Duchenne Muscular Dystrophy (DMD): Duchenne muscular dystrophy is monogenic disease linked to chromosome X that affects a newborn every 3,500 male, is characterized by alteration of gene dystropin. This alteration cause , at the level of the membrane muscles, a wrong complex formed by dystrophin and from glycoproteins associated with it causing the loss of cell function and a tissutal replacement by fibrotic tissue.
There are only limited therapeutic prospects in this field. The evidence that the stem cells from muscle transplanted in a mouse with a congenital dystrophinopathies partly restored the normal production of distrophinin in muscles and in the heart during the life postnatal has spurred further research in this sense.
In these years the work of our group has concentrated on the characterization of the myogenic potential of different cellular populations isolated from muscular tissue and from blood human; particularly we focus on the cellular fraction that has the antigen of membrane CD133, marker typically expressed from the stem cells. The evaluation of the capacity myogenic potential of these cells was conduct with experimental approaches is both in vitro and in vivo, using a murine model of muscular dystrophy immunedepressed, the mouse scid/mdx. Some ours recent articles support the theory according to which the stem cells could contribute to put right muscular damages and to reconstruct, after an injection intra-artery, the pool of satellite cells presents in the muscle. These obviousnes advise how this technology would be able to be especially advantageous and useful in the treatment of the muscular dystrophies.
In this sense, ,to evaluate the migrant capacity of the stem cells circulating CD133 + we analyzed to the citofluorimeter expression of adherence molecules on their surface and chemochine receptors. In this phase of the study the CD133+ co-expressing integrins expressed by eosinofile and basophile cells, and more of 50% of the stem cells circulating express adesion molecules, involved in the migration of the lymphocytes across the rolling and the homing of the lymphocytes T.
Generally in the leukocytes expression of chemochine receptors and of adherence molecules is regulate by inflammatory environment, while the cells CD133 positive obtained from the blood do not seem to be influenced in significant manner from these conditions. The circulating cells CD133+ express an entire pattern of chemochine receptors and of adherence molecules responsible of their capacity of to migrate across the vessel also in basal conditions, they don’t require stimulus of activation.
By the use of the intravital microscopy were analyzed also the vessels of the mouse scid/mdx for the antiligand expression of the adherence molecules. These results show, therefore, as the expression of adherence molecules, in particulary VCAM, on the endothelium of a dystrophic muscle represents a key mechanism for the capacity of migrate some cells CD133 + and increase manner the potential of a therapy for the muscular dystrophy that base itself on the administration of stem cells across the arterial district.
In a work, maked in collaboration with the Department of Sciences Applied, Politecnic University of the Marche, has proceeded to mark the cells CD133 + with nanoparticles of iron oxide, using ENDOREM, a tracing vital and inert, already used in clinic. In this study they showned like the microtomography to X-rays was efficent to visualize to high definition and resolution human stem cells so marked and transplanted intra-artery in a mouse scid-mdx. Furthmore, this technology offers the possibility to quantify the number of cells that can migrate from the circulatory torrent inside of the muscular tissue and allows the three-dimensional display of their distribution in the receiver tissue. The marking with Endorem is an promising approach to be able to visualize stem cells in vivo and would be able to be a significant help to understand the proceeded of base that regulate their homing and migrant their capacity.
In collaboration with the group of search directed by the Professor Cossu at Hospital San Raffaele of Milan we wanted to investigate the migrant potential of mesangioblasts, a population of stem cells associated to the blood vessel able to differentiate in different cellular types derived from mesoderm, including also the skeletal muscle. The reasons of partial effect of the mesangioblasts in this model of dystrophy would be due to their limited capacity to accede and to colonize the muscle that depends from an incomplete adherence to endothelial tissue. Therefore the aim to increase the process of restoration the muscular damage using the mesangioblasts becomes essential increase their migrant capacity in the muscular district. In a recent work they is shown in fact that the mesangioblastsis if exposed to different citokyne, among which the most important are SDF 1 and TNF., they purchase the ability to migrate in vitro and in alive after transplantation intra-muscle in distrophic mouse models meaningful the migratory abilities of these cells both in vitro and in the distrophic muscle with the consequent production of new fibers that express the normal copy of the changed gene.
In the last years some searches have shown that the animal model that reproduces in specific way the alterations of the gene of the distrophinin and everything the elapsed of the human pathology it is the canine model golden retriver. These sick animals introduce a single mutation to level of the exon 6 that cause absence of distrophinin and a severe form of distrophy, that compromise all the muscles, also faringeal muscles, lungs, heart. To test the efficacy of a gene therapy and/or cellular therapy we effected another study the collaboration with Professor Cossu. We isolated and the characterizated mesangioblastsis gotten by muscles of distrophic dogs. We injected cells derived from health donor in dogs after immunosupression using rapamycin and ciclosporin to evaluate the efficence of autologo transplant and allotransplant..
The results have shown that the dogs submitted eterologous transplantation are meaningfully improved, while the cells genetically correct autologhe has given less efficient results.
In our laboratory we have a considerable experience reegard biologic caracteristic of adult somitic stem cells used in a neuromuscular disease. Derived muscle stem cells ,transplanted in mdx mouse, restablish normal production of dystrophinin.
In this search it has been demonstrated that from the transplant of mesangioblasts in distrophinic dogs the distrophinin is possible to obtain an extensive reconstruction of expressing fibers and an increment of the contraction force, let alone, in many cases, preservarne the ability to walk.

It is possible make future clinical trial using stem cells captured from a isto-compatible donor, under immunosubpression. The search in fact has already demonstrated that muscular progenitors as the myoblast they can be fused with the cells of the host after transplant in damaged weaving or sick in which the cells of the host as an example go encounter to regeneration. Although the transplant of myoblast normal in a distrophinin-deficient rat is in a position to restoring the expression of the distrophinin, the identification of a stem cells population less commissioned and its use in clinical field could improve this type of therapeutic approach. For this reason we have executed a clinical study of Phase in order to demonstrate not the toxicity of these stem cells once transplanted in a distrophinic muscle. The result of this study has allowed us to confirm the emergency of the cellular product and its muscular potentiality. In fact two of the transplanted distrophinic children have shown an improvement of the vascular structures of muscles transplanted with consequent benefit in sense of muscular force. The next objective is that one to succeed to favor the production of the protein distrophinin from these stem cells. As corrective factor of the DNA of the distrophinin could be had use of of a methodical one recently documented on the scientific review Science that takes the name of exon-skipping. In fact, the majority of the mutations to the base of the DMD is caused from deletions that can generate a codone of premature stop or a proteica sequence does not work them in terms of attack to the complexes associated to the distrophinin.
The approach of the exon-skipping is developed in order to reconstitute to a picture of reading corrected at the moment of the transcription of the gene codifying for a protein distrophinin works them. In effects, numerous deletion are in a position to being brought back to a correct picture of reading as a result of the abolition or of various ones other hexones of the interest region. The acquaintance of the sequence of the hexones of the gene of the distrophinin allows to recognize the hexones easy that they are in the corrected position regarding those badly placed. These mechanisms of skipping occasionally are introduced spontaneously and been responsible of the said fiber appearance “revertant”, observed by means of immunoistochemical tests on muscle sections mdx. The first applications of the exon-skipping in order to correct the distrophin phenotype have used of the oligoribonucleotyde sequences antisense that mask in specific way the containing site the codon of premature stop allowing the progression of the reading of the other hexones of the gene of the distrophinin. The reading machine therefore “is forced” to execute a reading wider alternative. These oligoribonucleotyde modified have initial state employed with a sure one happening in the myoblast of rat mdx, later on for intramuscular trasfection in the rats mdx using of the liposomal complexes or the lipofectin. Recently the efficiency of exon-skipping has been increased thanks to the connection of the oligoribonucleotyde with a little one snRNA, the U7snRNA. In clinical perspective, the technique of the exon-skipping must be able to interest an immense muscular territory for having a therapeutic truth. The intramuscular injections give confirmation on the method of approach, but the effects remain too much limited to the dealt muscular territory. The insertion of sequences antisense in carriers turns having them a strong muscular trophism allows an important advance in terms of spread of the treatment. In effects, the approach of the genic therapy has lead to the construction and the injection of carriers AAV (Adeno-Associated Virus) for sistemic way (intra-venous and intra-arterial) in the rats mdx. Spectacular result of the king-expression of a distrophinin work them in an immense muscular territory, like the recovery of specific strength, have carried the group of Luis Garci'a of the Genethon institute to apply this approach in the canine model of DMD. Preliminary result from this group demonstrate that to the injection in the femoral vein of AAV U7snRNA permit the production than 2000 positive fibers distrophinin for muscle section in great part of muscles of the dealt limb. Unfortunately the approach by means of AAV places the problem of the of immunity answer, that it comes so much more evidenced in the event than repeated injections of the carrier. To this point it can be assumed to re-establish the picture of reading of the gene of the distrophinin by means of exon-skipping in as an example isolated stem cells from the blood circulation or the muscle of patients DMD. Therefore the combination of cellular and genic treatment would carry to alive the former correction of sick human cells and to the re-expression of the distrophinin in alive after their transplant. Therefore we have begun to work in this direction collaborating directly with the group of Luis Garci'a. In these experiments, thanks I use to it of lentiviral carriers we have obtained the expression of the distrophinin from isolated stem cells positive CD133 from the blood of subjects with deletion 48-50 and 49-50. These cells have been then directly injected in muscles of distrophinic rats and immunodeprexed scid/mdx. After 2 months from the intramuscular transplant the correct cells have supplied the test of the principle that can express the distrophinin and participate actively to regeneration of the muscular woven one of distrophinic rats. Now we are trying to obtain the same result with isolated cells from subjects with other types of deletion. These data must be considered preliminary and needy of ulterior confirmations before being able to think next to future clinical applications.

Neurodegenerative diseases

The able neuronal progenitors of giving life to neuronal and gliali cells have been isolated from the embryonic cerebral woven one. These cells are also ritrovabili in minor amount in some cerebral areas (periventricolari and of the olfattorio bulb) of brains adults. These neuronal progenitors are in a position to proliferare in vitro in presence of some factors of increase giving life to a pool of cells always available for eventual transplants. The gliomi they constitute a group of tumors that, although the diagnostic and therapeutic progresses, continue above all to having infaust prognosis because of the elevated heterogeneity of the same tumor that constitutes the main difficulty in the classification and the clinical management. For this reason during the last few years a search aimed at the identification of the genetic alterations expressed from the gliomi has been developed in the attempt better to define eventual target therapeutic that concur a greater effectiveness of the treatments. In this within various they are the tipologie of genetic anomalies inside of tumors to the same clinical stadiation thus like associations between some of they that they concur to define the degree of tumorale progression. The astrocyte one me of low degree chromosome delezioni of 17 and mutation of the gene are characterized from p53 that it resides in the region deleta. Since p53 it codifies for a regolatrice protein of the cellular cycle, its mutation cause a uncontrolled replication of the cells with consequent accumulate of ulterior mutations. Although this type of mutation is present in 65% of the astrocyte one me of low degree, it is found also in anaplastic patients with astrocyte and GBM to me, suggesting the possibility that these last ones are the outcome of a progression from a low degree. The successive stage, the astrocytoma anaplastic, introduces for the majority alterations of geniuses been involved in the regulation of the cellular cycle. More you attend are involved the Rb gene, present in 40% of the patients, and proteins of control of the cellular cycle to it associated which CDK4 and INK4A.
The amplification of the EGFR gene resorts in the 40-50% of the gliomi to high degree while in gliomi of degree smaller often its mutation it promotes the progression to GBM; the changed receiver cannot be tied to the ligand, but constituent it is activated and it promotes the cellular proliferation, the migration and the invasion. It seems moreover that the mutation in the EGFR expression is the cause of the resistance to the radius chemotherapy of some GBM. The conversion G: C-> To: T in the CpG island and the metilazione of the promotor of this gene causes in the GBM the loss of expression of the protein and therefore a greater answer to the therapy. Recent data have moreover evidenced the presence of stem cells in the tumors of glial origin (1,2,3). In these studies the oncogenic ability from isolated stem cells from glioblastoma expressing several markers which has been evidenced the CD133 and the Thy1. In fact, the injection of these cells in immunodeficent rats has determined the formation of a new gliale tumor of same istologica characteristic. Moreover, the isolated cells from the gliali tumors maintain stem potentialities in neurale sense as demonstrated from experiments based on the expansion they clones and on astrocytes, neurons and oligodendrocytes the differentiation of the off-spring in the three neural lineage. Still the effective role has not been gotten rid that these cells cover in the oncogenesi. In the event in which the or staminale member of the tumor to introduce genomic or chromosomic alterations of for if, it can be assumed that they are it cause of the tumorale insorgence as a result of mechanisms that of it induce the disregolazione in the increase. In alternative it could be the tumor to exercise metabolic infuences on these cells being induced them to modifications in neoplastico sense that persist also as a result of the system of the cell in a healthy atmosphere. This suggests that also the gliomi they could find origin in the proliferation of neurali stem cells that would be come to delineate like new and a more effective target therapeutic one. The discovery of the existence of these cells has moved therefore the attention of the search towards more deepened analysis of their molecular and genetic behavior, finalized to the understanding of the characteristics that cause the maintenance of the clone tumorale and the progression of the same tumor. In the Laboratory of Stem Cells of the Department of neurological Sciences of the IRCCS Policlinico of Milan we have isolated stem cells and progenitors from biopsies of gliomi to the aim obtaining neurosfere CD133+, representative of tumors of various degree and istologia, that they come then characterized from the citogenetic and genomic point of view. In fact those, various, morphologically present are not still in a position to determining which are the able cells to support the increase of the tumor between all in the tumorale mass. The genetic analysis, we have estimated the regeneration abilities and of differentiation in the three lineage neurali the eventual insorgence of new genomic alterations is in vitro that in alive, after the system of the cells in rats knots. From this study an analysis detailed of the pattern is wanted to be obtained genetic of isolated cells CD133+ from bioptici champions of gliomi of various degree and continuations after the espianto or in alive that in vitro to the aim clearing which role covers in the insorgence and the maintenance of the tumor. Morphologic studies have concurred an only partial acquaintance of the clinical behavior of the gliali tumors; similar phenotypes, in fact can have different prognoses and different answers to the treatments. The instability and the genomic heterogeneity of the tumor in toto render in fact its finer analysis in the molecular and genetic aspects difficult while the genetic study of the neurosphere from it can turn out simpler originated between which it could be discriminated patogenetiche aberrations more easy. To verify that these cells can be the cause of the insorgence of the gliomi constitutes a point of departure important in order to deepen the understanding of the mechanisms that sottostanno to the enormous genetic heterogeneity, and consequently to an extreme phaenotipic complexity, observed in these tumors. The identification of cells that cover a role inside key of the tumorale population and their genomic characterization us can concur to define with more precision the characteristics of the single case and to preview the clinical behavior of the tumor. Moreover the analysis can make to emerge the role of various been involved cellular factors in the tumorale increase that can be candidare as it upgrades them new target therapeutic more specific and more effective.