WO2007138577A2 - Methods of generating a neural tissue using muscle-derived cells - Google Patents

Abstract

Use of human myogenic satellite cells for the manufacture of a medicament identified for treating a medical condition of the CNS is provided.

Description

METHODS OF GENERATING A NEURAL TISSUE USING MUSCLE- DERIVED CELLS

FIELD AND BACKGROUND OF THE INVENTION The present invention relates to cloned, slowly-adherent, muscle derived cells having a MyoD+/ Pax-7+ expression profile which can be used to generate a neuronal tissue in vivo, and more particularly, to methods of using such cells for treating pathologies associated with disease, degenerated or injured neural tissues.

Recent studies have indicated the prevalence and importance of adult stem cells in the development, maintenance and regeneration of various tissues and raised hopes for using them for the therapy of degenerative diseases and/or injured tissues. The conversion of one cell type to another via reprogramming or trans- differentiation is the key process in which tissue progenitor cells can be used for regeneration and/or repair of other cells. Thus, mesenchymal stem cells, which are present in various tissues including bone marrow and which are known to differentiate into bone, cartilage, smooth muscle and skeletal muscle, were also reported to transdifferentiate into skin, liver and brain cells (neurons and glia) (reviewed by Krabbe C, et al., 2005). However, other studies showed that the incorporation of donor cells into different host tissues, is merely the result of fusion between host and donor cells (reviewed in Wagers and Weissman, 2004).

Muscle progenitor cells are of special interest as excellent, easy accessible cells, with well-characterized markers and transcription factors associated with their various differentiation stages. These cells are easily cloned and manipulated in culture, thus offering a convenient model system which can be utilized for treating muscle degenerative diseases and perhaps as a source for replacement of other cell types.

Therapeutic approaches for skeletal muscle degeneration, by cell transplantation have been hindered by poor cellular survival rates, and the limited spread of the injected cells [reviewed in 1-4]. Major efforts were made to identify the most suitable cells for transplantation.

Skeletal muscle myoblasts plated in cell culture adhere to the culture plates slower than fibroblasts. This offered a simple and very efficient method for a significant enrichment of muscle cell suspension for myogenic cells. In this procedure, the heterogeneous cell population obtained from trypsinized skeletal muscle is preplated in untreated cell culture plates. Following the adherence of the "fibroblastic" cells, the unattached cells are collected and plated in gelatin coated plates to enable the adherence of the myoblasts. The myoblasts proliferate as adherent cells and in the appropriate culture conditions fuse into multinucleated muscle fibers which express muscle specific proteins and become contractile. Serial passaging of such cultures, using differential plating and cell cloning, resulted in the establishment of myogenic cell lines [5-7],

Following several reports on the possible existence of slower adherent stem cells in skeletal muscle cell cultures [8,9], the present inventors have reexamined the differential plating procedure and assayed the content of the medium, which was previously used to discard, during the medium replacement following the myogenic cells adherence to the plate. These cells were isolated and were found capable of proliferating for extended periods as floating clusters (i.e., slowly adherent cells) of rounded cells (myospheres) consisting of virtually a pure population of MyoD positive cells (David Yaffe, 2003 FASEB conference, Skeletal muscle satellite and stem cells, Tucson, Arizona). However, their ability to differentiate into muscle cells and to be used for muscle tissue regeneration and/or repair has not been reported yet.

Prior studies demonstrated the isolation of human skeletal-muscle stem cells which consisted of populations of adherent and non-adherent cells. When such cells were induced to differentiate in vitro towards the neuronal cell lineage, the cells expressed neuronal markers such as GFAP and βlll-tubulin but not markers of intermediate stage of differentiation such as neurfilaments, nestin and synaptophysin. Moreover, when the cells were engrafted in vivo into an animal spinal-cord injury, the cells did not express any typical neuronal marker, demonstrating their inability to transdifferentiate in vivo (Alessandri G., et al., 2004).

Other studies demonstrated that in vitro differentiation of bone marrow stromal cells (MSC) by chemical induction (e.g., using DMSO, β-mercaptoethanol, butylated hydroxyanisol or actin filament-depolymerizing agents) results in neurite- like processes which are devoid of essential motility and lack the expression of essential neuronal markers, and therefore represent process artifacts and not truly differentiated neuronal cells (Neuhuber B., et al., 2004; Lu P., et al., 2004). Another study tested the capacity of multipotent stem cells isolated from adult muscle to survive and respond to migratory and differentiating cues when transplanted into the subventricular zone of adult rats. However, since prior to their transplantation these cells exhibited neuronal markers such as doublecoritn and βlll- tubulin, they are not typical muscle progenitor cells. In addition, after transplantation, the donor cells failed to express markers typical to migrating neuroblasts such as βlll- tubulin and doublecortin (Mignon et al., 2005).

Other studies demonstrated the isolation of desmin⁺/CD34⁺/Bcl-2⁺ cells from muscle cells using the serial preplating technique (i.e., a serial of 6-7 plating of supernatant-containing cells which are non-adherent following 1-24 hours) which gave rise to isolated desmin⁺/Flk-l⁺/Sca-l⁺ clones which, when allowed to adhere, formed multinucleated myotubes (Lee, JY et al., 2000, 150:1085-1099). In addition,

U.S. Pat. Appl. No. 2005/0238625 to Chancellor MB., et al. discloses using such primary cells (following a series of 6 preplating passages) for enhancing survival and regeneration of damaged or injured nerve tissue.

Another subset of muscle-derived cells [named muscle-derived stem cells (MDSC) or long-term proliferating (LTP)] display unique characteristics associated with noncommitted progenitor cells (e.g., most of the cells are desmin and MyoD negative) and are capable of proliferating in vitro for 30 passages (Qu-Petersen Z., et al., 2002, The Journal of Cell Biology, 157: 851-864; Urish K., et al., 2005, Current Topics in Developmental Biology, 68:263-280; Oshima H., et al., 2005, Molecular Therapy 12:1130-1141).

However, to date, there is no report on committed muscle progenitor cells which can be used to generate true neuronal cells in vitro or in vivo. There is thus a widely recognized need for, and it would be highly advantageous to have, a method of generating neuronal cells from muscle progenitor cells devoid of the above limitations.

SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided a use of human myogenic satellite cells for the manufacture of a medicament identified for treating a medical condition of the CNS. According to further features in preferred embodiments of the invention described below, the human myogenic satellite cells are characterized by MyoD+/Pax-7+ expression profile.

According to still further features in the described preferred embodiments the human myogenic satellite cells are formulated for local administration.

According to still further features in the described preferred embodiments the human myogenic satellite cells are formulated for systemic administration.

According to still further features in the described preferred embodiments the human myogenic satellite cells are of a single clone. According to still further features in the described preferred embodiments the human myogenic satellite cells proliferate in vivo.

According to still further features in the described preferred embodiments the human myogenic satellite cells express at least one neuronal marker following administration. According to still further features in the described preferred embodiments the medical condition of the CNS is a neurodegenerative disease or disorder.

According to still further features in the described preferred embodiments the medical condition of the CNS is selected from the group consisting of a brain injury, a spinal cord injury, cerebral pulsy, a spinal muscular atrophy, a motion disorder, a dissociative disorder, a mood disorder, an affective disorder, an addictive disorder and a convulsive disorder.

According to still further features in the described preferred embodiments the neurodegenerative disorder is selected from the group consisting of Parkinson's, multiple sclerosis, epilepsy, amyatrophic lateral sclerosis, stroke, autoimmune encephalomyelitis, diabetic neuropathy, glaucatomus neuropathy, Alzheimer's disease, Down's syndrome, dementia, Gaucher disease, dementia associated with Lewy bodiesand Huntingdon's disease.

According to still further features in the described preferred embodiments the human myogenic satellite cells are autologous cells. According to still further features in the described preferred embodiments the human myogenic satellite cells are non-autologous cells.

According to still further features in the described preferred embodiments the human myogenic satellite cells are obtained by: (a) generating a single cell culture from a human muscle; and

(b) culturing the single cell culture under conditions which allow cell proliferation.

According to still further features in the described preferred embodiments the human myogenic satellite cells are immortalized.

According to still further features in the described preferred embodiments the culturing is effected for 3-4 weeks.

According to still further features in the described preferred embodiments the single cell culture comprise cells which adhere to a matrix within 2-18 hours. According to still further features in the described preferred embodiments the human myogenic satellite cells are encapsulated.

The present invention successfully addresses the shortcomings of the presently known configurations by providing methods of generating neural tissues using cloned, muscle derived progenitor cells. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION QF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the drawings:

FIGs. la-f depict Cultures of myospheres. (a) Myospheres grown in suspension (c) Outgrowth of myogenic cells from adherent myospheres (c)

Myospheres cells grown as an adherent monolayer. Myosphere cells adhere to the plate as rounded or spindle shaped cells, (d) A monolayer of a clone Rl#al, derived from ROSA26 myospheres cells. Note the formation of a network of predominantly thin mononucleated fibers, (e) Magnification of D. (f) After several passages of the adherent cells, foci of cells fusing into thick multinucleated fibers start to appear. The figure shows a fusing subline isolated from such focus. A-C: Phase contrast. D-F: Giemsa staining. Bars:.Bars: (a,c-e) 50 μm; (b) 100 μm; (d) 250 μm;

FIG. 2 depict Myogenic markers expressed by myospheres. Uncloned myosphere population (cultured for one week) grown in the proliferation medium and immunostained with the indicated antibodies. Most of the cells (>90%) express MyoD and desmin, indicating that they belong to the skeletal muscle lineage. Few cells express myogenin (arrow head), which may explain the contraction of single cells within the myosphere. Most of the cells (-70%) express the satellite cell marker, Pax- 7, suggesting that myosphere cells derive from satellite cells. Bars: 25 μm;

FIGs. 3a-d depict Single myosphere cells express MHC, without cell fusion.

When myospheres were grown in differentiation medium (10HI), most of the cells express MHC (green), either within the myospheres (a), or as single mononucleated adherent cells (b). This is in contrast to the pattern of differentiation in previously described myogenic cell lines, in which MHC appears after cell fusion, as shown here in the C2 cells (c). The myosphere clone R4#c that differentiates directly to multinucleated fibers, express MHC only in the multinucleated fibers, similarly to C2 cells (d). Nuclei were stained with DAPI (blue). Bars: (a,b,d) 25 μm; (c) 50 μm;

FIG. 4. LIF dramatically increases the proportion of cells expressing Sca-1.

LIF was added to the proliferation medium of both uncloned and cloned myospheres cells populations. The effect of continuous exposure to LIF on Sca-1 expression was determined by FACS analysis 7 days later (upper panel). Lower panel: A graph summarizing 3 independent experiments;

FIGs. 5a-d depict participation of myospheres cells in muscle regeneration, (a) Gastrocnemius muscle of nude mice was injured by injection of cardiotoxin. One day later, the muscle was injected with 10⁶ myospheres cells derived from a ROS A26 mouse, harboring a β-gal transgene. Six weeks later the muscle was fixed, X-gal stained and made transparent (by Benzyl Alcohol and Benzyl Benzoate). (b) The injured muscle was fixed and stained 2 months post injection, embedded in paraffin, sliced and counterstained with eosin. The blue cross-striations indicate that myospheres cells are able to regenerate injured muscle, (c) Higher magnification of (b). (d) A transverse section showing the distribution of donor cells. The differences in the intensity of X-gal staining suggest variability in the proportion of host and donor nuclei in single fibers. Bars: (a) 500 μm; (b) 150 μm; (c) 25 μm; (d) 60 μm; FIGs. 6a-c depict multipotential capacity of myosphere cells, (a) In the appropriate culture conditions (high density of differentiated muscle cells) cells in some clones spontaneously differentiated into adipocytes colonies, detected by Oil Red O staining. Exposure to bone morphogenic protein-4 (BMP-4) induced, in the myosphere derived clones, the expression of the osteogenic marker, alkaline- phosphatase (b), and osteocalcin (c). Bars: (a) 25 μm; (b) 50 μm;

FIGs. 7a-c depict myosphere cells did not trans-differentiate into hematopoietic cell, (a) Uncloned and cloned myosphere cells were cultured for 7 days in Methocult GF medium, and the percentage of cells expressing CD45 was analyzed by FACS. Bone marrow cells were used as a positive control. Freshly prepared uncloned population cultured only 5 days prior to treatment (5d) contain stem cells that give rise to high level of CD45+ cells. In contrast, uncloned population that was cultured for a long period prior to treatment (9Od) lost its ability to give rise to hematopoietic cells. The indicated clones gave rise to various levels of CD45 positive cells, however, these cells did not morphologically resemble hematopoietic cells, nor did they continue to proliferate, (b) Freshly prepared myosphere cells (up to 7 days in culture) contain 30%-40% CD45+ cells. The graph represents FACS analysis of one such preparation. Right panel: FITC only, left panel: anti-CD45-FITC. (c) Injection of myosphere cells directly into the bone marrow did not rescue lethally irradiated mice. Injection of freshly prepared, uncloned myosphere cells (myospheres) into the BM (I.BM) of lethally irradiated mice did not improve their survival. Mice that were injected with BM cells as a control for the I.BM injection procedure (BM), survived the irradiation; FIGs. 8a-d depict that cells that differentiate as needles express p27kip when cultured in the growth medium. Immunostaining of p27kip in cells cultured in the growth medium (GM) indicated that -50% of the cells from the clone Rl#al (in which the cells differentiate as needles) express this protein (a) while C2 cells do not (b). After 48h in 10HI medium, the Rl#al cells expressed p27kip in the mononucleated needles (c), while C2 cells express p27kip in the multinucleated fibers (d). Nuclei were stained with DAPI (right panel). Bars: (a-c) 25 μm; (d) 50 μm;

FIG. 9 depict that myosphere cells fuse with C2 cells. Co-culture of cells from a myosphere clone (Rl#al) derived from ROS A26 mice together with C2 myoblasts, in 10HI medium resulted in the formation of thick multinucleated blue fibers. Almost all mononucleated cells are X-gal negative, indicating preferential incorporation of the myosphere cells into the fusing fibers. Bar: 25 μm;

FIG. 10 depict that myosphere cells injected into injured muscle form both uniform and variegated fibers. Muscle was treated as described in Figures 5a-d. A magnification of 40Ox shows both uniform labeled blue fibers, indicating fusion between donor myoblasts, and variegated fibers, which suggest fusion between host and donor cells (arrows);

FIG. 11 is a photomicrograph depicting the expression of nestin by myosphere cells. Cloned populations of myosphere cells, grown in the proliferation medium were immunostained with anti-nestin antibody. Note the green staining in most of the cells (> 90 %) indicating nestin expression by the myosphere cells. Cell nuclei were stained with DAPI (blue);

FIGs. 12a-c are photomicrographs depicting X-GaI (5-bromo-4-chloro-3- indolyl-beta-D-galactopyranoside) staining of cloned myosphere cells following injection into recipient brains. Brains of newborn mice were injected with cloned populations of myosphere cells, derived from ROSA26 mice (which ubiquitously express β-galactosidase). Mice were sacrificed at 2 days, 4 days or 9 days post injection and their brains were removed. X-GaI staining was performed on either the whole brain (2 days post-injection, Figure 12a), or on brain slices derived at 4 days (Figure 12b) or 9 days (Figure 12c) post-injection. Note that two days after the injection, the cells were still localized in the ventricle, in a compact cluster, while few cells started to migrate out of the ventricle (Figure 12a). Four days after the injection, the cells already migrated out of the ventricle, and started to disperse, still in the vicinity of the injected site (Figure 12b). After nine days, the cells were detected in several brain regions, identified as single blue cells, including the cerebellum (Figure 12c). Magnifications: Figure 12a - XlOO; Figure 12b-c - X200; FIGs. 13a-h are photomicrographs depicting the in vivo expression of

Doublecortin in myosphere cells following injection into recipient brains. Cloned MyoD+ myosphere cells, obtained from ROSA26 mice, were injected into the lateral ventricles of newborn mice. Seven days after injection the brains were, removed, fixed and sliced (25 μm). Representative slices (every fifth slice) were stained with X-GaI and brain areas containing X-GaI stained cells were selected for further immunofluorescence studies using double labeling with both anti-β-gal (β- galactosidase; which labels donor cells derived from ROSA26 mice) and anti- Doublecortin (a marker for immature neurons). Figures 13a-d are adjacent slices in which the β-gal positive cells were observed in the corpus callosum. Arrows indicate cells that were stained with both markers; arrowhead indicates a muscle fiber that was formed by the injected cells and expressed only β-gal. Figure 13a - X-GaI staining; Figure 13b - Doublecortin staining; Figure 13c - β-gal staining; Figure 13d — a merged image of Doublecortin and β-gal. Figures 13e-h are confocal microscopy images of a single neuronal cell. Figure 13e - Doublecortin staining; Figure 13f - β- gal staining; Figure 13g — merged image of Doublecortin and β-gal; Figure 13h - Nomarski imaging. Note the neuronal morphology of the β-gal positive cells (i.e., which are derived from the donor myosphere cells) which also express Doublecortin, demonstrating the capacity of the donor cells (which were preprogrammed to a muscle cell) to develop into neuronal cell; FIGs. 14a-e are photomicrographs depicting the in vivo expression of βlll- tubulin in myosphere cells following injection into recipient brains. Injected brains were treated as described in Figures 13a-h, and were immunostained with both anti-β- gal (red) and anti-βlll-tubulin (TUJl, green). Double-labeled cells were observed mostly in the corpus callosum, and few cells were observed also in the CAl region of the hippocampus (arrow). Figure 14a - β-gal staining; Figure 14b - Tujl staining; Figure 14c - a merged image of β-gal and TUJl stainings; Figure 14d - X-GaI staining; Figure 14e (inset in Figure 14d) - higher magnification of the merged image of the cell indicated by arrow in Figure 14c;

FIGs. 15a-d are photomicrographs depicting the expression of markers of mature neurons in myosphere cells following injection into recipient brains. Brains were injected as described in Figures 3a-h, and collected after 2 weeks. Figure 15a - Double- immunostaining with anti-β-gal (red) and anti-NF-160 (green) revealed a proportion of cells that expressed both markers (arrows). Arrowheads indicate cells that express β-gal and not NF- 160. Figures 15b-d - X-GaI stained slices (blue) were incubated with anti-NeuN antibody, and labeling was detected using peroxidase staining (brown). Arrows indicate cells that express both NeuN and β-gal. The image in Figure 15d is taken from the cerebellum; and

FIGs. 16a-d are photomicrographs depicting the activation of a neurospecific transgene in donor cells following the injection of myosphere cells into the brain of C57bl newborn mice. Cloned myosphere cell populations obtained from mice carrying neuron specific Thy 1 -YFP transgene were injected into the brains of newborn mice. The brains were removed one week after the injection, sliced, and selected slices were screened using fluorescence microscope for the expression of YFP (green). Counterstaining was performed using DAPI (blue). A substantial proportion of the injected cells expressed YFP, indicating that the neuron specific transgene was activated in those donor cells. The pattern of the cells distribution was very similar to the pattern observed with either X-GaI or immunoflourescence stainings. Nuclei were stained with DAPI. Figure 16a- corpus- callosum, Figure 16b- cortex, Figure 16c- hippocampus, Figure 16d- magnification of Figure 16c. FIGs. 17a-b are photomicrographs showing cultures of human muscle cells.

Shown are human pre-plated muscle cells mass culture (Figure 17a) and a clone of myogenic cell population (Figure 17b, higher magnification). The cultures were grown for 10 days in the growth medium (BioAmf-2, Biological Industries, IL), and then induced to differentiate by changing to 10HI medium. After about 7 days the cells were fixed with methanol and stained with Giemsa.

FIGs. 18a-f are photomicrographs showing the ability of human myogenic cells to spread in the brain of new-born mice, and to express neuronal markers. Cloned human myogenic cells were labeled with Hoechst dye, and injected to the lateral ventricles of new-born mice. Brains were removed 9 days following the injection, fixed and sliced. Slices containing Hoechst stained nuclei were immunostained with NF-70, which specifically recognizes human neurofilament. Cells were localized mostly in the cortex, near the injection site; (Figures 18a,c,e), in the SVZ (Figures 18a,b), and in the corpus callosum (Figures 18a,d,f). A- Low magnification (4x) of merged image of the bright field and Hoechst labeling. Figure 18b- the SVZ (6Ox) Figures 18c,d-20x, Figures 18e,f-higher magnifications of Figures 18c,d (8Ox). IS- injection site, SVZ- subventricular zone, CC- corpus callosum. FIGs. 19a-c are photomicrographs depicting incorporation of human myogenic cells in brains of GFP expressing mice. Human myogenic progenitor cells, labeled with Hoechst (H), were injected into the brain of mice ubiquitously expressing GFP. The brains were analyzed 9 days after the injection. The injected cells were mostly localized along the corpus-callosum (cc), and at the sub- ventricular zone. Figures 19a- b are merged images of the bright field of the endogenous host tissue and H labeled donor cells, showing the spreading of the cells from the injected site. Figures 19c-d are merged images of the GFP expressing host cells and H labeled donor cells. Figures 19d and e are higher magnifications of the inset shown in Figure 19c. Figure 19e shows only the GFP host tissue, the arrow indicates vacuoles in which H labeled cells are localized. Figure 19f is a merged image of another region along the cc, showing a similar localization of H labeled cells in vacuoles of endogenous tissue.

FIGs. 20a-c are photomicrographs showing donor injected human cells do not fuse with endogenous tissue. Human myogenic progenitor cells were injected into brains of GFP expressing mice. Following 9 days the brains were sliced and immunostained with a human specific anti-NF-70 antibody. Group of donor cells, in several brain regions expressed human NF-70 protein, and merge images revealed that most of these cells do not express the host GFP protein (Figure 20a). Figures 20b and c show a group of cells expressing human NF-70 at the injection vicinity. Arrows in Figure 20c point at two cells that may represent rare fusion events between host and donor cells. DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of pharmaceutical compositions and uses of same for the treatment of medical conditions of the CNS.

The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

Neurodegenerative disorders which are characterized by loss of neuronal functions, such as CNS injury or degeneration [e.g., Parkinson's disease and multiple sclerosis (MS)[, cannot be efficiently treated using conventional drug therapy since such drugs have no effect on the underlying disease process which is typically caused by neuronal degeneration. Consequently, drug therapy can not fully compensate for the increasing loss of neuronal cells.

While conceiving the present invention, the present inventors have surprisingly uncovered that satellite cells of a muscle tissue can in vivo differentiate into neural and even glial tissue suggesting their safe and effective use in cell-replacement therapy of , degenerated or injured neural tissues. These cells can be easily harvested, cultured and transplanted to produce neural cells capable of synthesizing neurotransmitters, such as dopamine, in response to environmental stimuli.

As is illustrated hereinbelow and in the Examples section which follows, the present inventors have shown that Pax 7+ cells (satellite cells) obtained from murine muscle tissue can form myospheres in vitro. Such microspheres proliferate in culture into cell mass which comprise myogenic progenitor cells (MyoD+). These cells can be cultured for several months to obtain a sufficient amount of cells suitable for transplantation. Such cells are of sufficient sternness (i.e., not terminally differentiated) to differentiate to muscle tissue, adipocye and bone cells (mesodermanl lineage) in vitro.

Cloned myospheres (or heterogeneous populations of same) can differentiate in vivo to neural cells. As shown in Example 2 of the Examples section which follows, cells were injected into the lateral ventricles of new born mice. Immunohistochemistry and innumofluorescence analysis revealed marker expression in accord with neuronal differentiation. Cell scattering was noted in the cortex, corpus-collasum, hippocampus, thalamus, cerebellum, rostral migratory stream and the olfactory bulb. Cell differentiation was proven be eliminating cell fusion (see Examples 2-4). Similarly to the murine cells, human satellite cells obtained from muscle tissue exhibited similar characteristics, exhibiting unprecedented integration in a host brain and ability to differentiate in vivo to neural tissue (see Examples 5-6 of the Examples section which follows). These results place the cells of the present invention as a primary source of cells in therapy of medical conditions of the central nervous system (CNS).

Thus, according to one aspect of the present invention there is provided a method of treating a medical condition of the CNS in a subject in need thereof

As used herein the term "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of the medical condition of the CNS.

As used herein the phrase "subject in need thereof refers to a mammal (e.g., human) who has been diagnosed or is predisposed to a medical condition of the CNS.

The method comprising administering myogenic satellite cells to the subject, thereby treating the medical condition of the CNS.

As used herein, the phrase "a medical condition of the CNS" refers to any disorder, disease or condition (e.g., injury) of the central nervous system which may be treated with the cells of the present invention.

Representative examples of CNS diseases or disorders that can be beneficially treated with the cells described herein include, but are not limited to, a brain injury, cerebral pulsy, a spinal muscular atrophy, a pain disorder, a motion disorder, a dissociative disorder, a mood disorder, an affective disorder, a neurodegenerative disease or disorder and a convulsive disorder.

More specific examples of such conditions include, but are not limited to, Parkinson's, amyatrophic lateral sclerosis (ALS), stroke, Multiple Sclerosis,

Huntingdon's disease, autoimmune encephalomyelitis, diabetic neuropathy, glaucomatous neuropathy, macular degeneration, action tremors and tardive dyskinesia, panic, anxiety, depression, alcoholism, insomnia, manic behavior, Alzheimer's disese, Down's syndrome, dementia, Gaucher disease, dementia associated with Lewy bodies and epilepsy.

As used herein the phrase "myogenic satellite cells" refers to satellite cells of a fetal or adult muscle tissue. Satellite cells are precursors to primary myoblasts. Myogenic satellite cells are commited stem cells of adult and fetal skeletal muscle. Their major function is to repair, revitalize, and mediate skeletal muscle tissue and growth by differentiating into myocytes. Satellite cells posses plasticity and are normally non proliferative. They do become active in vivo, however, when skeletal muscle tissue is injured or heavily used during activities such as weight lifting or running. Satellite cells are located at the surface of the basal lamina of the myofiber. MyoD is only expressed when satellite cells are activated to proliferate and differentiate into primary myoblasts, which will then differentiate into cells of the myofibers. Satellite cells express the homeobox protein Pax-7. The exact role of Pax- 7 in terms of satellite cells is unknown, however, one thing is clear: when without Pax-7, there are no satellite cells.

As mentioned, satellite cells of the present invention are obtained from adult or fetal skeletal muscle cells.

The surgical procedure to obtain a muscle biopsy is relatively simple and poses little risk to the subject, but it is a specialized procedure and must be performed properly to optimize yield for the benefit of the patient. Biopsy is typically effected under local or general anaesthesia. Biopsy is effected by open excision or by needle biopsy. Open biopsies are preferred since provide larger specimens.

Once isolated (from the muscle tissue) satellite cells of the present invention express Pax7 (Pax7+). However, expression of Pax7 may decline concomitant with the appearance of markers which are characteristic of more commited differentiation state. These include MyoD, Myogenin and Desmin. According to a preferred embodiments cells of this aspect of the present invention are MyoD+ Pax7+Desmin+. Typically, single cell cultures are effected by mechanic and enzymatic dissociation such as described in the Examples section which follows. Once these are obtained, the cells are allowed to adhere to a matrix such as a tissue culture plate (e.g., for 2-18 hours). The adherent cell population is collected and cultured under conditions which allow cell proliferation. Such conditions are described at length in the Examples section which follows. Single clones may be used or heterogeneous cell populations. Cloning may be effected by methods which are well known in the art of cell culturing and include but not limited to cell separation by FACS or by cell dilution. Cells are continued to culture until a sufficient number of cells is at hand to allow transplantation. Once isolated and possibly cloned that satellite cells of the present invention become myogenic committed cells as evidenced by their MyoD positive expression and possibly desmin positive expression.

Cells of the present invention can be used for preparing a medicament (interchangeably referred to as pharmaceutical composition), whereby such a medicament is formulated for treating a CNS disease or disorder.

In any of the methods described herein the cells may be obtained from any autologous or non-autologous (i.e., allogeneic or xenogeneic) human donor. For example, cells may be isolated from a human cadaver or a donor subject.

Cells of the present iavention may be naϊve cells or genetically modified to express an exogenous polynucleotide for improving treatment.

The cells of the present invention can be administered to the treated individual using a variety of transplantation approaches (i.e., local or systemic), the nature of which depends on the site of implantation.

The term or phrase "transplantation", "cell replacement" or "grafting" are used interchangeably herein and refer to the introduction of the cells of the present invention to target tissue. The cells can be derived from the recipient or from an allogeneic or xenogeneic donor.

The cells can be grafted into the central nervous system or into the ventricular cavities or subdurally onto the surface of a host brain. Conditions for successful transplantation include: (i) viability of the implant; (ii) retention of the graft at the site of transplantation; and (iii) minimum amount of pathological reaction at the site of transplantation. Methods for transplanting various nerve tissues, for example embryonic brain tissue, into host brains have been described in: "Neural grafting in the mammalian CNS", Bjorklund and Stenevi, eds. (1985); Goldman et al. Cell replacement therapy in neurological disease. Philos Trans R Soc Lond B Biol Sci. 2006 Sep 29;361(1473): 1463-75. These procedures include intraparenchymal transplantation, i.e. within the host brain (as compared to outside the brain or extraparenchymal transplantation) achieved by injection or deposition of tissue within the host brain so as to be opposed to the brain parenchyma at the time of transplantation.

Intraparenchymal transplantation can be effected using two approaches: (i) injection of cells into the host brain parenchyma or (ii) preparing a cavity by surgical means to expose the host brain parenchyma and then depositing the graft into the cavity. Both methods provide parenchymal deposition between the graft and host brain tissue at the time of grafting, and both facilitate anatomical integration between the graft and host brain tissue. This is of importance if it is required that the graft becomes an integral part of the host brain and survives for the life of the host. Alternatively, the graft may be placed in a ventricle, e.g. a cerebral ventricle or subdurally, i.e. on the surface of the host brain where it is separated from the host brain parenchyma by the intervening pia mater or arachnoid and pia mater. Grafting to the ventricle may be accomplished by injection of the donor cells or by growing the cells in a substrate such as 3 % collagen to form a plug of solid tissue which may then be implanted into the ventricle to prevent dislocation of the graft. For subdural grafting, the cells may be injected around the surface of the brain after making a slit in the dura. Injections into selected regions of the host brain may be made by drilling a hole and piercing the dura to permit the needle of a microsyringe to be inserted. The microsyringe is preferably mounted in a stereotaxic frame and three dimensional stereotaxic coordinates are selected for placing the needle into the desired location of the brain or spinal cord. The cells may also be introduced into the putamen, nucleus basalis, hippocampus cortex, striatum, substantia nigra or caudate regions of the brain, as well as the spinal cord.

The cells may also be transplanted to a healthy region of the tissue. In some cases the exact location of the damaged tissue area may be unknown and the cells may be inadvertently transplanted to a healthy region. In other cases, it may be preferable to administer the cells to a healthy region, thereby avoiding any further damage to that region. Whatever the case, following transplantation, the cells preferably migrate to the damaged area. For transplanting, the cell suspension is drawn up into the syringe and administered to anesthetized transplantation recipients. Multiple injections may be made using this procedure. Cells of the present invention may also be administered systemically such as by intra-venous (i.v) or intra-peritoneal (i.p) injections, provided that the blood-brain- barrier (BBB) is penetratable. This may be done by artificial modification using methods which are well known in the art. Alternatively, in some medical conditions, like MS, in which the BBB is impaired, the administration of the cells by i.v or i.p injection has major advantages. In addition to the simplicity of these injections, (compared to intra-cranial injections), the cells can spread or migrate to widespread damaged sites in the spinal cord and the brain.

The cellular suspension procedure thus permits grafting of the cells to any predetermined site in the brain or spinal cord, is relatively non-traumatic, allows multiple grafting simultaneously in several different sites or the same site using the same cell suspension, and permits mixtures of cells from different anatomical regions. Multiple grafts may consist of a mixture of cell types. Preferably from about 10⁴ to about 10 (e.g., 10 to about 5x10 ) cells are introduced per graft. For transplantation into cavities, which may be preferred for spinal cord grafting, tissue is removed from regions close to the external surface of the central nerve system (CNS) to form a transplantation cavity, for example as described by Stenevi et al. (Brain Res. 114:1-20., 1976), by removing bone overlying the brain and stopping bleeding with a material such a gelfoam. Suction may be used to create the cavity. The graft is then placed in the cavity. More than one transplant may be placed in the same cavity using injection of cells or solid tissue implants. Preferably, the site of implantation is dictated by the CNS disorder being treated. Thus, for example, as mentioned above, treating multiple sclerosis is preferably effected by transplanting the cells systemically, while treating PD will be preferably effected by transplanting the cells into the injured site to replace the dopaminergic neurons.

Since non-autologous cells are likely to induce an immune reaction when administered to the body several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells in immunoisolating, semipermeable membranes before transplantation.

Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).

Methods of preparing microcapsules are known in the arts and include for example those disclosed by Lu MZ, et al., Cell encapsulation with alginate and alpha- phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng. 2000, 70: 479-83, Chang TM and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. MoI Biotechnol. 2001, 17: 249-60, and Lu MZ, et al., A novel cell encapsulation method using photosensitive poly(allylamine alpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245- 51.

For example, microcapsules are prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 μm. Such microcapsules can be further encapsulated with additional 2-5 μm ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S. M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide (Sambanis, A. Encapsulated islets in diabetes treatment. Diabetes Technol. Ther. 2003, 5: 665-8) or its derivatives. For example, microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smaller capsules are used. Thus, the quality control, mechanical stability, diffusion properties, and in vitro activities of encapsulated cells improved when the capsule size was reduced from 1 mm to 400 μm (Canaple L. et al., Improving cell encapsulation through size control. J Biomater Sci Polym Ed. 2002; 13:783-96). Moreover, nanoporous biocapsules with well-controlled pore size as small as 7 run, tailored surface chemistries and precise microarchitectures were found to successfully immunoisolate microenvironments for cells (Williams D. Small is beautiful: microparticle and nanoparticle technology in medical devices. Med Device Technol. 1999, 10: 6-9; Desai, T. A. Microfabrication technology for pancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

Examples of immunosuppressive agents include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE^®), etanercept, TNF- alpha blockers, a biological agent that targets an inflammatory cytokine, and Nonsteroidal Anti-Inflammatory Drug (NS AIDs). Examples of NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

In any of the methods described herein, the cells can be administered either per se or, preferably as a part of a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the chemical conjugates described herein, with other chemical components such as pharmaceutically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to a subject.

Hereinafter, the term "pharmaceutically acceptable carrier" refers to a carrier or a diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered compound. Examples, without limitations, of carriers are propylene glycol, saline, emulsions and mixtures of organic solvents with water.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.

Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

According to a preferred embodiment of the present invention, the pharmaceutical carrier is an aqueous solution of saline. Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Suitable routes of administration include direct administration into the tissue or organ of interest. Thus, for example the cells may be administered directly into the brain as described hereinabove or directly into the muscle as described in Example 2 hereinbelow.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. Preferably, a dose is formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. For example, 6-OHDA-lesioned mice may be used as animal models of Parkinson's. In addition, a sunflower test may be used to test improvement in delicate motor function by challenging the animals to open sunflowers seeds during a particular time period.

Transgenic mice may be used as a model for Huntingdon's disease which comprise increased numbers of CAG repeats have intranuclear inclusions of huntingtin and ubiquitin in neurons of the striatum and cerebral cortex but not in the brain stem, thalamus, or spinal cord, matching closely the sites of neuronal cell loss in the disease.

Transgenic mice may be used as a model for ALS disease which comprise SOD-I mutations.

The septohippocampal pathway, transected unilaterally by cutting the fimbria, mimics the cholinergic deficit of the septohippocampal pathway loss in Alzheimers disease. Accordingly animal models comprising this lesion may be used to test the cells of the present invention for treating Alzheimers. Survival and rotational behavior (e.g. on a rotarod) of the animals may be analyzed following administration of the cells of the present invention.

Experimental Autoimmune Encephalomyelitis (EAE), also called Experimental Allergic Encephalomyelitis, is an animal model of Multiple Sclerosis. The animals are injected with the whole or parts of various proteins that make up myelin, the insulating sheath that surrounds nerve cells (neurons). These proteins induce an autoimmune response in the animals - that is the animal's immune system mounts an attack on its own myelin as a result of exposure to the injection. The animals develop a disease process that closely resembles MS in humans. Several proteins or parts of proteins (antigens) are used to induce EAE including: Myelin Basic Protein (MBP), Proteolipid Protein (PLP), and Myelin Oligodendrocyte Glycoprotein (MOG). Disease score is evaluated several days following induction (e.g., day 14). Incorporation of the cells in the tissue may be evaluated using methods which are well known in the art. Thus for example, cells may be characterized for a neuronal or glial marker such as listed infra; Activin RIIA, A2B5, AP-2 Alpha, BMP- 3b/GDF-10 , ChAT, CNPase, Coronin IA, GAD1/GAD67, GAP43 (Growth- Associated Protein, 43 kDa), GFAP, Jaggedl, Mashl, MAP2, (Microtubule assoc. protein 2), MOG, NCAM-Ll, NSE (Neuron-Specific Enolase), Nestin, Netrin-1, Netrin-4, Neurofilament NF-H, Neurofilament NF-L, Neurofilament NF-M, Neurofilament alpha- internexin/NF66, Notch 1, Notch3, OHg 1,2,3, Oligodendrocyte Marker 01, Oligodendrocyte Marker O4, Pl 1/Calpactin I LC/Annexin II), Peripherin, PGP9.5, huPGP9.5 175-191, huPGP9.5 176-191, PLP (Proteolipid Protein), Po, RAGE, ROBOl, SlOOB, Semaphorin, Synapsin, Synaptophysin, Tuj 1 (Neuron- specific class IH beta-tubulin), Tuj 1 (Neuron-specific class HI beta-tubulin), phospho-Tyrosine Hydroxylase (Ser40), Tyrosine Hydroxylase, Vimentin. Antibodies for immunohistochemistry are available from Neuromics (Edina, NM).

The data obtained from these animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition, (see e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l). For example, Parkinson's patient can be monitored symptomatically for improved motor functions indicating positive response to treatment. For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.

Dosage amount and interval may be adjusted individually to levels of the active ingredient which are sufficient to effectively regulate the neurotransmitter synthesis by the implanted cells. Dosages necessary to achieve the desired effect will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, be dependent on the individual being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. The dosage and timing of administration will be responsive to a careful and continuous monitoring of the individual changing condition. For example, a treated Parkinson's patient will be administered with an amount of cells which is sufficient to alleviate the symptoms of the disease, based on the monitoring indications. The cells of the present invention may be co-administered with therapeutic agents useful in treating neurodegenerative disorders, such as gangliosides; antibiotics, neurotransmitters, neurohormones, toxins, neurite promoting molecules; and antimetabolites and precursors of neurotransmitter molecules such as L-DOPA. Additionally, the cells of the present invention may be co-administered with other cells capable of synthesizing a neurotransmitter. Such cells are described in U.S. Pat. Appl. No. 20050265983 to the present inventors.

Following transplantation, the cells of the present invention preferably survive in the diseased area for a period of time (e.g. at least 6 months), such that a therapeutic effect is observed.

As used herein the term "about" refers to ± 10 %. Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al, (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., Ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (Eds.) "Genome Analysis: A Laboratory Manual Series", VoIs. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., Ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., Ed. (1994); Stites et al. (Eds.), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (Eds.), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., Ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., Eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., Ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLE 1

REGENERATION AND TRANS-DIFFERENTIATION POTENTIAL OF MUSCLE DERIVED STEM CELLS PROPA GA TED AS MYOSPHERES

Materials and methods Cell culture

Myosphere preparation: Primary muscle cultures were prepared from 3-4 weeks old mice using a modified version of the previously described preplating procedure [5-7,18, incorporated herein by reference]. The hind-limb muscles of mice were isolated and the fat and bones discarded. The muscle was minced with scissors, and enzymatically dissociated at 37°C with 0.05% trypsin-EDTA for 30 minutes, and then centrifuged at 2500 rpm for 5 min. The cells were collected and the trypsinization of the remaining undigested tissue was repeated three more times by adding fresh trypsin solution. On the fourth time, the cells were incubated for 30 min with 0.25% trypsin-EDTA. After centrifugation, the cells were suspended in the proliferation medium, BIO-AMF-2 (Biological Industries), which contains fetal calf serum, steroids, bFGF, insulin, glutamine and antibiotics, either with or without LIF (10 ng/ml, CytoLab). As indicated in the Results, different populations of myoblasts were isolated based on their adhesion characteristics.

Myospheres were serially passaged by allowing them to sediment by gravitation to the bottom of a test tube, the old medium was removed by decantation followed by careful suspension of the myospheres in fresh medium and plating them in uncoated cell culture plates.

Adherent monolayer of myospheres derived cells were grown in gelatin coated plates, in the proliferation medium. When the cultures reached confluence, the medium was changed to differentiation enhancing medium, 10HI (DMEM containing

10% carefully selected horse serum, 0.04 units/ml insulin, 0.5% chick embryo extract and penicillin-streptomycin). Where indicated, the cells were isolated as described by Qu-Peterson et al. [9]:

The trypsinized cells were serially passaged as non-adherent cells for 4 days. On the fifth day, the cells that adhered to the plate were collected either as uncloned cell population, or were sorted by FACS, to isolate single Sca-1 positive cells for clonization as described below.

C2 cells [18]: were kept frozen in -80⁰C and amplified in culture in DMEM medium containing 20% fetal calf serum and penicillin (100 units/ml)-streptomycin (O.lmg/ml) in gelatin coated plates. To avoid uncontrolled cell fusion, the cells were split before reaching confiuency, and not more than 2-3 days after plating. Intensive cell fusion was induced by changing the medium to 10HI

FACS analysis: The percent of cells expressing the following antigenes was analyzed by FACScan; Sca-1 (eBioscience, PE conjugated), CD45 (eBioscience, FITC conjugated), CD34 (BD PharMingen, FITC conjugated). Cells were washed once with PBS, and resuspended in 0.1ml of cold PBS. Mouse serum, (S igma-Aldrich, 1:10) and Fc block (rat anti mouse CD16/CD32, BD PharMingen) were added, and the suspensions were incubated for 10 min on ice. Each sample was divided into two halves; one half was incubated with the Ab (diluted according to the manufacture instructions) and the other half was incubated with the conjugated flourescin only (PE/FITC). The tubes were incubated at 4⁰C, for 30 min, and then washed twice with cold PBS. For the sterile sorting and collection of Sca-1 positive cells, the cells were stained with Sca-1 -PE as above, and the cells expressing Sca-1 were collected by FACSVantage.

Immunocytochemistty: Adherent cells were grown on gelatin or fibronectin coated glass coverslips. Intact myosphere cells were fixed on a glass slide using a cytospine centrifuge. The cells were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 10 min and permeabilized with 0.2% Triton X- 100 in PBS for 5 min. For blocking, the cells were incubated in PBS containing 0.1% Triton and 3% bovine serum albumin for 30 min at room temperature. For immunostaining, the cells were incubated for Ih with the following monoclonal antibodies, diluted in the blocking solution; MyoD (1:100, Dako), myogenin (1:1, a kind gift from WE Wright), desmin (1:100, DE-U-10, Sigma), MHC (1:20, MF-20, DSHB), Pax-7 (1:100, DSHB). After three washes with PBS containing 0.1% Triton, the cells were stained for 30 min at room temperature with Alexa-488-labeled goat anti-mouse Abs (Molecular Probes, 1:150), followed by 5 min of 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining (lOμg/ml). The cells were mounted with elvanol, and viewed under a Nikon fluorescence microscope at a magnification of x200/x400. Pictures were taken with a 1310 digital camera (DVC).

Muscle regeneration: To induce muscle injury, cardiotoxin (0.1ml of lOμM, sigma) was injected into the gastrocnemius muscle of nude mice. Myosphere cells (~10⁶ cells) were injected to the injured muscle the following day. All injections were done using a 27G1/2 needle. The mice were sacrificed at the indicated time points, and the injected muscle was removed, together with a control non-injected gastrocnemius muscle and subjected to X-gal staining, as described below.

X-GaI staining: the muscles were fixed for 60-90 min (according to their size) in 4% paraformaldehyde, washed with PBS and stained overnight with X-gal [55]. Where indicated, dehydrated muscles were cleared in benzyl alcohol:benzylbenzoate (BABB) solution (1:2). For sectioning, stained muscles were post- fixed overnight in 4% PFA, dehydrated, embedded in paraffin and cut on a microtome (5-7μM). The slices were deparafinized with xylene (2 min), rehydrated (from 95% to 25% ethanol), counterstained with eosin and mounted. BMP treatment and osteogenic markers: 293T cells expressing BMP-4 were provided by Dr. Eldad Tzahor from the Weizmann Institute of Science, Rehovot, Israel. The cells were grown in culture for 4-5 days (until confluence), and the growth medium (DMEM with 10% FCS) was collected, filtered and used as the source for BMP-4. The detection of Alkaline-Phosphatase and osteocalcin was done according to Wada et al., [52].

Transplantation of cells to lethally irradiated mice: Mouse bone-marrow (BM) cells were flushed from femurs using a 271/2 gauge needle attached to a ImI syringe and suspended in phosphate-buffered saline (PBS). Myosphere cells that grew as a monolayer were trypsinized and suspended in PBS while those that grew as non- adherent cells were collected, separated by pipetting and suspended in PBS.

For the I. V. injections, recipient mice were exposed to two gamma irradiation doses of 600rad from a Cs source (with a 3h separation between the two irradiations). Myosphere cells were injected into the tail veins of the mice 4-5h after irradiation. The amount of cells injected in each experiment is indicated in table 3. For the injections of cells into the BM, mice were irradiated with 950rad, 24h before transplantation. The mice were anaestheised prior to the injection, and their knee was flexed to 90°. Either myosphere cells (10⁶ cells/mouse) or BM cells (5xlO⁵ cells/mouse) were injected into the bone marrow cavity of the tibia using a 271/2 - gauge needle.

Mice were kept in sterile conditions, and antibiotics (Cyproxine 100) were added to their water. All animal studies were approved by the Institutional Animal Care and Use

Committee of the Weizmann Institute of Science.

Abbreviations list: BM, bone-marrow; BMP, bone morphogenetic protein; LBM, intra bone-marrow; MHC, myosin heavy chain. Experimental Results Most myospheres are formed mainly by clonal proliferation of single cells -

Twenty four hours after plating of freshly isolated adult mouse skeletal muscle cells, most living cells are attached to the bottom of the plate. The nutrient medium contains mostly cells debris. However, careful examination reveals the presence of very few small rounded cells suspended in the medium. Some of them are in clusters of 2-4 cells. One or two days later, many clusters containing up to 10-15 cells, are very conspicuous. In the next few days, these myospheres continued to grow in size and number. These cells could be maintained, by serial passages, as suspended myospheres, for at least several months without loosing their proliferation capacity (Fig. IA). To determine whether the myospheres were formed by cell aggregation or by clonal cell proliferation, muscle cells isolated from wild-type (wt) mice of the strain 129 SVJ were dissociated and co-cultivated together with muscle cell populations derived from muscles of ROSA26 mice, which contain a transgene encoding a ubiquitously expressed bacterial β-galactosidase [10]. After 4 days the myospheres were collected by decantation of the medium and stained for β-galactosidase activity. The majority of myospheres consisted exclusively of either blue cells or of unstained cells. Only 12% of the myospheres contained both stained and unstained cells, often clustered separately within the myosphere, suggesting fusion between myospheres (Table 1). It is therefore concluded that the myospheres were formed mainly by clonal proliferation of single cells that stayed together. However, there is also fusion between myospheres.

Table 1. Myospheres are formed mainly by clonal cell proliferation

Table 1: Myospheres were prepared as described in materials and methods, from ROSA26 and 129 SVJ mice. After trypsinization, the cells were counted and plated together in a 1: 1 ratio. After 4 days, the plates were fixed and stained with X-gal as described. Myospheres containing at least 10 cells were counted

Cells from the myospheres spread out and form a myogenic monolayer -

When myospheres grown in gelatin coated plates are left for several days in the same plate, many of them adhere to the plate and start to spread out and to shed cells which adhere to the plate as single rounded cells or as spindle shaped cells (Fig. IB₅C). Later, many of these cells elongate and form very thin myogenin positive fibers (needles). Most of these muscle fibers are mononucieated or containing 2-3 nuclei (Fig. 1D,E) and many of them are contractile. Growing these cells in the differentiation stimulating medium 10HI, enhance the process of cell elongation as needles. Slow cell fusion and formation of a network of multinucleated fibers occurs in aged cultures, indicating fusion between differentiated needles. In addition, serial passages of the cells for extended periods, as adherent monolayers, resulted in increased proportion of cells fusing into multinucleated fibers in response to 10HI medium. Clonal analysis indicated that this transition is stably inherited, suggesting an epigenetic change (Fig. IF). Isolation and characterization of myogenic cell lines derived from myospheres - Closer examination of freshly isolated uncloned myospheres revealed that in many of them some of the rounded cells twitched. Immunostaining revealed that most or all of the cells expressed MyoD, and in a small proportion of the cells within the myospheres the nuclei were myogenin positive, confirming their differentiated nature (Fig. 2). Most of the cells express Pax-7, suggesting their origin from muscle reserve cells (satellite cells) (Fig. 2). Serial passages and cloning of the proliferating attached cells described above, or of dissociated myosphere cells, resulted in the isolation of several myogenic clones. These clones proliferated as a mixture of rounded and spindle shaped mononucleated cells (Fig. 1). Immunofluorescence staining with antibodies against MyoD, myogenin, desmin, and Pax-7 revealed intrinsic differences between clones. Most or all of the cells in all the clones expressed MyoD. Some clones did not express MyoD, or expressed it in a low percentage of the cells, when grown as myospheres. However, when these same clones were grown as a monolayer of adherent cells, most of the cells expressed MyoD. All clones tested were desmin positive, and expressed myogenin and skeletal muscle MHC after induction of myogenic differentiation. A small percentage of the cells in some clones retained the expression of Pax-7, however, at much lower frequency than the uncloned myospheres cells population (not shown). When induced to differentiate, most of the clones formed contractile needles, as described above, while in some clones the cells fused and formed multinucleated fibers without prior differentiation into mononucleated needles. All the clones were CD45 and CD34 negative, indicating that they do not belong to the hematopoietic or endothelial lineage (Table 2).

Table 2. Characterization of myosphere cell clones (partial list)

MyoD myogenin desmin MHC CD45 CD34 differentiation

Rl#al ++ + ND ++ needles

R4#c + ++ ND ++ Multi-nucleated

R21#4 + + ++ + needles

R21#8 ++ + ++ + Multi-nucleated

R23#l + + -H- ND Mixed population

Mdx#8 adherent + + ++ ND needles

Mdx#8 spheres +/- ++ ND needles Table 2: A representative list of clones that were isolated either as non-adherent myosphere cells (Rl#al, R4#c, Mdx#8), or as adherent cells, as described in Material and methods (R21#4, R21#8, R23#l). The cells were subjected either to immunocytochemistry (with anti- MyoD, myogenin, desmin and MHC) or to FACS analysis (using anti-CD45 and CD34). -, no expression; +/-, <30% positive cells; +, 30-70% positive cells; ++, >70% positive cells; ND, not determined. R=ROSA26

Thus, the pathway of differentiation of most of the myospheres derived cells differs from that of the previously established myogenic cell lines. While the common myogenic differentiation follows cell fusion, most of the myospheres derived cells differentiate into contractile myogenin and myosin expressing muscle cells without cell fusion (Fig. 3 A-C). Lin et al. [11] reported that in primary chick muscle cultures, myoblasts initiate synthesis of MHC prior to cell fusion. This was studied in rat and mouse primary myoblasts cultures isolated from muscle by differential plating. Using immunostaining it was found that while in mouse cultures, a significant proportion of the mononucleated myoblasts express MHC at the onset of cell fusion, in rat myoblast cultures, the expression of MHC is confined mostly to the multinucleated fibers (not shown). Interestingly, in the mouse, myospheres derived cell lines of which the spindle shaped myoblasts fuse into multinucleated fibers without first forming needles (Table 2, hereinabove), staining for MHC is confined almost exclusively to the multinucleated fibers (Fig. 3D). Thus, it seems that the differences in the sequence of events are determined by genetic and epigenetic factors. It should be noted that unlike the situation with primary mouse myoblasts, in which a fraction of the myoblast shaped cells express MHC, in the myosphere derived cultures, almost the entire cell population differentiate into needle shaped contractile mononucleated fibers. It was recently shown that the expression of p27Kipl controls the differentiation of C2 (C2C12) cells, by up-regulating MyoD levels in undifferentiated cells. Exogenous expression of p27Kipl allowed low-density cultured C2 cells to differentiate and express MHC as single cells [12]. Our examination of endogenous p27kipl expression revealed that C2 cells and myosphere derived clones that differentiate directly to multinucleated fibers express p27kipl only after their induction to differentiate by 10HI medium. In contrast, clones that differentiate as needles express p27kipl already when cultured in the growth medium (Figs. 8a-d). These results indicate a difference in the sequence of signal transductions leading to terminal differentiation of muscle cells. Leukemia inhibitory factor (LIF) enhances Sca-1 expression and support cell proliferation - Sca-1 is a stem cell marker of both hematopoietic and myogenic stem cells [13]. The percentage of Sca-1 positive cells in uncloned myospheres cell populations, one week after their isolation from the muscle, ranged between 4% to 20% (Figs. 4a-c). Using fluorescence activated cell sorter (FACS), myosphere clones, which originated from single Sca-1 positive cells were isolated. Analysis of Sca-1 in these clones revealed that after amplification, the cells had a similar expression pattern of Sca-1 as the original unsorted cell population. Thus, using the regular growth medium and cloning, it was not possible to obtain Sca-1 enriched cell populations. LIF is known to inhibit the differentiation of mouse embryonic stem (ES) cells, and of isolated multipotent adult progenitor cells (MAPC) [14,15]. It was found that including LIF in the growth medium significantly enhanced the proliferation of Sca-1+ isolated clones. After propagation of these clones in medium containing LIF, they could continue to proliferate also without the addition of LIF. The addition of LIF to either the uncloned myosphere cell populations or to the Sca-1+ isolated clones greatly increased the percentage of Sea- 1 positive cells (from 15% to 80%) (Figs. 4a- c). Moreover, the addition of LIF to Sca-1 negative cells for 7 days induced the expression of Sca-1 in 15% of the cells (not shown). Interestingly, in some of the clones the addition of LIF and enrichment of Sca-

1 positive cells reduced the percentage of cells expressing MyoD. However, changing to the differentiation-inducing medium, 10HI, enhanced MyoD expression and cell differentiation. Chulman et al. [16] showed that LIF reduced MyoD expression and cell fusion in the myogenic cell line C2. However, using primary muscle cultures, Vakakis et al. [17] showed that LIF effect on myoblasts differentiation was reversible, thus withdrawal of LIF from the medium, and the replacement of the medium to 10HI resulted in myogenic differentiation. It was found that the addition of LIF to primary uncloned population of myoblasts did not inhibit their differentiation; the cells fused and differentiated in the presence of LIF (not shown). Myosphere cells fuse with C2 cells - To reveal whether mononucleated myosphere cells that form needles can fuse with myoblasts that form multinucleated fibers, a myosphere clone, derived from ROSA26 mouse, that differentiates as mononucleated cells (Rl#al) was mixed with C2 myoblasts, which form a network of large multinucleated fibers [18]. Exposure to 10HI medium resulted in the formation of many multinucleated fibers. Most of the fibers stained blue by X-gal, showing that the myospheres cells participated in the formation of the multinucleated fibers (Fig. 9). This suggests that C2 cells provide a factor, which accelerate cell fusion, and that this factor is deficient in pure populations of myosphere derived cells. Interestingly, in these mixed cultures almost all mononucleated cells were LacZ negative, suggesting that the myospheres- derived cells were preferentially incorporated into the fibers.

Replacement of the C2 cells with a fibroblastic feeder-layer also resulted in the acceleration of cell fusion of myospheres cells (not shown). However, using conditioned medium from either cultures of C2 or fibroblasts feeder-layer did not affect the myospheres cell fusion, suggesting that the effect is mediated by cell contact.

Participation of myospheres cells in regeneration of damaged muscle - To test the capacity of myosphere cells to participate in muscle regeneration, cloned myospheres cells derived from ROSA26 mice were injected into cardiotoxin injured gastrocnemius muscle of nude mice (106 cells/muscle). Treated mice were sacrificed at various times post injection, followed by X-gal staining of the treated muscle. The regenerating, stained muscle was then sectioned. Microscopic examination of the sections revealed that the participation of myosphere cells in muscle regeneration was progressive. Sections of muscle taken 2-3 weeks after treatment showed small and local stained areas with little cross-striation. The stained area was much larger at 6 weeks post injection, while sections of muscle taken 2-3 months after injection contained large bundles of blue cross- striated fibers in the regenerating areas (Figs. 5a-d). Some of the fibers were variegated, suggesting fusion between host and donor myogenic cells (Fig. 5B-D, and Fig. 10). Transverse sections revealed the distribution of donor derived fibers, organized both in clusters as well as dispersed single blue fibers (Fig. 5D).

Testing the capacity of myosphere cells to trans-differentiate into osteogenic and hematopoietic cells - When left for a long period in differentiation medium, two clones (R21#4 and R21#8) spontaneously formed foci of adipocytes (Fig. 6A). Spontaneous trans-differentiation of satellite cells to adipocytes was recently described also by Shefer et al. [19].

To investigate the potential of myosphere cells to trans-differentiate to other mesodermal cell lineages, we exposed the myosphere cell clones were exposed to BMP-4. This resulted in acquisition of fibroblastic morphology, suppression of the expression of MyoD, inhibition of myogenic differentiation, and expression of osteogenic markers such as alkaline-phosphatase (AP) and osteocalcin (Fig. 6B,C) in all cells, in all the tested clones (which are described in Table 2, hereinabove). As reported for BMP treated C2 cells [20], removal of the BMP resulted in regaining of the myogenic phenotype, and disappearance of the osteogenic markers.

The growth of cloned myospheres in medium enriched with factors that support hematopoietic cells proliferation (Methocult GF, Stem Cell Technologies) often resulted in enrichment of the cell population with cells expressing CD45 (Fig. 7A). However, these CD45 positive cells enlarged in size, did not morphologically resemble hematopoietic cells, nor did they continue to proliferate (not shown). On the other hand, a great enrichment for CD45 positive cells (up to 95%) was observed when a very early passage of uncloned myosphere cell populations was effected (cultured for only 5 days prior to treatment) (Fig. 7A). This was probably due to CD45 positive cells that exist in the early plating stage, as was shown by McKinney- Freeman et al. [21], and Polesskaya et al. [22]. Indeed, FACS analysis showed that myosphere populations harvested 5 days after the primary preparation contain 30%- 40% CD45+ cells (Fig. 7B). The capability of the myosphere cells to express CD45 upon exposure to Methocult GF medium decreased with subsequent passaging (Fig. 7A). Moreover, separation of the cells that express CD45 from those that were CD45 negative by FACS, and culturing these populations separately in Methocult GF medium yielded hematopoietic colonies only in the culture of the CD45 expressing cells (data not shown). These results indicate that myosphere cells which do not express CD45 can not trans-differentiate to hematopoietic cells, in-vitro, and that the origin of the hematopoietic cells that were grown in Methocult GF is in the CD45 expressing cells that reside in the muscle.

To test the capacity of myosphere cells to participate in hematopoiesis, the cells were injected into lethally irradiated mice. C3H and C57bl female mice were lethally irradiated, and 6h-12h later the mice were injected LV. with dissociated myospheres derived from C3H or C57bl male mice, respectively (Table 3). In addition, since it was reported that muscle stem cells obtained from mix mice rescued lethally irradiated SJL mice [23], lethally irradiated SJL mice were injected with myospheres derived from either mdx, SJL, or ROSA26 mice (that were inbred on the C57bl background which is the genetic background of mdx). In 19 injection experiments that were made, no advantage of the injected mice was observed. In two experiments, there was a delay of 7 to 8 days in the mortality of some of the injected mice, compared to un-injected irradiated mice (Table 3). Irradiated mice that were injected with bone-marrow (BM) cells served as a positive control that survived the irradiation (not shown).

Table 3. Survival after lethal irradiation followed by I. V. cell transplantation. Table 3: Cell of the indicated myospheres strains were injected LV. into mice 4-5h post lethal irradiation. The cells were collected from cultures grown as floating myospheres (myospheres) or as adherent monolayer (adherent).

Host strain Myospheres strain Survival after irradiation (days) (no. of injected mice) (no. of donor cells)

Untreated Treated

C3H ( 3 ) C3H spheres (2- 10⁶) 15±2 14

C3H ( 6 ) C3H adherent (2- 10⁶) 16±3

C3H ( 7 ) C3H adherent (2- 10⁶) 16±2 15±2

C3H ( 5 ) C3H adherent (0.75- 10⁶) 17±1

C3H ( 5 ) C3H n.b adherent 14±2 15±2 (1.3-10⁶)

C57 ( 5 ) C57 adherent (2 10⁶) 10±2 l l±l

C57 ( 5 ) C57 adherent (MO⁶) 15±3 14±2

SJL (5) mdx3#8 adherent (0.5-10⁶) l l±l 14±3

SJL (5) mdx#8 spheres (0.4- 10⁶) 16±3

SJL (5) mdx#4 spheres (0.4- 10") 14±2

SJL (5) mdx3#4 adherent (0.5- 10") 13±2

SJL (6) mdx3#2 adherent (0.5- 10⁶) 12±2 12±2

SJL (5) SJL adherent (0.3- 10⁶) 12±1 12±1

SJL (6) SJL adherent(1 10⁶) 1 1±2 l l±l

SJL (4) mdx#3 (0.5-10⁶) 9 12

SJL (5) mdx3#8 adherent (0.5- 10⁶) 15±2 15±2

SJL (3) mdx#8 spheres (0.5- 10⁶) 15±2

SJL (4) ROSA 23#1 adherent (0.5- 10⁶) 12±2 13

SJL (4) mdx#8 spheres (0.5- 10") 13±3

Table 4. Summary of intra-muscular injections of myospheres derived cells

Table 4: Mice were injected intra-muscularly as described in materials and methods. The table shows a representative list of injected clones. Each clone was injected to at least 3 mice. To evaluate the amount of blue fibers, representative slides from each injected muscle were chosen and the blue fibers were counted. All the injected clones were CD45 negative, and only those treated with LIF expressed Sca-1 by most of the cells.

In addition, C3H female mice were sub-lethally irradiated and injected with myospheres obtained from C3H male mice. PCR analysis of muscle, BM, peripheral blood, spleen, lungs, kidneys, and colon obtained from the injected mice did not detect the presence of the injected cells.

To bypass a possible inefficient homing of the LV. injected myosphere cells to the BM, 10⁶ cells of either uncloned myospheres population cultured for 5 days (and contain -40% CD45+ cells) or myosphere clones (which do not express CD45), were injected directly into the BM (I.BM) of isogenic lethally irradiated mice. As a positive control for the procedure, irradiated mice were injected with 5xlO⁵ BM cells. Nevertheless, the mice injected with the myosphere cells (either uncloned early population or clones) died after 10-12 days, as did the uninjected irradiated mice. All mice that were injected I.BM with BM cells survived the effect of irradiation (Fig. 7C).

Taken together, these experiments did not provide evidence for trans- differentiation of the myosphere cells to hematopoietic cells. Moreover, although primary muscle cultures contain hematopoietic stem cells, which can give rise to hematopoietic cells, both in-vitro and in-vivo ([21] and Figs. 7a-c), they could not efficiently reconstitute the hematopoietic system of lethally irradiated mice. Analysis and Discussion The investigation reported here describes the reproducible isolation of myogenic cells derived from mouse skeletal muscle that can be propagated in suspension culture as myospheres for extended period. The myosphere cells retain their capacity to differentiate in vitro and to participate efficiently in muscle regeneration. The selection of those cells is based in principle on the old observation, that skeletal muscle and cardiac myogenic cells adhere to the cell culture plate surface slower than most non-myogenic cells that are obtained during the conventional preparation of muscle cell culture (5). A partially similar approach was taken by Qu- Peterson et al. [9] who showed that cells, which do not adhere to the plate during the first 5 days, yield myogenic clones that differ from early adherent myoblasts in their capacity to repopulate degenerated muscle. Those cells were collected after 5 days and plated for further amplification as adherent cells. In the study reported here, it was found that it is possible to propagate suspended cultures for at least several months without loosing their capacity to proliferate, differentiate and to participate in muscle regeneration.

Interestingly, unlike previously described rat and mouse myogenic cell lines, this procedure selected for cells that differentiate, mostly without cell fusion, into very thin elongated mononucleated myogenin and myosin positive contractile cells. The formation of mononucleated contractile cells may indicate that these cells represent an earlier stage of myogenic differentiation, reminiscent of the mononucleated muscle cells of early somite [24]. It is possible that the continued proliferation of nonadherent cells selects for cell surface properties that leads to terminal differentiation without or prior to cell fusion. One possible candidate that enables terminal differentiation in single myogenic cells is p27kipl. This cyclin-dependent kinase inhibitor was shown to play a critical role in the N-cadherin-dependent signaling during myogenesis, and its forced expression in C2 cells resulted in their differentiation as single cells [12]. It is shown here that myosphere derived cells express p27kipl already when grown in the proliferation medium. It is possible that growing the cells as myospheres select indirectly for cells expressing p27kipl (e.g. perhaps by selecting for cells expressing altered levels of N-cadherin). Thus, conceivably, myosphere cells that express p27kipl when cultured in the growth medium, as single cells, may bypass the requirement for cell-cell contact in order to differentiate.

As shown, the serial passages of uncloned mass-cultures of myospheres resulted in a very efficient selection of myospheres consisting virtually of MyoD positive myogenic precursor cells. Cloning of single cells revealed mild heterogeneity between clones, with regard to several markers, including expression of MyoD and the tendency of the cells to fuse and form multinucleated fibers or to differentiate first as mononucleated needles. Using the same experimental procedure with rat muscle cells myosphere like clusters of cells were obtained that after their adherence, most of them differentiated into a mixture of multinucleated muscle fibers and colonies of adipocytes (unpublished results). As shown here, some cells of the mouse myosphere clones spontanously trans-differentiated to adipocytes (fig. 6A), suggesting that the rat adipocytes were derivatives of myogenic cells.

Several groups reported on the isolation of stem cells derived from various other tissues, and their proliferation in suspension as spheres [25-27]. Neurospheres cells were shown to have pluripotent capability, giving rise to cells from all three germ layers, and participate in skeletal muscle and hematopoietic regeneration [28,29]. Adult BM derived cells have been shown to integrate into diverse adult tissues, including skeletal and cardiac muscle [30-33]. Several other stem cells were isolated and proliferated as adherent cells, which had either multipotent or pluripotent capability [15,23,34-36]. Despite these investigations, the issue of trans- differentiation of cells is still a matter of controversy, since other findings indicated the inability of stem cells to trans-differentiate and into other cell types [37-42].

In contrast to previously described pluripotent stem cells, which are immature and do not express proteins associated with specific differentiation (e.g neurospheres, mesenchymal adult progenitor cells), the myosphere cells described here express the myogenic marker MyoD, and are conceivably already more committed.

C2 cells and primary satellite cells respond to BMP by expressing AP and osteocalcin, and can also give rise to adipocytes [20,48-54]. BMP treatment induced the expression of AP and osteocalcin in all the cloned myosphere cell populations (Fig. 6B,C). These results suggest the potential of the myospheres to give rise to other mesodermal cell lineages. In accordance with the results obtained with C2 cells [20], the effect of BMP on myosphere cells was dependent on its continuous presence in the medium; withdrawal of BMP resulted in re-expression of MyoD and myogenic differentiation. The capacity of myosphere-derived cells to participate in regeneration of injured muscle was also demonstrated and is of clinical importance.

Altogether, these results suggest the use of the cloned myosphere cells for in vivo generation of muscle tissue. EXAMPLE 2 MYOSPHERES EXPRESS NESTIN, A NEURONAL STEM CELL MARKER

As described in Example 1, hereinabove, the present inventors isolated and propagated a sub-population of myogenic cells from mouse skeletal muscle, which proliferate, for at least several months as suspended clusters of cells (myospheres). These cells express the myogenic markers: MyoD and desmin (an intermediate filament protein present in smooth muscle cells, striated muscle cells and myocardium), and a subset of them also express myogenin, indicating their belonging to the myogenic lineage (Sarig et al., Stem Cells. 2006 Mar 30; [Epub ahead of print]). To test if these myogenic cells are also capable of expressing nestin, a marker for neuronal stem cells, the cloned myosphere cells were subjected to immunofluorescence analysis using the anti-nestin antibody, as follows. Materials and Experimental Methods Cell culture - Cells were prepared as previously described (Sarig et al., Stem

Cells. 2006 Mar 30; [Epub ahead of print]). Briefly, primary muscle cultures were prepared from 3-4 weeks old ROSA26 mice, which contain a transgene encoding a ubiquitously expressed bacterial β-galactosidase (Zambrowicz et al., 1997). The hind-limb were isolated and the fat and bones discarded. The muscle was minced with scissors, enzymatically dissociated for 30 minutes at 37 °C with 0.05 % trypsin- EDTA followed by 5-minute centrifugation at 2500 rpm. The cells were collected and trypsinization of the remaining undigested tissue was repeated three more times by adding a fresh trypsin solution. On the fourth time, the cells were incubated for 30 minutes with 0.25 % trypsin-EDTA. After centrifugation, the cells were suspended in the proliferation medium, which is a basal medium, containing fetal calf serum, steroids, bFGF, insulin, glutamine and antibiotics (BIOAMF-2, Biological Industries, Beth H'emek, Israel) either with or without leukemia inhibitor factor (LIF) (10 ng/ml, CytoLab, Rehovot, Israel). The trypsinized cells were serially passaged (for one or two times) as non-adherent cells for 4 days. On the fifth day, the cells that attached to the plate were collected either as uncloned cell population, or were further sorted by FACS, to isolate single Sca-1 [a stem cell marker of both hematopoietic and myogenic stem cells (Mitchell PO, et al., 2005, Dev. Biol. 283:240-252)] positive cells for clonization as described below. Adherent monolayer of myospheres derived cells were grown in gelatin coated plates, in the proliferation medium. Cells that did not attach to the plates on the fifth day were serially passaged as non-adherent cells (myospheres). Myospheres were serially passaged by allowing them to sediment by gravitation to the bottom of a test tube, the old medium was removed by decantation followed by careful suspension of the myospheres in fresh medium and plating them in uncoated cell culture plates. Changing to differentiation enhancing medium, 10HI (DMEM containing 10 % carefully selected horse serum, 0.04 units/ml insulin, 0.5 % chick embryo extract and penicillin-streptomycin; Gibco), was done when the cultures approached confluency. Immunochemistry of cells: Adherent cells were grown on gelatin or fϊbronectin coated glass coverslips. The cells were fixed for 10 minutes with 4 % PFA in PBS and permeabilized for 5 minutes with 0.2 % Triton X-100 in PBS. For blocking, the cells were incubated for 30 minutes at room temperature in PBS containing 0.1% Triton and 3 % bovine serum albumin. For immunostaining, the cells were incubated for 1 hour with the following monoclonal antibodies, diluted in the blocking solution; MyoD (1:100, DAKO Corp, Carpenteria, CA, USA), MHC (myosin heavy chain) (1 :20, MF-20, DSHB), Nestin (1:5, Rat-401, DSHB). After three washes with PBS containing 0.1 % Triton, the cells were stained for 30 minutes at room temperature with Alexa-488-labeled goat anti-mouse Abs (1:150; Molecular Probes, Inc., Eugene, OR, USA), followed by 5 minute incubation in a solution of 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining (10 mg/ml). The cells were mounted with elvanol, and viewed under a Nikon fluorescence microscope at a magnification of x200/x400. Pictures were taken with a 1310 digital camera (DVC). Experimental Results

Myospheres express nestin, a neuronal stem cell marker - Cloned populations of myosphere cells, grown in the proliferation medium were immunostained with anti-nestin antibody. As is shown in Figure 11, most of the cells (> 90 %) express nestin (green). These results demonstrate, for the first time, that in addition to the myogenic markers, myospheres, a sub-population of myogenic cells, also express the neuronal stem cell marker, nestin.

Nestin, an intermediate filament protein is the most common marker that is used to isolate neuronal stem cells (since it is expressed by immature cells of the nervous system), however, it was also shown to be expressed in myogenic cells and other tissues (reviewed in Michalczyk and Ziman, 2005). In addition, it was recently demonstrated that the expression of nestin by mesenchymal stem cells is a prerequisite for their trans-differentiation to astrocytes or neurons (Wislet-Gendebien et al., 2005; 2003).

Altogether, these results demonstrate that a cloned population of myosphere cells express nestin and suggest their further use in transdifferentiation into the neuronal cell lineage.

EXAMPLE 3

CLONED MYOSPHERE CELLSARE CAPABLE OF TRANSDIFFERENTIATLONINTO THE NEURONAL CELL LINEAGE

To further determine the potential of myospheres to transdifferentiate into the neuronal cell lineage in vivo, the present inventors have used the developing mouse brain as the optimal microenvironment to enable such a phenomenon. Since the brains of newborn mice keep developing in the first 2-3 weeks, it may provide the necessary factors and supportive tissue for reprogramming of the cells. Hence, the present inventors have injected cloned myosphere cell populations into the lateral ventricles of brains of newborn mice, and followed their fate. Since the cloned cells were obtained from ROSA26 mice, which ubiquitously express β-gal, the location of the injected cells was identified by X-gal staining. In addition, the injected brains were subjected to a comprehensive immunofluorescence and immunohistochemistry analyses using various neuronal markers, as follows. Materials and Experimental Methods Cells - as described in Example 2, hereinabove.

FACS analysis - Cells were washed once with phosphate-buffered saline (PBS), and resuspended in 0.1 ml of cold PBS. Mouse serum (Sigma-Aldrich, 1:10) and Fc block (rat anti mouse CD16/CD32, BD PharMingen) were added, and the suspensions were incubated for 10 minutes on ice. Each sample was divided into two halves; one half received anti-Sea- 1 (eBioscience, PE conjugated), and the other half received the conjugated flourescin only (PE). The tubes were incubated for 30 minutes at 4 °C, and then washed twice with cold PBS. The cells expressing Sca-1 were collected by FACSVantage. Transplantation of cells into the brain of mice — Slow-adherent Cells (Sca- l+/MyoD+ were collected, washed with PBS, and re-suspended in a concentration of 10⁵ cells/1.5 ml in cold PBS. Three days old C57bl mice were anaesthetized, and 10⁵ cells were injected into their lateral ventricles, with a Hamilton syringe. Mice were sacrificed at the indicated time points, and their brains were removed for further analysis. Mice of at least 14 days-old were perfused with 2.5 % cold paraformaldehyde (PFA) prior to brain removal. Mice younger than 14 days-old were not subject to PFA perfusion prior to brain removal. The brains were fixed for 2-3 hours with 2.5 % PFA, transferred to a solution of 1 % PFA containing 15 % sucrose and incubated for at least 16 hours at 4 °C. Brain slices of 25-40 μm were collected using a floating microtom.

X-GaI staining - Brain slices were washed with PBS and stained overnight at 37 °C with X-GaI (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) solution, containing: PBS at pH:7.3-7.4, 5 mM K3Fe(CN)₆, 5 mM K4Fe(CN)₆, 0.02 % NP-40, 2 mM MgCl₂, 0.1 % X-GaI . Slices were washed with PBS, and counterstained with nuclear fast red.

Immunochemistty of brain slices - Sections adjacent to X-GaI positive slices were chosen for immunohistochemical analysis. Slices were blocked and stained using M.O.M kit solutions (Vector). The slices were incubated over-night, at room temperature (RT), with anti-β-gal (1:200) together with one of the following antibodies: Doublecortin (1:100, Santa-Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Tujl (1:400, Covance), NF-160 (1:800, abeam, Cambridge, UK), NeuN (1:200, Chemicon Intnl, Inc. Temecula, CA, USA). Secondary antibodies were either used for direct detection (for the β-gal labeling Cy3 anti-mouse,), or with biotin- streptavidin-FITC (Jackson ImmunoResearch Lab.) for the neuronal markers. Slices were stained for 2 minutes with DAPI solution, air-dried and mounted as above. For the detection of NeuN, X-gal stained slices were labeled with anti-NeuN and detected with Vectastain ABC Kit (Vector). Experimental Results Cloned myosphere cells are extensively scattered in the brains of the implanted newborn mice - Brains of newborn mice were injected with cloned populations of myosphere cells (10⁵ cells per brain), derived from ROS A26 mice which ubiquitously express β-gal and the brains were removed from mice sacrificed at 2 days, 4 days or 9 days post injection. The presence of the myosphere cells in the injected brains was visualized by X-gal staining. As is shown in Figures 12a-c, two days after the injection, the cells were still localized in the ventricle, in a compact cluster, while few cells started to migrate out of the ventricle (Figure 12a). Four days after the injection, the cells already migrated out of the ventricle and started to disperse, still in the vicinity of the injected site (Figure 12b). Extensive scattering of the cells was observed already one-week post injection. After nine days, the cells were detected in several brain regions, including the cortex, corpus-callosum, hippocampus, thalamus, cerebellum, rostral migratory stream and the olfactory bulb (Figure 12c).

Myosphere cells implanted into the brains of newborn mice express doublecortin, a marker for young neurons — As is further shown in Figures 13a-h, seven days after injection of the cloned MyoD+ myosphere cells into the lateral ventricles of newborn mice, a proportion of the implanted myosphere cells expressed both β-gal (which identifies the cells as donor cells that are derived from the ROSA26 mice which ubiquitously express β-gal) and doublecortin (a marker for young neurons) (see Figures 13a-d for positive cells observed in the corpus callosum). In addition, these cells, which express both β-gal and doubcortin exhibited a neuronal cell morphology as observed using confocal microscopy (Figures 13e-h).

Myosphere cells implanted into the brains of newborn mice express β- tubulin III, a marker for immature neurons - As is further shown in Figures 14a-e, immunofluorescence analysis using both anti-β-gal and anti-β-tubulin III (TUJl) antibodies demonstrated that the implanted myosphere cells express both markers. Such double-labeled cells were observed mostly in the corpus callosum, and few cells were observed also in the CAl region of the hippocampus (Figures 14a-e). These results demonstrate, for the first time, the in vivo expression of β-tubulin III, a marker for immature neurons, from myogenic MyoD+ cells in the brain.

Myosphere cells implanted into the brains of newborn mice express NF-160 and NeuN, markers for mature neurons - Brains of mice injected with the myosphere cells were further subjected to double immunofluorescence analysis using anti-β-gal and anti-NF-160 (a marker for mature neurons) antibodies. As is shown in Figure 15 a, a proportion of cells expressed both NF- 160 and β-gal, demonstrating the ability of implanted donor cells (β-gal positive cells) to express a marker of mature neuronal cells. Further analysis using X-GaI staining and NeuN immunohistochemistry (using the anti-NeuN antibody conjugated to peroxidase) revealed that in certain brain regions (e.g., the cerebellum) a proportion of myosphere donor cells (which are X-gal positive) which express NeuN, a mature neuron - specific marker. Additional markers observed include, GFAP and Tuj-1 indicating the presence of glial cells. These results unequivocally show the ability of the cells of the present invention to form glial tissue in vivo. The expression of neuronal markers in donor myosphere cells is likely to result from transdifferentiation - The percentage of β-gal positive cells expressing Doublecortin was -15 % (Figures 13a-h). Of them, only ~2 % had an aberrant nucleus and the rest of them (about 98 %) contained only one normal looking nucleus. This suggests that the expression of the neurogenic markers was not a result of cell fusion between the donor (e.g., myosphere cells) and host cells. Moreover, many cells expressing β-gal and immature markers appeared in brain regions atypical of immature neurons (e.g. corpus callosum and specific hippocampal regions), while the result of fusion events in these regions would have probably been the expression of mature neuronal markers. Altogether, these results suggest that the injected cloned myogenic cells trans- differentiated into neuronal cells. The same results were obtained using 3 independently isolated clones.

EXAMPLE 4 MYOGENIC PROGENITOR CELLS OBTAINED FROM YFP-2.2 TRANSGENIC MICE EXPRESS YFP IN THE BRAIN OF NEW-BORN MICE

To further demonstrate the ability of cloned myosphere cells to transdifferentiate into neuronal cell lineage, the present inventors have isolated several myogenic clones from skeletal muscles of transgenic mice, which express yellow fluorescent protein (YFP) under the control of specific regulatory elements of the Thyl gene, which confer specificity of YFP expression to a subset of neurons (B6.Cg-Tg(Thyl-YFPH)2Jrs/J, Feng et al., 2000). In general, a nucleic acid construct containing a YFP gene under a transcriptional control of regulatory elements derived from the mouse thyl gene was injected into fertilized bόcbafl mouse eggs. Regulatory elements are composed of a 6.5 bb fragment obtained from the 5' portion of the THYl gene, extending from the promoter to the intron following exon 4. Exon 3 and its flanking introns are absent. The deleted sequences are required for expression in non-neural cells but not in neurons. The remainder of the sequence is required for neuronal expression. The cloned cells (which, like all muscle cells, did not express YFP prior to their injection), were injected into the lateral ventricles of new-born C57bl mice. Following one week, the unstained slices of brains were examined for the expression of YFP. The pattern of distribution of injected cells that expressed YFP was very similar to that of the neuronal marker and β-gal expressing myosphere cells described above. This further supports the conclusion that myogenic progenitor cells are induced to express neuronal genes, in the developing mouse brain

In vivo activation of a neurospeciβc transgene in donor cells following the injection of myosphere cells into the brain of newborn mice - As is shown in Figures 16a-d, a very similar pattern of distribution of injected cells that express YFP. The cells were mostly in the corpus collasum, the cortex, the thalamus and the hippocampus. It should be noted that the clones did not express YFP prior to their injection into the brains of newborn mice (data not shown). Altogether, these results confirm the ability of myogenic progenitor cells to differentiate into the neuronal cell lineage and thus to express neuronal markers. These results suggest the use of the cloned myosphere cells of the present invention for in vivo generation of neural tissues (neurons and/or glial cells). It will be appreciated that for treating a subject in need thereof, the cells can be part of a pharmaceutical composition and be formulated with a pharmaceutical acceptable carrier.

Analysis and Discussion

The present inventors have shown, for the first time, that cloned myogenic cells, which are programmed to differentiate to the mesodermal cell lineage and express myogenic markers such as MyoD can be reprogrammed to differentiate into the ectodermal cell lineage and express neuronal cell markers.

In a recent study (Sarig et al., 2006) the present inventors have identified very slow adherent mouse skeletal muscle cells, which can be propagated as floating colonies. In the present study, these myosphere cells were tested for their ability to be reprogrammed, in the appropriate microenvironment, into neurogenic cells. The findings presented herein demonstrate that muscle progenitor cells injected into the brains of newborn mice changed their morphology to neuron-like cells, migrated to several brain regions, and expressed immature and mature neuronal markers. Most of the injected cells expressing neuronal markers contained only one nucleus. These results suggest the use of these cells for cell therapy and/or gene therapy approaches for neurodegenerative diseases (e.g. multiple sclerosis, Parkinson, Alzheimer, etc.), based on the reprogramming of muscle progenitor cells, preferably from autologous source. The present application is concerned with the developing of reproducible efficient methods for in- vivo reprogramming of myogenic cells for such approaches.

EXAMPLE 5

HUMAN MYOGENIC CELLS ARE CAPABLE OF DIFFERENTIA TING INTO NEURAL TISSUE IN VIVO

Materials and Experimental Procedures

In-vitro generation of human myogenic cells - Human muscle biopsies (Vastus lateralis) taken from young and adult patients, during surgical operations, were obtained by Drs. Ben Itshak and Panski of Kaplan Hospital. The biopsies were dissociated at 37 °C with 0.05 % trypsin-EDTA for 30 minutes, and then centrifuged at 2500 rpm for 5 min. The cells were collected and suspended in the growth medium (BIO-AMF-2, Biological Industries). The trypsinization of the remaining undigested tissue was repeated two more times by adding fresh trypsin solution. The cells were serially passaged, and several fractions were collected in accordance with their adhesion properties. The fraction with major proliferative capacity was the one that adhered to the plates at 2-18h. For clonization, cells were diluted at 2-3 cells/cm², single cell colonies were collected, and propagated.

Transplantation of human myogenic cells into mouse brains - Cells were collected, washed with PBS, and re-suspended in a concentration oflO⁵ cells/1.5μl in cold PBS. Three days old C57bl mice were anaesthetized, and 10⁵ cells were injected into their lateral ventricles, with Hamilton syringe. Human myogenic cells were labeled with 2OmM Hoechst prior to injection, at 37°C, for 10 min. Mice were sacrificed at the indicated time points, and their brains were removed for further analysis. The brains were fixed with 2.5 % PFA for 2-3 hours, transferred to 15 % sucrose solution with 1% PFA, and incubated at 4°C for at least 16 hr. Brain slices of 25-40μm were collected using floating microtom.

Immunocytochemistty - Brain slices containing Hoechst labeled human cells were immunostained with a human specific antibody to NF-70 (1 : 100, Chemicon). Results

Almost pure populations of myogenic cells proliferating as a myoblasts monolayer were obtained from primary muscle mass cultures. The cells later formed by cell fusion a network of multinucleated fibers (Figures 17a-b). Most of these cells expressed MyoD, and upon induction of myogenic differentiation with 10HI medium

(described in example 1) they express dystrophin.

These cultures were used to test the capacity of human derived myoblasts to give rise to neurogenic cells, following inoculation into the brain of new-born mice. The cells were labeled with Hoechst dye prior to injection, and as described for mouse myosphere cells, the inoculated brains were fixed and sectioned 5 and 9 days following injections. Sections with Hoechst labeling were stained with anti-NF-70, which is specific for human neurons. As shown in Figures 18a-f, the injected cells were mostly localized around the ventricles, in the subventricular zone (SVZ), and in the corpus callosum. Most of the NF-70 positive cells (with Hoechst labeled nuclei) were found in the corpus callosum, and in the cortex, near the injected site. Nine days after the injection there were many more NF-70 expressing cells that also changed their morphology resembling neuronal cells, than in the brains collected 5d after the injection.

These results indicate that human myogenic cells are also able to integrate, in vivo, in the developing mouse brain and to express a neurogenic marker, suggesting their use in brain regeneration. EXAMPLE 6

HUMAN MYOGENIC CELLS GENERATED ACCORDING TO THE

TEACHINGS OF THE PRESENT INVENTION INCORPORATE INTO HOST

BRAINBUTDONOTFUSE WITHCELLS THEREOF

Human myogenic progenitor cells generated as described hereinabove were labeled with Hoechst (H), and injected into the brain of mice ubiquitously expressing GFP. The brains were analyzed 9 days after the injection. The injected cells were mostly localized along the corpus-callosum (cc), and at the sub- ventricular zone (Figures 19a-f). Thus, cell spreading in the host brain was noted indicating the suitability of the cells for use in clinical applications.

As shown in Figures 20a-c donor injected human cells do not fuse with endogenous tissue. Human myogenic progenitor cells were injected into brains of GFP expressing mice. Following 9 days the brains were sliced and immunostained with a human specific anti-NF-70 antibody. Group of donor cells, in several brain regions expressed human NF-70 protein, and merge images revealed that most of these cells do not express the host GFP protein (Figure 20a). Figures 20b and c show a group of cells expressing human NF-70 at the injection vicinity. Arrows in Figure 20c point at two cells that may represent rare fusion events between host and donor cells.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

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Claims

WHAT IS CLAIMED IS:

1. Use of human myogenic satellite cells for the manufacture of a medicament identified for treating a medical condition of the CNS.

2. The use of claim 1, wherein said human myogenic satellite cells are characterized by MyoD+/Pax-7+ expression profile.

3. The use of claim 1, wherein said human myogenic satellite cells are formulated for local administration.

4. The use of claim 1, wherein said human myogenic satellite cells are formulated for systemic administration.

5. The use of claim 1, wherein said human myogenic satellite cells are of a single clone.

6. The use of claim 1, wherein said human myogenic satellite cells proliferate in vivo.

7. The use of claim 1, wherein said human myogenic satellite cells express at least one neuronal marker following administration.

8. The use of claim 1, wherein said at least one neuronal marker comprise NF-70.

9. The use of claim 1, wherein said medical condition of the CNS is a neurodegenerative disease or disorder.

10. The use of claim 9, wherein said medical condition of the CNS is selected from the group consisting of a brain injury, a spinal cord injury, cerebral pulsy, a spinal muscular atrophy, a motion disorder, a dissociative disorder, a mood disorder, an affective disorder, an addictive disorder and a convulsive disorder.

11. The use of claim 10, wherein said neurodegenerative disorder is selected from the group consisting of Parkinson's, multiple sclerosis, epilepsy, amyatrophic lateral sclerosis, stroke, autoimmune encephalomyelitis, diabetic neuropathy, glaucatomus neuropathy, Alzheimer's disease, Down's syndrome, dementia, Gaucher disease, dementia associated with Lewy bodiesand Huntingdon's disease.

12. The use of claim 1, wherein said human myogenic satellite cells are autologous cells.

13. The use of claim 1, wherein said human myogenic satellite cells are non-autologous cells.

14. The use of claim 1, wherein said human myogenic satellite cells are obtained by:

(a) generating a single cell culture from a human muscle; and

(b) culturing said single cell culture under conditions which allow cell proliferation.

15. The use of claim 1, wherein said human myogenic satellite cells are immortalized.

16. The use of claim 14, wherein said culturing is effected for 3-4 weeks.

17. The use of claim 14, wherein said single cell culture comprise cells which adhere to a matrix within 2-18 hours.

18. The use of claim 1, wherein said human myogenic satellite cells are encapsulated.