1
Restoration of Human Dystrophin Following Transplantation of Exon-Skipping-Engineered DMD Patient Stem Cells into Dystrophic Mice

Benchaouir R, Meregalli M, Farini A, D'Antona G, Belicchi M, Goyenvalle A, Battistelli M, Bresolin N, Bottinelli R, Garcia L, Torrente Y.

Duchenne muscular dystrophy (DMD) is a hereditary disease caused by mutations that disrupt the dystrophin mRNA reading frame. In some cases, forced exclusion (skipping) of as in single exon can restore the reading frame, giving rise to a shorter, but still functional, protein. In this study, we constructed lentiviral vectors expressing antisense oligonucleotides in order to induce an efficient exon skipping and to correct the initial frameshift caused by the DMD deletion of CD133+ stem cells. The intra­muscular and intra-arterial delivery of geneti­cally corrected CD133 expressing myogenic progenitors isolated from the blood and muscle of DMD patients results in a significant recov­ery of muscle morphology, function, and dystrophin expression in scid/mdx mice. These data demonstrate that autologous engrafting of blood or muscle-derived CD133+ cells, previously genetically modified to reexpress a functional dystrophin, represents a promising approach for DMD.

2
T and B lymphocyte depletion has a marked effect on the fibrosis of dystrophic skeletal muscles in the scid/mdx mouse.

Farini A, Meregalli M, Belicchi M, Battistelli M, Parolini D, D'Antona G, Gavina M, Ottoboni L, Constantin G, Bottinelli R, Torrente Y.

Fondazione IRCCS Ospedale Maggiore Policlinico of Milan, Department of Neurological Sciences, Dino Ferrari Center, University of Milan, Italy.

Abnormal connective tissue proliferation following muscle degeneration is a major pathological feature of Duchenne muscular dystrophy (DMD), a genetic myopathy due to lack of the sarcolemmal dystrophin protein. Since this fibrotic proliferation is likely to be a major obstacle to the efficacy of future therapies, research is needed to understand and prevent the fibrotic process in order to develop an effective treatment. Murine muscular dystrophy (mdx) is genetically homologous to DMD, and histopatological alterations are comparable to those of the muscles of patients with DMD. To investigate the development of fibrosis, we bred the mdx mouse with the scid immunodepressed mouse and analysed fibrosis histologically; we used ELISA analysis to determine TGF-beta1 expression. Significant reduction of fibrosis and TGF-beta1 expression was found in the muscles of the scid/mdx mice. However, we observed similar centrally located nuclei, necrosis, muscle degeneration and muscle force compared to the mdx animals. These data demonstrate a correlation between the absence of B and T lymphocytes and loss of fibrosis accompanied by reduction of TGF-beta1, suggesting the importance of modulation of the immune system in DMD.

3
Effect of human skin-derived stem cells on vessel architecture, tumor growth, and tumor invasion in brain tumor animal models.

Pisati F, Belicchi M, Acerbi F, Marchesi C, Giussani C, Gavina M, Javerzat S, Hagedorn M, Carrabba G, Lucini V, Gaini SM, Bresolin N, Bello L, Bikfalvi A, Torrente Y.

Stem Cell Laboratory, Department of Neurological Science, Centro Dino Ferrari, University of Milan, Milan, Italy.

Glioblastomas represent an important cause of cancer-related mortality with poor survival. Despite many advances, the mean survival time has not significantly improved in the last decades. New experimental approaches have shown tumor regression after the grafting of neural stem cells and human mesenchymal stem cells into experimental intracranial gliomas of adult rodents. However, the cell source seems to be an important limitation for autologous transplantation in glioblastoma. In the present study, we evaluated the tumor targeting and antitumor activity of human skin-derived stem cells (hSDSCs) in human brain tumor models. The hSDSCs exhibit tumor targeting characteristics in vivo when injected into the controlateral hemisphere or into the tail vein of mice. When implanted directly into glioblastomas, hSDSCs distributed themselves extensively throughout the tumor mass, reduced tumor vessel density, and decreased angiogenic sprouts. In addition, transplanted hSDSCs differentiate into pericyte cell and release high amounts of human transforming growth factor-beta1 with low expression of vascular endothelial growth factor, which may contribute to the decreased tumor cell invasion and number of tumor vessels. In long-term experiments, the hSDSCs were also able to significantly inhibit tumor growth and to prolong animal survival. Similar behavior was seen when hSDSCs were implanted into two different tumor models, the chicken embryo experimental glioma model and the transgenic Tyrp1-Tag mice. Taken together, these data validate the use of hSDSCs for targeting human brain tumors. They may represent therapeutically effective cells for the treatment of intracranial tumors after autologous transplantation.

4
Skin-derived stem cells transplanted into resorbable guides provide functional nerve regeneration after sciatic nerve resection.

Marchesi C, Pluderi M, Colleoni F, Belicchi M, Meregalli M, Farini A, Parolini D, Draghi L, Fruguglietti ME, Gavina M, Porretti L, Cattaneo A, Battistelli M, Prelle A, Moggio M, Borsa S, Bello L, Spagnoli D, Gaini SM, Tanzi MC, Bresolin N, Grimoldi N, Torrente Y.

Fondazione IRCCS Ospedale Maggiore Policlinico-Mangiagalli e Regina Elena of Milan, Stem Cell Laboratory, Department of Neurological Sciences, Centro Dino Ferrari, University of Milan, Italy.

The regeneration in the peripheral nervous system is often incomplete and the treatment of severe lesions with nerve tissue loss is primarily aimed at recreating nerve continuity. Guide tubes of various types, filled with Schwann cells, stem cells, or nerve growth factors are attractive as an alternative therapy to nerve grafts. In this study, we evaluated whether skin-derived stem cells (SDSCs) can improve peripheral nerve regeneration after transplantation into nerve guides. We compared peripheral nerve regeneration in adult rats with sciatic nerve gaps of 16 mm after autologous transplantation of GFP-labeled SDSCs into two different types of guides: a synthetic guide, obtained by dip coating with a L-lactide and trimethylene carbonate (PLA-TMC) copolymer and a collagen-based guide. The sciatic function index and the recovery rates of the compound muscle action potential were significantly higher in the animals that received SDSCs transplantation, in particular, into the collagen guide, compared to the control guides filled only with PBS. For these guides the morphological and immunohistochemical analysis demonstrated an increased number of myelinated axons expressing S100 and Neurofilament 70, suggesting the presence of regenerating nerve fibers along the gap. GFP positive cells were found around regenerating nerve fibers and few of them were positive for the expression of glial markers as S-100 and glial fibrillary acidic protein. RT-PCR analysis confirmed the expression of S100 and myelin basic protein in the animals treated with the collagen guide filled with SDSCs. These data support the hypothesis that SDSCs could represent a tool for future cell therapy applications in peripheral nerve regeneration.

5
Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells.

Dellavalle A, Sampaolesi M, Tonlorenzi R, Tagliafico E, Sacchetti B, Perani L, Innocenzi A, Galvez BG, Messina G, Morosetti R, Li S, Belicchi M, Peretti G, Chamberlain JS, Wright WE, Torrente Y, Ferrari S, Bianco P, Cossu G.

Stem Cell Research Institute, San Raffaele Scientific Institute, 58 Via Olgettina, 20132 Milan, Italy.

Cells derived from blood vessels of human skeletal muscle can regenerate skeletal muscle, similarly to embryonic mesoangioblasts. However, adult cells do not express endothelial markers, but instead express markers of pericytes, such as NG2 proteoglycan and alkaline phosphatase (ALP), and can be prospectively isolated from freshly dissociated ALP(+) cells. Unlike canonical myogenic precursors (satellite cells), pericyte-derived cells express myogenic markers only in differentiated myotubes, which they form spontaneously with high efficiency. When transplanted into severe combined immune deficient-X-linked, mouse muscular dystrophy (scid-mdx) mice, pericyte-derived cells colonize host muscle and generate numerous fibres expressing human dystrophin. Similar cells isolated from Duchenne patients, and engineered to express human mini-dystrophin, also give rise to many dystrophin-positive fibres in vivo. These data show that myogenic precursors, distinct from satellite cells, are associated with microvascular walls in the human skeletal muscle, may represent a correlate of embryonic 'mesoangioblasts' present after birth and may be a promising candidate for future cell-therapy protocols in patients.

6
Autologous transplantation of muscle-derived CD133+ stem cells in Duchenne muscle patients.

Torrente Y, Belicchi M, Marchesi C, Dantona G, Cogiamanian F, Pisati F, Gavina M, Giordano R, Tonlorenzi R, Fagiolari G, Lamperti C, Porretti L, Lopa R, Sampaolesi M, Vicentini L, Grimoldi N, Tiberio F, Songa V, Baratta P, Prelle A, Forzenigo L, Guglieri M, Pansarasa O, Rinaldi C, Mouly V, Butler-Browne GS, Comi GP, Biondetti P, Moggio M, Gaini SM, Stocchetti N, Priori A, D'Angelo MG, Turconi A, Bottinelli R, Cossu G, Rebulla P, Bresolin N.

Fondazione IRCCS Ospedale Maggiore Policlinico of Milan, Department of Neurological Sciences, Dino Ferrari Center, University of Milan, Italy. torrenteyvan@hotmail.com

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive muscle disease due to defect on the gene encoding dystrophin. The lack of a functional dystrophin in muscles results in the fragility of the muscle fiber membrane with progressive muscle weakness and premature death. There is no cure for DMD and current treatment options focus primarily on respiratory assistance, comfort care, and delaying the loss of ambulation. Recent works support the idea that stem cells can contribute to muscle repair as well as to replenishment of the satellite cell pool. Here we tested the safety of autologous transplantation of muscle-derived CD133+ cells in eight boys with Duchenne muscular dystrophy in a 7-month, double-blind phase I clinical trial. Stem cell safety was tested by measuring muscle strength and evaluating muscle structures with MRI and histological analysis. Timed cardiac and pulmonary function tests were secondary outcome measures. No local or systemic side effects were observed in all treated DMD patients. Treated patients had an increased ratio of capillary per muscle fibers with a switch from slow to fast myosin-positive myofibers.

7
Induction of neurotrophin expression via human adult mesenchymal stem cells: implication for cell therapy in neurodegenerative diseases.

Pisati F, Bossolasco P, Meregalli M, Cova L, Belicchi M, Gavina M, Marchesi C, Calzarossa C, Soligo D, Lambertenghi-Deliliers G, Bresolin N, Silani V, Torrente Y, Polli E.

Fondazione IRCCS Ospedale Maggiore, Department of Neurological Sciences, Stem Cell Laboratory, Dino Ferrari Center, University of Milan, Milan, Italy.

In animal models of neurological disorders for cerebral ischemia, Parkinson's disease, and spinal cord lesions, transplantation of mesenchymal stem cells (MSCs) has been reported to improve functional outcome. Three mechanisms have been suggested for the effects of the MSCs: transdifferentiation of the grafted cells with replacement of degenerating neural cells, cell fusion, and neuroprotection of the dying cells. Here we demonstrate that a restricted number of cells with differentiated astroglial features can be obtained from human adult MSCs (hMSCs) both in vitro using different induction protocols and in vivo after transplantation into the developing mouse brain. We then examined the in vitro differentiation capacity of the hMSCs in coculture with slices of neonatal brain cortex. In this condition the hMSCs did not show any neuronal transdifferentiation but expressed neurotrophin low-affinity (NGFR(p75)) and high-affinity (trkC) receptors and released nerve growth factor (NGF) and neurotrophin-3 (NT-3). The same neurotrophin's expression was demonstrated 45 days after the intracerebral transplantation of hMSCs into nude mice with surviving astroglial cells. These data further confirm the limited capability of adult hMSC to differentiate into neurons whereas they differentiated in astroglial cells. Moreover, the secretion of neurotrophic factors combined with activation of the specific receptors of transplanted hMSCs demonstrated an alternative mechanism for neuroprotection of degenerating neurons. hMSCs are further defined in their transplantation potential for treating neurological disorders.

8
Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs.

Sampaolesi M, Blot S, D'Antona G, Granger N, Tonlorenzi R, Innocenzi A, Mognol P, Thibaud JL, Galvez BG, Barthélémy I, Perani L, Mantero S, Guttinger M, Pansarasa O, Rinaldi C, Cusella De Angelis MG, Torrente Y, Bordignon C, Bottinelli R, Cossu G.

San Raffaele Scientific Institute, Università Vita e Salute, Stem Cell Research Institute, Via Olgettina 58, 20132 Milan, Italy.

Duchenne muscular dystrophy remains an untreatable genetic disease that severely limits motility and life expectancy in affected children. The only animal model specifically reproducing the alterations in the dystrophin gene and the full spectrum of human pathology is the golden retriever dog model. Affected animals present a single mutation in intron 6, resulting in complete absence of the dystrophin protein, and early and severe muscle degeneration with nearly complete loss of motility and walking ability. Death usually occurs at about 1 year of age as a result of failure of respiratory muscles. Here we report that intra-arterial delivery of wild-type canine mesoangioblasts (vessel-associated stem cells) results in an extensive recovery of dystrophin expression, normal muscle morphology and function (confirmed by measurement of contraction force on single fibres). The outcome is a remarkable clinical amelioration and preservation of active motility. These data qualify mesoangioblasts as candidates for future stem cell therapy for Duchenne patients.

9
High-resolution X-ray microtomography for three-dimensional visualization of human stem cell muscle homing.

Torrente Y, Gavina M, Belicchi M, Fiori F, Komlev V, Bresolin N, Rustichelli F.

Department of Neurological Sciences, Centro Dino Ferrari, University of Milan, Padiglione Ponti, Ospedale Policinico, Via Francesco Sforza 35, 20122 Milan, Italy. torrenteyvan@hotmail.com

In the perspective of clinical translation of stem cell research, it would be advantageous to develop new techniques to detect donor cells after transplantation to track their fate and thus better understand their role in regeneration of damaged and diseased tissues. In this study we use X-ray computed microtomography for three-dimensional visualization of stem cells that were labeled with magnetic nanoparticles and transplanted via intra-arterial infusion. We show that X-ray computed microtomography offers the possibility to detect with high definition and resolution human cells after transplantation, and opens new possibilities for both experimental stem cell research.

10
VCAM-1 expression on dystrophic muscle vessels has a critical role in the recruitment of human blood-derived CD133+ stem cells after intra-arterial transplantation.

Gavina M, Belicchi M, Rossi B, Ottoboni L, Colombo F, Meregalli M, Battistelli M, Forzenigo L, Biondetti P, Pisati F, Parolini D, Farini A, Issekutz AC, Bresolin N, Rustichelli F, Constantin G, Torrente Y.

Stem Cell Laboratory, Department of Neurological Sciences, IRCCS, Centro Dino Ferrari, University of Milan, Ospedale Maggiore Policlinico, via Francesco Sforza 35, 20122 Milan, Italy.

Recently our group demonstrated the myogenic capacity of human CD133(+) cells isolated from peripheral blood when delivered in vivo through the arterial circulation into the muscle of dystrophic scid/mdx mice. CD133(+) stem cells express the adhesion molecules CD44, LFA-1, PSGL-1, alpha4-integrins, L-selectin, and chemokine receptor CCR7. Moreover these cells adhere in vitro to VCAM-1 spontaneously and after stimulation with CCL19. Importantly, after muscle exercise, we found that the expression of VCAM-1 is strongly up-regulated in dystrophic muscle vessels, whereas the number of rolling and firmly adhered CD133(+) stem cells significantly increased. Moreover, human dystrophin expression was significantly increased when muscle exercise was performed 24 hours before the intra-arterial injection of human CD133(+) cells. Finally, treatment of exercised dystrophic mice with anti-VCAM-1 antibodies led to a dramatic blockade of CD133(+) stem cell migration into the dystrophic muscle. Our results show for the first time that the expression of VCAM-1 on dystrophic muscle vessels induced by exercise controls muscle homing of human CD133(+) stem cells, opening new perspectives for a potential therapy of muscular dystrophy based on the intra-arterial delivery of CD133(+) stem cells.

11
Galectin-1 induces skeletal muscle differentiation in human fetal mesenchymal stem cells and increases muscle regeneration.

Chan J, O'Donoghue K, Gavina M, Torrente Y, Kennea N, Mehmet H, Stewart H, Watt DJ, Morgan JE, Fisk NM.

Institute of Reproductive and Developmental Biology, Imperial College London, Hammersmith Campus, London, United Kingdom. jerrychan@nus.edu.sg

Cell therapy for degenerative muscle diseases such as the muscular dystrophies requires a source of cells with the capacity to participate in the formation of new muscle fibers. We investigated the myogenic potential of human fetal mesenchymal stem cells (hfMSCs) using a variety of stimuli. The use of 5-azacytidine or steroids did not produce skeletal muscle differentiation, whereas myoblast-conditioned medium resulted in only 1%-2% of hfMSCs undergoing muscle differentiation. However, in the presence of galectin-1, 66.1% +/- 5.7% of hfMSCs, but not adult bone marrow-derived mesenchymal stem cells, assumed a muscle phenotype, forming long, multinucleated fibers expressing both desmin and sarcomeric myosin via activation of muscle regulatory factors. Continuous exposure to galectin-1 resulted in more efficient muscle differentiation than pulsed exposure (62.3% vs. 39.1%; p < .001). When transplanted into regenerating murine muscle, galectin-1-exposed hfMSCs formed fourfold more human muscle fibers than nonstimulated hfMSCs (p = .008), with similar results obtained in a scid/mdx dystrophic mouse model. These data suggest that hfMSCs readily undergo muscle differentiation in response to galectin-1 through a stepwise progression similar to that which occurs during embryonic myogenesis. The high degree of myogenic conversion achieved by this method has relevance for the development of therapies for muscular dystrophies.

12
Complete repair of dystrophic skeletal muscle by mesoangioblasts with enhanced migration ability.

Galvez BG, Sampaolesi M, Brunelli S, Covarello D, Gavina M, Rossi B, Constantin G, Torrente Y, Cossu G.

Stem Cell Research Institute, San Raffaele Hospital, 20132 Milan, and Department of Experimental Medicine, Human Anatomy Institute, University of Pavia, Italy.

Efficient delivery of cells to target tissues is a major problem in cell therapy. We report that enhancing delivery of mesoangioblasts leads to a complete reconstitution of downstream skeletal muscles in a mouse model of severe muscular dystrophy (alpha-sarcoglycan ko). Mesoangioblasts, vessel-associated stem cells, were exposed to several cytokines, among which stromal- derived factor (SDF) 1 or tumor necrosis factor (TNF) alpha were the most potent in enhancing transmigration in vitro and migration into dystrophic muscle in vivo. Transient expression of alpha4 integrins or L-selectin also increased several fold migration both in vitro and in vivo. Therefore, combined pretreatment with SDF-1 or TNF-alpha and expression of alpha4 integrin leads to massive colonization (>50%) followed by reconstitution of >80% of alpha-sarcoglycan-expressing fibers, with a fivefold increase in efficiency in comparison with control cells. This study defines the requirements for efficient engraftment of mesoangioblasts and offers a new potent tool to optimize future cell therapy protocols for muscular dystrophies.

13
Human skin-derived stem cells migrate throughout forebrain and differentiate into astrocytes after injection into adult mouse brain.

Belicchi M, Pisati F, Lopa R, Porretti L, Fortunato F, Sironi M, Scalamogna M, Parati EA, Bresolin N, Torrente Y.

Department of Neurological Sciences, Stem Cell Laboratory, Centro Dino Ferrari, IRCCS Ospedale Maggiore Policlinico, University of Milan, Milan, Italy.

Recent evidence indicates that neural stem cell properties can be found among a mammalian skin-derived multipotent population. A major barrier in the further characterization of the human skin-derived neural progenitors is the inability to isolate this population based on expression of cell surface markers. Our work has been devoted to purified human skin-derived stem cells that are capable of neural differentiation, based on the presence or absence of the AC133 cell surface marker. The enriched skin-derived AC133(+) cells express the CD34 and Thy-1 antigens. These cells cultured in a growth medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) proliferate, forming spheres, and differentiate in vitro into neurons, astrocytes, and rarely into oligodendrocytes. Single cells from sphere cultures initiated from human purified AC133(+) cells were replated as single cells and were able to generate new spheres, demonstrating the self-renewing ability of these stem cell populations. Brain engraftment of cells obtained from human purified AC133(+)-derived spheres generated different neural phenotypes: immature neurons and a most abundant population of well differentiated astrocytes. The AC133-derived astrocytes assumed perivascular locations in the frontal cortex. No donor-derived oligodendrocytes were found in the transplanted mouse brains. Several donor small, rounded cells that expressed endothelial markers were found close to the host vessel and near the subventricular zone. Thus, mammalian skin AC133-derived cells behave as a multipotent population with the capacity to differentiate into neural lineages in vitro and, prevalently, endothelium and astrocytes in vivo, demonstrating the great plasticity of these cells and suggesting potential clinical application. Copyright 2004 Wiley-Liss, Inc.

14
Human circulating AC133(+) stem cells restore dystrophin expression and ameliorate function in dystrophic skeletal muscle.

Torrente Y, Belicchi M, Sampaolesi M, Pisati F, Meregalli M, D'Antona G, Tonlorenzi R, Porretti L, Gavina M, Mamchaoui K, Pellegrino MA, Furling D, Mouly V, Butler-Browne GS, Bottinelli R, Cossu G, Bresolin N.

Stem Cell Laboratory, Department of Neurological Science, Instituto di Ricovero e Cura a Carattere Scientifico Ospedale Maggiore Policlinico, Centro Dino Ferrari, University of Milan, Italy. torrenteyvan@hotmail.com

Duchenne muscular dystrophy (DMD) is a common X-linked disease characterized by widespread muscle damage that invariably leads to paralysis and death. There is currently no therapy for this disease. Here we report that a subpopulation of circulating cells expressing AC133, a well-characterized marker of hematopoietic stem cells, also expresses early myogenic markers. Freshly isolated, circulating AC133(+) cells were induced to undergo myogenesis when cocultured with myogenic cells or exposed to Wnt-producing cells in vitro and when delivered in vivo through the arterial circulation or directly into the muscles of transgenic scid/mdx mice (which allow survival of human cells). Injected cells also localized under the basal lamina of host muscle fibers and expressed satellite cell markers such as M-cadherin and MYF5. Furthermore, functional tests of injected muscles revealed a substantial recovery of force after treatment. As these cells can be isolated from the blood, manipulated in vitro, and delivered through the circulation, they represent a possible tool for future cell therapy applications in DMD disease or other muscular dystrophies.

15
Fate of autologous dermal stem cells transplanted into the spinal cord after traumatic injury (TSCI).

Gorio A, Torrente Y, Madaschi L, Di Stefano AB, Pisati F, Marchesi C, Belicchi M, Di Giulio AM, Bresolin N.

Laboratory of Pharmacology, Department of Medicine, Surgery and Dentistry, Faculty of Medicine, University of Milan, Via A di Rudinì 8, Milano 20142, Italy. alfredo.gorio@unimi.it

Rat dermis is a source of cells capable of growing in vitro and, in appropriate conditions, forming floating spheres constituted by nestin-positive cells. We have clonally grown these spheres up to the 15th generation. These spheres can be dissociated into cells that differentiate in vitro under appropriate conditions, these cells are labeled by antibodies to immature neuron markers such as nestin and beta-tubulin III and, later, to mature neuron markers such as microtubule-associated protein 2 and neurofilaments. However, most cells are positive to the astroglial marker glia fibrillary acidic protein (GFAP). When sphere-derived cells are transplanted into the spinal cord after traumatic injury, their migration into the lesion cavity is optimal but their differentiation is dependent upon the time interval between lesioning and cell transplantation. Injection of skin-derived stem cell within 30 min from injury yields mainly membrane activated complex-1 (MAC-1), cluster of differentiation-4 (CD-4) and CD-8 positive cells, that 60-90 days later undergo apoptosis. However, when transplantation is performed 7 days after injury, most cells (65% of total) are positive to staining with antibodies to GFAP, others (16%) to neurofilaments, and a smaller amount (2%) to the endothelial marker, platelet endothelial cell adhesion molecule. Thus our study shows that delayed transplantations of dermis-derived stem cells yield healthy cells that do not die, migrate to the lesion site, and there differentiate mainly in cells expressing glia and neuronal markers. On the other hand there is the possibility of dye transfer from labeled cells to endogenous cells, and this might influence the data.

16
Identification of a putative pathway for the muscle homing of stem cells in a muscular dystrophy model.

Torrente Y, Camirand G, Pisati F, Belicchi M, Rossi B, Colombo F, El Fahime M, Caron NJ, Issekutz AC, Constantin G, Tremblay JP, Bresolin N.

Department of Neurological Sciences, Stem Cell Laboratory, University of Milan, Padiglione Ponti, Ospedale Policlinico, via Francesco Sforza 35, 20122 Milan, Italy. torrenteyvan@hotmail.com

Attempts to repair muscle damage in Duchenne muscular dystrophy (DMD) by transplanting skeletal myoblasts directly into muscles are faced with the problem of the limited migration of these cells in the muscles. The delivery of myogenic stem cells to the sites of muscle lesions via the systemic circulation is a potential alternative approach to treat this disease. Muscle-derived stem cells (MDSCs) were obtained by a MACS(R) multisort method. Clones of MDSCs, which were Sca-1+/CD34-/L-selectin+, were found to adhere firmly to the endothelium of mdx dystrophic muscles after i.v. or i.m. injections. The subpopulation of Sca-1+/CD34- MDSCs expressing L-selectin was called homing MDSCs (HMDSCs). Treatment of HMDSCs with antibodies against L-selectin prevented adhesion to the muscle endothelium. Importantly, we found that vascular endothelium from striate muscle of young mdx mice expresses mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a ligand for L-selectin. Our results showed for the first time that the expression of the adhesion molecule L-selectin is important for muscle homing of MDSCs. This discovery will aid in the improvement of a potential therapy for muscular dystrophy based on the systemic delivery of MDSCs.

17
Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts.

Sampaolesi M, Torrente Y, Innocenzi A, Tonlorenzi R, D'Antona G, Pellegrino MA, Barresi R, Bresolin N, De Angelis MG, Campbell KP, Bottinelli R, Cossu G.

Stem Cell Research Institute, H. S. Raffaele, Via Olgettina 58, 20132 Milan, Italy.

Preclinical or clinical trials for muscular dystrophies have met with modest success, mainly because of inefficient delivery of viral vectors or donor cells to dystrophic muscles. We report here that intra-arterial delivery of wild-type mesoangioblasts, a class of vessel-associated stem cells, corrects morphologically and functionally the dystrophic phenotype of virtually all downstream muscles in adult immunocompetent alpha-sarcoglycan (alpha-SG) null mice, a model organism for limb-girdle muscular dystrophy. When mesoangioblasts isolated from juvenile dystrophic mice and transduced with a lentiviral vector expressing alpha-SG were injected into the femoral artery of dystrophic mice, they reconstituted skeletal muscle in a manner similar to that seen in wild-type cells. The success of this protocol was mainly due to widespread distribution of donor stem cells through the capillary network, a distinct advantage of this strategy over previous approaches.

18
Tumor necrosis factor-alpha (TNF-alpha) stimulates chemotactic response in mouse myogenic cells.

Torrente Y, El Fahime E, Caron NJ, Del Bo R, Belicchi M, Pisati F, Tremblay JP, Bresolin N.

Centro Dino Ferrari, Institute of Clinical Neurology, University of Milan, Milan, Italy.

Migration of transplanted myogenic cells occurs during both embryogenesis and regeneration of skeletal muscles and is important for successful myoblast transplantation, but little is known about factors that promote chemotaxis of these cells. Tumor necrosis factor-alpha (TNF-alpha) is known to induce chemotactic effect on several cell types. In this study, we investigated its influence on the in vitro and in vivo motility of C2C12 and primary myoblasts. In the in vitro test performed in the blind-well Boyden chambers, we showed that TNF-alpha (50-400 U/ml) significantly enhanced the ability of myogenic cells to migrate. The dose-response curve for this factor was bell shaped, with maximum activity in the 200 U/ml range. In the in vivo test, intramuscular administration of TNF-alpha was performed by an Alzet pump connected to a perforated polyethylene microtube inserted in the tibialis anterior (TA) of CD1 mice. In these experiments, myoblasts were injected under the muscle epimysium. The recipient mice were immunosuppressed with FK506. Our results showed that, 5 days after myoblast transplantation, cells migrated further in the muscles infused with TNF-alpha than in the muscles not exposed to TNF-alpha. TNF-alpha not only has a chemotactic activity but may also modify cell migration via its action on matrix metalloproteinase (MMP) expression. The proteolytic activities of the MMPs secreted in the muscles were thus also assessed by gelatin zymography. The results showed an increased of MMP-2 and MMP-9 transcripts in the TNF-alpha-infused muscles injected with myogenic cells. Myoblast migration during transplantation may be enhanced by overlapping gradients of several effector molecules such as TNF-alpha, interferon-gamma (INF-gamma), and interleukins, released at the site of muscle injury. We propose that TNF-alpha may promote myoblast migration directly through chemotactic activity and indirectly by enhancing MMP activity at the site of muscle injury.

20
Alternative sources of neurons and glia from somatic stem cells.

Torrente Y, Belicchi M, Pisati F, Pagano SF, Fortunato F, Sironi M, D'Angelo MG, Parati EA, Scarlato G, Bresolin N.

IRCCS Ospedale Maggiore Policlinico, Italy.

Stem cell populations have been shown to be extremely versatile: they can generate differentiated cells specific to the tissue in which they reside and descendents that are of different germ layer origin. This raises the possibility of obtaining neuronal cells from new biological source of the same adult human subjects. In this study, we found that epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) cooperated to induce the proliferation, self-renewal, and expansion of neural stem cell-like population isolated from several newborn and adult mouse tissues: muscle and hematopoietic tissues. This population, in both primary culture and secondary expanded clones, formed spheres of undifferentiated cells that were induced to differentiate into neurons, astrocytes, and oligodendrocytes. Brain engraftment of the somatic-derived neural stem cells generated neuronal phenotypes, demonstrating the great plasticity of these cells with potential clinical application.

21
High-efficiency gene transfer into adult fish: a new tool to study fin regeneration.

Tawk M, Tuil D, Torrente Y, Vriz S, Paulin D.

Laboratoire de biologie moléculaire de la différenciation, Université Paris 7, Paris, France.

Zebrafish represents an excellent model to study the function of vertebrate genes (e.g., well-developed genetics, large number of mutants, and genomic sequencing in progress), inasmuch as we have tools to manipulate gene expression. Recent use of injected morpholinos in eggs provides a good method to " knockdown " gene expression in early development (Nasevicius and Ekker, 2000), and the "caged" RNA injected in eggs allows to overexpress a gene in a specific set of cells (Ando et al., 2001). However, a method to specifically modify gene expression in the juvenile or in the adult is still missing. Such a method would be a very powerful tool to understand gene function in differentiated tissues. We describe here an electroporation-based approach, which allows gene transfer in adult tissues. Its efficiency was assessed using a GFP (green fluorescent protein) dependent assay. We then used this method to disrupt the Fgf signalling pathway during the process of regeneration. Copyright 2002 Wiley-Liss, Inc.

22
Biodistribution studies of 99mTc-labeled myoblasts in a murine model of muscular dystrophy.

Colombo FR, Torrente Y, Casati R, Benti R, Corti S, Salani S, D'Angelo MG, DeLiso A, Scarlato G, Bresolin N, Gerundini P.

IRCCS Ospedale Maggiore, Institute of Nuclear Medicine, Milan, Italy. colombof@policlinico.mi.it

The purpose of this study was twofold: first, to evaluate the myoblast labeling of various 99mTc complexes and to select the complex that best accomplishes this labeling, and second to evaluate the biodistribution of myoblasts labeled with this complex using mice with MDX muscular dystrophy (the murine homologue of Duchenne's muscular dystrophy). The following ligands were used to prepare the corresponding 99mTc complexes: hexakis-methoxy-isobutyl-isonitrile (MIBI), bis(2-ethoxyethyl)diphosphinoethane (Tf), (RR,SS)-4,8-diaza-3,6,6,9-tetramethyl-undecane-2,10-dione-bisoxime (HM-PAO), bis(N-ethyl)dithiocarbamate (NEt), and bis(N-ethoxy, N-ethyl)dithiocarbamate (NOEt).One million murine myoblasts were incubated for 30-60 minutes with 5 mCi of each of the 99mTc complexes prepared from the above ligands. Viability was assessed by microscopic counting after trypan blue staining, and the radioactivity absorbed in the cells was measured after centrifugation. The compound with the highest uptake in cellular pellets was [99mTc]N-NOEt. The biodistribution of myoblasts labeled with this complex was evaluated after intraaortic injection in dystrophic mice. Such an approach has the potential of effecting widespread gene transfer through the bloodstream to muscles lacking dystrophin.

23
Beta-enolase deficiency, a new metabolic myopathy of distal glycolysis.

Comi GP, Fortunato F, Lucchiari S, Bordoni A, Prelle A, Jann S, Keller A, Ciscato P, Galbiati S, Chiveri L, Torrente Y, Scarlato G, Bresolin N.

Istituto di Clinica Neurologica, Università degli Studi di Milano, IRCCS, Ospedale Maggiore Policlinico, Italy. gpcomi@mailserver.unimi.it

A severe muscle enolase deficiency, with 5% of residual activity, was detected in a 47-year-old man affected with exercise intolerance and myalgias. No rise of serum lactate was observed with the ischemic forearm exercise. Ultrastructural analysis showed focal sarcoplasmic accumulation of glycogen beta particles. The enzyme enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. In adult human muscle, over 90% of enolase activity is accounted for by the beta-enolase subunit, the protein product of the ENO3 gene. The beta-enolase protein was dramatically reduced in the muscle of our patient, by both immunohistochemistry and immunoblotting, while alpha-enolase was normally represented. The ENO3 gene of our patient carries two heterozygous missense mutations affecting highly conserved amino acid residues; a G467A transition changing a glycine residue at position 156 to aspartate, in close proximity to the catalytic site, and a G1121A transition changing a glycine to glutamate at position 374. These mutations were probably inherited as autosomal recessive traits since the mother was heterozygous for the G467A and a sister was heterozygous for the G1121A transition. Our data suggest that ENO3 mutations result in decreased stability of mutant beta-enolase. Muscle beta-enolase deficiency should be considered in the differential diagnosis of metabolic myopathies due to inherited defects of distal glycolysis.

24
In vitro and in vivo tetracycline-controlled myogenic conversion of NIH-3T3 cells: evidence of programmed cell death after muscle cell transplantation.

Del Bo R, Torrente Y, Corti S, D'Angelo MG, Comi GP, Fagiolari G, Salani S, Cova A, Pisati F, Moggio M, Ausenda C, Scarlato G, Bresolin N.

Dino Ferrari Center, Institute of Clinical Neurology, University of Milan, Italy.

Ex vivo gene therapy of Duchenne muscular dystrophy based on autologous transplantation of genetically modified myoblasts is limited by their premature senescence. MyoD-converted fibroblasts represent an alternative source of myogenic cells. In this study the forced MyoD-dependent conversion of murine NIH-3T3 fibroblasts into myoblasts under the control of an inducible promoter silent in the presence of tetracycline was evaluated. After tetracycline withdrawal this promoter drives the transcription of MyoD in the engineered fibroblasts, inducing their myogenesis and giving rise to beta-galactosidase-positive cells. MyoD-expressing fibroblasts withdrew from the cell cycle, but were unable to fuse in vitro into multinucleated myotubes. Five days following implantation of engineered fibroblasts in muscles of C57BL/10J mice we observed a sevenfold increase of beta-galactosidase-positive regenerating myofibers in animals not treated with antibiotic compared with treated animals. After 1 week the number of positive fibers decreased and several apoptotic myonuclei were detected. Three weeks following implantation of MyoD-converted fibroblasts in recipient mice, no positive "blue" fiber was observed. Our results suggest that transactivation by tetracycline of MyoD may drive an in vivo myogenic conversion of NIH-3T3 fibroblasts and that, in this experimental setting, apoptosis plays a relevant role in limiting the efficacy of engineered fibroblast transplantation. This work opens the question whether apoptotic phenomena also play a general role as limiting factors of cell-mediated gene therapy of inherited muscle disorders.

25
Intracellular delivery of a Tat-eGFP fusion protein into muscle cells.

Caron NJ, Torrente Y, Camirand G, Bujold M, Chapdelaine P, Leriche K, Bresolin N, Tremblay JP.

Unité de Recherche en Génétique Humaine, Centre de Recherche du Centre Hospitalier de l'Université laval, Ste-Foy, Quebec, Canada G1V 4G2.

The Tat protein from HIV-1, when fused with heterologous proteins or peptides, can traverse biological membranes in a process called "protein transduction," delivering its cargo into cells. A Tat-eGFP fusion protein was purified from bacteria to study the transduction kinetics of Tat fusion proteins into cultured myoblasts and in the muscle tissue. Correctly folded Tat-eGFP reaches a maximum intracellular level in nearly 30 min, while its endogenous fluorescence is first detected only after 14 h. The nuclear localization signal from the basic domain of Tat was not sufficient to confer nuclear localization to Tat-eGFP, suggesting that the nuclear import pathway used by the exogenously added Tat-eGFP might be sensitive to the folding state of eGFP. In mice, the direct delivery to the muscle tissue using subcutaneous injections or the intra-arterial pathway led to few positive fibers in the muscle periphery or surrounding the blood vessels. Muscles injected with Tat-eGFP showed intense labeling of the extracellular matrix (ECM), suggesting that, although Tat fusion proteins can transduce muscle fibers, their binding by components of the ECM surrounding myofibers could interfere with the intracellular transduction process.

26
Intraarterial injection of muscle-derived CD34(+)Sca-1(+) stem cells restores dystrophin in mdx mice.

Torrente Y, Tremblay JP, Pisati F, Belicchi M, Rossi B, Sironi M, Fortunato F, El Fahime M, D'Angelo MG, Caron NJ, Constantin G, Paulin D, Scarlato G, Bresolin N.

Instituto de Ricovero e Cura a Carattere Scientifico (IRCCS) Ospedale Maggiore Policlinico, 20122 Milan, Italy.

Duchenne muscular dystrophy is a lethal recessive disease characterized by widespread muscle damage throughout the body. This increases the difficulty of cell or gene therapy based on direct injections into muscles. One way to circumvent this obstacle would be to use circulating cells capable of homing to the sites of lesions. Here, we showed that stem cell antigen 1 (Sca-1), CD34 double-positive cells purified from the muscle tissues of newborn mice are multipotent in vitro and can undergo both myogenic and multimyeloid differentiation. These muscle-derived stem cells were isolated from newborn mice expressing the LacZ gene under the control of the muscle-specific desmin or troponin I promoter and injected into arterial circulation of the hindlimb of mdx mice. The ability of these cells to interact and firmly adhere to endothelium in mdx muscles microcirculation was demonstrated by intravital microscopy after an intraarterial injection. Donor Sca-1, CD34 muscle-derived stem cells were able to migrate from the circulation into host muscle tissues. Histochemical analysis showed colocalization of LacZ and dystrophin expression in all muscles of the injected hindlimb in all of five out of five 8-wk-old treated mdx mice. Their participation in the formation of muscle fibers was significantly increased by muscle damage done 48 h after their intraarterial injection, as indicated by the presence of 12% beta-galactosidase-positive fibers in muscle cross sections. Normal dystrophin transcripts detected enzymes in the muscles of the hind limb injected intraarterially by the mdx reverse transcription polymerase chain reaction method, which differentiates between normal and mdx message. Our results showed that the muscle-derived stem cells first attach to the capillaries of the muscles and then participate in regeneration after muscle damage.

27
T-antigen regulated expression reduces apoptosis of tag-transformed human myoblasts.

Corti S, Salani S, Del Bo R, Torrente Y, Strazzer S, Belicchi M, Paganoni S, Li Z, Comi GP, Bresolin N, Paulin D, Scarlato G.

Institute of Clinical Neurology, IRCCS Ospedale Maggiore Policlinico, University of Milan, Italy. ticor@tin.it

The generation of human myogenic cell lines could potentially provide a valuable source for cell transplantation in myopathies. The dysregulation of proliferative-differentiative signals by viral oncogenes can result in the induction of apoptosis. Whether apoptosis occurred in myogenic cells expressing large T antigen (Tag) from SV40 upon differentiation was unknown. Human muscle satellite cells were transfected with two different constructs, containing either an origin-defective SV40 genome or Tag under vimentin promoter control. When differentiation was triggered, Tag expression reduced the formation of myotubes and dead cells showing apoptotic features were present. However, the cells expressing SV40 Tag under vimentin promoter control retained their capacity to form myotubes and expressed the myofibrillar proteins as myosin heavy chain and dystrophin when Tag expression was silent. Their apoptotic rate was similar to that of untransfected cells. The observation that apoptosis can be prevented by the down-regulation of Tag suggests that the programmed cell death induced in transformed cells can be reversed, and confirms the regulatory efficiency of the human vimentin promoter.

28
In vivo migration of transplanted myoblasts requires matrix metalloproteinase activity.

El Fahime E, Torrente Y, Caron NJ, Bresolin MD, Tremblay JP.

Unité de Recherche en Génétique Humaine, Centre Hospitalier de l'Université Laval, Ste.-Foy, Québec, Canada.

Muscle cell migration and extracellular matrix remodeling are essential aspects of muscle development and regeneration. In this study, using a new technique to assess in vivo myoblast migration, we have confirmed previous results showing that the C(2)C(12) myoblast cell line exhibits a higher migratory capacity than primary myoblasts. To test the hypothesis that matrix metalloproteinases (MMPs) are required for the migration of C(2)C(12) myoblasts, we determined whether a synthetic metalloproteinase inhibitor, BB94 (Batimastat), inhibited this process in vivo. Pretreatment with BB94 for 3 days decreased the C(2)C(12) migration at 2 days after cell injection. Since MMP expression is thus necessary for myoblast migration, we have undertaken the identification and characterization of the MMPs expressed by the C(2)C(12) cell line. An RT-PCR assay was used to determine the pattern of MMP mRNA expression by the C(2)C(12) cell line. The proteolytic activities of the MMPs secreted in the culture medium were also assessed by gelatin zymography. The results showed that MMP2 (gelatinase A, 72-kDa type IV collagenase) and MT1-MMP transcripts were expressed by this cell line; however, only MMP2 was secreted and was able to be activated in the extracellular environment. This cell line failed to express