B) folowing exposure to Cre in adult cels. This observation sugests that the population of cels that express the pre-excision reporter may be the only cels capable of undergoing Cre-mediated activation of the post-excision reporter in adult cels. Admitedly, this model is largely based on corelative data. In order to establish a causative relationship, the methylation status of the Z/AP and Z/EG loci would ned to be manipulated dynamicaly. However, at present it is not possible to alter the  102 methylation status of a single locus. Thus, the treatment required to manipulate DNA methylation would certainly result in an alteration of the expression level of many genes and therefore this experiment and would not directly link the methylation status of reporter loci to the eficiency with which they are recombined. Ultimately, this research provides a caveat for the future use of the Z/AP and Z/EG reporters as surogate markers of excision or in lineage tracing experiments. However, a number of groups have employed the Z/EG reporter to investigate the role of fusion in the formation of donor-derived tissue in a maner similar to our initial specific aim[19, 20]. These reports sugest that fusion does not play a role in the formation of donor-derived epithelia or in the contribution of embryonic stem cels to chimeras and are based on the lack of EGFP expression folowing transplantation. While these findings may be valid, they should be reconsidered in the light of our data. Our final specific aim was to increase the contribution of bone marow derived cels to skeletal muscle repair. Although our own eforts to identify the factors involved in the generation of donor derived muscle were precluded by the technical limitations described above, contemporary studies revealed that this phenomenon is primarily due to fusion of inflamatory cels with damaged myofibers[21, 2]. Unfortunately, despite its involvement in the formation and maintenance of a number of tissues including the placenta, osteoclasts and skeletal muscle itself, the process of cel fusion remains insuficiently understod to facilitate its enhancement for therapeutic purposes. Therefore, in order to specificaly target and enhance the fusion of bone marow derived cels with skeletal muscle fibers, we adapted a  103 fusogenic membrane glycoprotein complex derived from easles virus. Measles virus has recently emerged as a promising vector for the treatment of malignancies[23]. This oncolytic efect is based in part on the ability of the Edmonston B strain of measles virus to preferentialy infect various human tumours based on elevated expression of CD46[24]. However, in an efort to further increase the specificity of this vector, Nakamura et al. have created a mutant measles hemaglutinin, H481A,53A, which is unable to bind its natural receptors and may be retargeted to virtualy any cel surface antigen by the adition of a suitable polypeptide to its carboxyl-terminus[25]. Measles viruses expressing chimeric hemaglutinin proteins based on H481A,53A, have ben shown to successfuly infect malignant cels expressing the target antigen, leading to regression in a number of tumour models[26].  The measles virus fusogenic membrane glycoprotein complex is capable of mediating cel fusion in the absence of viral infection[27]. Therefore, in order to utilize this system for our purposes, we generated a single chain antibody, which recognizes the muscle specific integrin, alpha7, and fused it to the carboxyl-terminus of H481A,53A. This reagent, Hα7, specificaly and eficiently mediated fusion of al cel types tested with skeletal muscle fibers in vitro and in vivo. When compared directly to polyethylene glycol, Hα7 consistently generated a ten to fiften fold increase in heterokaryon yield and induced insignificant levels of toxicity. We also demonstrated that Hα7-mediated fusion results in the generation of true heterokaryons, which retain the capacity for nuclear reprograming. Although our third specific aim was  104 originaly focused entirely on increasing the frequency of fusion for therapeutic purposes, the striking eficiency of Hα7-mediated fusion in vitro was worth pursuing. A number of significant discoveries in the field of nuclear reprograming have ben made via fusion of various cel types with diferentiated muscle in vitro[28, 29]. However, the low yield of existing fusogenic agents has generaly prohibited advances in our understanding of this phenomenon. We anticipate that the increased yield and quality of heterokaryons generated via Hα7 mediated fusion wil facilitate the systematic identification of the factors and mechanisms involved in the process of nuclear reprograming. Finaly, we performed prof of principle experiments demonstrating that Hα7 is able to increase the fusion of both mouse and human bone marow derived cels with skeletal muscle in vivo. Unfortunately, in the mouse bone-marow transplantation model, the magnitude of this efect was limited by the low eficiency of lentiviral transduction. We suspect that this result may be due to inadequacy of the lentiviral vector itself and not due to expression of the measles virus proteins. Barete et al. have demonstrated that ecotropic murine retroviruses may transduce mouse hematopoietic stem cels more eficiently than VSV-G pseudotyped lentiviral vectors[30]. Therefore, we are curently constructing MSCV-based retroviral vectors encoding Hα7 and F in an efort to increase the transduction eficiency of mouse bone marow. However, transduction of long-term repopulating hematopoietic stem cels may not be necessary to met the clinical goal. It is conceivable that a transient wave of circulating fusogenic cels derived from the transduction of short term hematopoietic progenitors may prove suficient to deliver healthy or ex vivo  105 corected nuclei to ailing myofibers. In fact, this strategy may even be preferable as it may avoid the risk of generating leukemias via insertional mutagenesis of hematopoietic stem cels[31].  Cel fusion itself has also ben proposed to be a potential cause of malignancy[32]. According to this theory, the proliferation of hybrid cels results in aberant chromosome segregation, genomic instability and stochastic loss of tumor supressor genes, eventualy leading to neoplastic transformation. However, empirical evidence for this model is lacking. While it is known that many tumor cels cel types are more fusogenic than their untransformed counterparts[3], it remains to be proven that cel fusion is an initiating event in tumorigenesis as oposed to a by product of the process. Furthermore, the Hα7-mediated fusion of bone marow derived cels to skeletal muscle is unlikely to result in tumorigenesis as myofibers are post-mitotic and the simultaneous loss of a tumor supressor from al syncytial myonuclei is extremely improbable. On the other hand, hybrid cels generated via Hα7 mediated fusion of bone marow derived cels and satelite cels may be prone to aneuploidy folowing proliferation. However, we have not investigated the extent to which this type of fusion occurs or the viability of resultant hybrids. Clearly, these and other experiments should be performed in order to resolve whether malignancy is a legitimate concern for therapies involving cel fusion. At present, a more pragmatic limitation of Hα7-mediated fusion therapy may be the inability to treat a subset of human myopathies. While diseases caused by the simple absence of a single gene such as Duchene muscular dystrophy would likely  106 be treatable by the integration of healthy nuclei into prexisting myofibers, treatment of other diseases with a dominant negative etiology including myotonic dystrophy[34] and oculopharyngeal muscular dystrophy[35] may require the generation of new myofibers exclusively composed of healthy nuclei. However, the autonomous generation of donor-derived myofibers in vivo has not ben observed with any myogenic progenitor including myoblasts and may represent an intrinsic limitation to cel based therapies of skeletal myopathies. Fortunately, skeletal myopathies exhibiting a dominant mode of inheritance are relatively rare and are associated with comparatively mild symptoms[36]. Therefore we remain confident that the creation of circulating myogenic progenitors via expression of Hα7 on inflamatory cels wil facilitate the development of novel cel and gene therapies for the majority of skeletal myopathies.   107 4.2  REFERENCES 1. Peault, B., et al., Stem and progenitor cels in skeletal muscle development, maintenance, and therapy. Mol Ther, 207. 15(5): p. 867-77. 2. Guerete, B., et al., Lymphocyte infiltration folowing alo- and xenomyoblast transplantation in mice. Transplant Proc, 194. 26(6): p. 3461-2. 3. Snow, M.H., The efects of aging on satelite cels in skeletal muscles of mice and rats. Cel Tissue Res, 197. 185(3): p. 399-408. 4. Neumeyer, A.M., D.M. DiGregorio, and R.H. Brown, Jr., Arterial delivery of myoblasts to skeletal muscle. Neurology, 192. 42(12): p. 258-62. 5. Karpati, G., et al., Myoblast transfer in Duchene muscular dystrophy. An Neurol, 193. 34(1): p. 8-17. 6. Gussoni, E., et al., Normal dystrophin transcripts detected in Duchene muscular dystrophy patients after myoblast transplantation. Nature, 192. 356(6368): p. 435-8. 7. Skuk, D., et al., Successful myoblast transplantation in primates depends on apropriate cel delivery and induction of regeneration in the host muscle. Exp Neurol, 199. 15(1): p. 2-30. 8. Montaras, D., et al., Direct isolation of satelite cels for skeletal muscle regeneration. Science, 205. 309(5743): p. 2064-7. 9. Sulivan, S. and K. Egan, The potential of cel fusion for human therapy. Stem Cel Rev, 206. 2(4): p. 341-9. 10. 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