Summary of experiments for testing the myogenesis potential of ESCs in NMD.
The neuromuscular disorders are a heterogeneous group of genetic diseases, causing a progressive loss of the motor ability.More than 30 genetically defined forms are recognized, and in the last decade, mutations in several genes have been reported, resulting in the deficiency or loss of function of different important muscle proteins.Biochemical and imunohistological analysis have localized these proteins in several compartments of the muscle fiber.The proteins dystrophin, sarcoglycans and dysferlin are sarcolemmal or peri-sarcolemmal proteins, α2-laminin and collagen VI are extracellular matrix proteins, telethonin and actin are sarcomeric proteins, calpain 3 and FKRP are cytosolic enzymes, and emerin and lamin A/C are nuclear proteins (Vainzof et al., 2008).
Defects in components of the dystrophin-glycoprotein complex (DGC) are known to be an important cause of different forms of muscular dystrophies (Ervasti & Campbell, 1993; Yoshida & Ozawa, 1990).The DGC is an oligomeric complex which connects the subsarcolemmal cytoskeleton to the extracellular matrix.The DGC consists of dystroglycan (α- and β-DG), sarcoglycan (α, β-, γ-, δ- and ε-SG) and syntrophin/dystrobrevin subcomplexes.The intracellular link of the DGC is the protein dystrophin, that plays an important structural role in muscle fibers.Mutations in the dystrophin gene cause the most common form of X-linked Duchenne muscular dystrophy (DMD) (Hoffman et al., 1987). Dystrophin binds its amino-terminal and rod domain to actin and with its carboxy terminal to the integral membrane protein β-DG.The sarcoglycan sub-complex is also linked to β-DG and includes α-SG, β-SG, γ-SG, and δ-SG, which are tightly associated and inserted into the membrane. Mutations in the genes coding the 4 SG proteins cause severe forms of limb-girdle muscular dystrophies type LGMD2D, 2E, 2C and 2F, respectively.The peripheral membrane glycoprotein α-DG, a receptor for the heterotrimeric basement membrane protein laminin-2, binds to β-DG and so completes the connection from the inside to the outside of the cell (Straub & Campbell, 1997). Mutations in the LAMA2 gene, encoding the α2 chain of laminin-2, cause α2-laminin deficiency, and a severe form of congenital muscular dystrophy (CMD1A) linked to chromosome 6q (Tomé et al., 1994). In addition, some forms of muscular dystrophy have recently been associated with genes encoding putative or known glycosyltransferases. Muscle protein analysis in these patients show a hypoglycoslation of α-dystroglycan and a consequent reduction of numerous ligands components of the extracellular matrix, such as laminin 2 (Muntoni et al., 2004).Other milder forms of muscular dystrophy are caused by mutations in genes coding the enzyme calpain 3 (LGMD2A), the sarcolemmal protein dysferlin (LGMD2B), and the sarcomeric protein telethonin (LGMD2G) (revision in Vainzof & Zatz, 2007).
Several animal models, manifesting phenotypes observed in neuromuscular diseases have been identified in nature or generated in laboratory.These models generally present physiological alterations observed in human patients, and can be used as important tools for genetic, clinic and histopathological studies (Vainzof et al., 2008)
Autosomal recessive limb-girdle MD forms models include the
Mouse models for congenital MD include the
The study of animal models for genetic diseases, in spite of the existence of differences in some phenotypescan provide important clues to the understanding of the pathogenesis of these disorders and are also very valuable for testing strategies for therapeutic approaches.
In all forms of muscular dystrophies, in the early phase of the disease, there is a continuous process of degeneration and regeneration. The ability of adult skeletal muscle to regenerate has been attributed to the special properties of satellite cells. In mature skeletal muscle satellite cells are normally in a dormant state. Upon injury, many growth factors are secreted at the site of the lesion that recruits satellite cells, triggering the process of regeneration. In the progression of the dystrophic process, however, the endogenous satellite cell pool becomes exhausted and degenerated muscle fibers are replaced by fibrotic and adipose tissues, responsible for the observed muscle weakness.
Therefore, stem cell therapy should be an ideal treatment for all forms of muscular dystrophies, in which the deficiency of a specific muscle protein leads to muscle degeneration. Stem cell transplantation would either prevent or break the cycles of degeneration and regeneration in the dystrophic process, by replacing the deficient protein ( Meng et al., 2011 ). The majority of the therapeutic trial under investigation are done using Duchenne muscular dystrophy as a model for, because it is the most common and severe mendelian form of muscular dystrophy.
2. Stem cell therapy for muscular dystrophies
The use of normal stem cells to rescue the effects associated with mutant tissue is a promising avenue of research. To correct the dystrophic phenotype healthy stem cells transplanted to the diseased muscles must first be attracted to the injured area, answer to the endogenous signals that stimulate muscle differentiation and, ultimately, fuse to dystrophic myofibers, or form new myotubes. Incorporated healthy nuclei would thus contribute to restore the normal levels of functional skeletal muscle proteins such as dystrophin.
According to a recent revision by Meng et al. (2011 ), the ideal stem cell for treating DMD should fulfill several criteria, and should be able to: expandable in vivo, maintaining stem cell properties, be immune-competent, be systemically-delivered reaching all muscles of the body, survive, proliferate and migrate upon arrival within the host muscle, differentiate into muscle fibres, reconstitute the satellite cell pool with functional stem cells, be capable of expressing the missing protein, lead to improvement in muscle strength.
The most appropriated type of stem cell for these therapies is still under investigation. Among adult stem cells, skeletal muscle satellite cells have been considered the only source of stem cells for post-natal muscle regeneration (Seale & Rudnicki, 2000). Other stem cells within the skeletal muscle have, however been identified, including muscle-derived stem cells, muscle side population cells, myogenic endothelial cells, and mesoangioblasts (pericytes) ( Meng et al, 2011 ). Human skeletal muscle-derived pericytes have been shown to form muscle fibers, after intra-arterial transplantation in the dystrophin deficiency host mice (Dellavalle et al., 2007). However, in contrast, muscle-derived cells (mdcs), which are very similar to the pericytes, did not contribute to muscle regeneration after systemic delivery in mdx nu/nu host ( Meng et al., 2011 b). These findings illustrate the opinion of several authors suggesting that further work is necessary to prepare pure cell populations from skeletal muscle that maintain their phenotype in culture and make a robust contribution to skeletal muscle regeneration after systemic delivery in dystrophic mouse models ( Meng et al., 2011 b)
Among the other non-muscle stem cells, previous attempts at developing stem cell therapies for the treatment of DMD were performed using bone marrow and blood-derived stem cells, mesenchimal stem cells from other origins, and pluripotent stem cells. Bone marrow-derived stem cells (BM) are a population of circulating cells with myogenic potential, present in the bone marrow, as already demonstrated as early as in the 1960s.Afterward, some authors confirmed that BM-derived cells can undergo myogenic differentiation and participate in muscle repair after injury, albeit at very low levels (Ferrari et al., 1998; Gussoni et al., 1999). Transplantation studies with BM cells injected into
Mesenchymal stem cells from other origins have been shown to regenerate muscle fibers, but at a very low efficiency, in mouse models. However, some therapeutic effects have been observed, such as by reducing inflammation in the dystrophin deficient muscle (Ichim et al., 2010).
Because of their pluripotency, much attention has been devoted to the potential applications of Embryonic Stem (ES) (Evans &Kaufman, 1981). However, to date, ES cells have not had a significant impact on the development of cell-based therapies to treat muscular dystrophy. While a few studies have been performed in
Thus, stem cell therapies for muscle disorders are still in the early days. There are many outstanding questions such as the immunogenic capacity of stem cells, whether local or systemic injections are the best route of administration in the diseased tissue, whether there is an optimal amount of cells to be administered, or whether the regenerative growth factors present in the dystrophic muscle are sufficient to promote the survival and the fusion of these cells to the damaged muscle (Bradley et al., 2002; Mimeault et al., 2007).
In order to answer at least some of the questions summarizedabove, we investigated the capacity of ES and EB cells to generate muscle in vivo, and to express the deficient protein dystrophin in the
3. In vivo experiments with ES cells and potential therapies for NMD
3.1. ES cell culture
The embryonic stem cell line USP-1 derived from 129/Sv mice (Sukoyan et al., 2002) was cultivated as described (Rohwedel et al., 1994).Briefly, cells grew on irradiated mouse feeder layer on gelatin-coated flasks (Nunc) in DMEM (Gibco) supplemented with 10% fetal bovine serum (Hyclone) and additives (Rohwedel et al., 1994). During the isolation and early stages of ES cell cultivation, the medium was supplemented with human recombinant leukemia inhibitory factor (hLIF) at 1000 units/ml (Chemicon) and was replaced every day. When reached the confluence (after 4 days in culture) these cells were trypsinized, and transferred to another culture flask (75 cm2) previously covered by alayer of mouse fibroblast feeders, plated at a density of 1.0 x 106 cells/cm2 for proliferation. For differentiation, aliquots of 20 µl cultivation medium containing 800 cells were placed on the lids of Petri dishes filled with PBS, based in protocols developed by Wobus et al. in 1988 (2002), which are based in previous differentiation by embryoid body formation and posterior treatment with 1% DMSO (dimethyldisulfoxide). The ES cell aggregates (Embryoid Bodies - EB) were cultivated in hanging drops for 2 days and subsequently in suspension on bacteriological Petri dishes for additional 3 days in a specific skeletal muscle differentiation medium (proliferation medium with 1% DMSO, but no LIF). At day 5, EBs were plated separately onto gelatin-coated 24 well-microwell plates for morphological analysis. EB were dissociated before injection and ES cells were marked with red dye Vybrant® Dil cell-labeling solution (Invitrogen) to facilitate their tracking in injected muscles. This dye efficiently label live cells, and is diluted and disappears with cells proliferation and cellular death.
3.2. Animals, immunosuppression and transplantation
Four to six-week-old
For the transplantation experiments, the
Each animal was injected with 1.0 x 106 ES or EB cells into the gastrocnemius muscle, or through intravenous injections in the tail vein.
Animals were euthanized using CO2 chamber 2 days, 1 week, 2 weeks, 4 weeks and 8 weeks post-transplantation, and muscles were analyzed for dystrophin expression, and identification of the presence of the injected stem cells.
3.3. Tissue processing and dystrophin analysis
Gastrocnemius muscles were dissected and collected from all mice and additionally, several other tissues such as tail, liver and spleen were collected from the mice injected systemically. Samples of the contra lateral muscles were also collected and used as controls in the intramuscular injected mice.All tissue were immediately frozen in liquid nitrogen.For histological analysis, frozen sections of 7 μm were prepared on slides using a cryostat (Zeiss, Jena, Germany).
Cryosections were stained with hematoxilin and eosin or used for immunohistochemistry to evaluate the expression of the muscle proteins. In brief, they were incubated with 1:100 diluted rabbit antibody anti-dystrophin AB 15277 (Abcam) and mouse antibody anti-fetal myosin NCL-MHCd (Novocastra). Cy3-conjugated anti-rabbit and anti-mouse (1:100) were used as secondary antibodies (Sigma-Aldrich). Samples were analyzed under fluorescence microscopy using appropriate filters for the fluorophore.
|Treatment: ES cells|
|Treatment: EB cells|
Total proteins were extracted from muscle and other tissues, separated by 6% SDS-PAGE polyacrylamide gel electrophoresis and were transferred onto nitrocellulose membrane (GE Healthcare) for 60 min at 0.35A at 4ºC. Membranes were then pre-stained in 0.2% Ponceau S, to ensure protein transfer and equal loading of the lanes with protein. Membranes were blocked with 5% nonfat milk in PBS, 0.1% Tween 20 (PBS-T) for 1 h and subsequently incubated with mouse antibody directed against dystrophin VP-D508 (Vector). After an overnight period of incubation with the primary antibody, membranes were washed three times with PBS-T for 10 min. The blots were then reacted with alkaline phosphatase-conjugated secondary antibody (1:1000) for 1 h at room temperature.The detection of protein was done using colorimetric reaction for the enzyme, using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrate (Vainzof et al., 1993).
Data on the injected cells (ES or EB cells), immunosupression and detection of cells both through red dye presence and DNA analysis, as well as screening for dystrophin expression in all injected animals are summarized in Table 3.
To check the ability of these cells to differentiate into new muscle fibers, and to express muscle proteins, we analyzed recipient muscles by immunohistochemistry using an anti-dystrophin antibody. The immunohistochemistry revealed no significant labeling, with only scattered fibers partially labeled, in a pattern observed also in non-injected
|time||red dye||DNA||Dystrophin||time||red dye||DNA||Dystrophin|
|Mdx 19||2 d||+++||-||-|
|Mdx 20||2 w||-||-||-|
|Mdx 21||4 w||-||-||-|
|Mdx 10||1 w||+++||-||-||Mdx 15||4 w||-||-||-|
|Mdx 11||2 w||++||+||-||Mdx 16||8 w||-||-||-|
|Mdx 12||4 w||+||-||-|
|time||red dye||DNA||Dystrophin||time||red dye||DNA||Dystrophin|
|Mdx 22||2 w||-||-||-|
|Mdx 23||4 w||-||-||-|
|Mdx 13||1 w||+ (tail)||-||-||Mdx 17||4 w||-||-||-|
|Mdx 14||2 w||+ (tail)||-||-||Mdx 18||8 w||-||-||-|
3.4. ES and EB cells identification
Genomic DNA was obtained from cells and muscle tissue of injected mice. Fragments of injected and not injected muscles were placed in an extraction solution (100mM Tris HCl pH 8.5; 5mM EDTA, 0.2% SDS; 200mM NaCl), containing 2.5 μl proteinase K (10mg/ml). These samples were kept overnight in dry bath at 55°C. The samples were subjected to centrifugation of 1000g for 10 minutes, and added 500 μl isopropanol in the supernatant. After the new centrifugation of 13500g for 15 minutes, the DNA was precipitated in TE buffer (TRIS-EDTA).The PCR primers used were from the same region of the genome of mice, which produce products of different sizes due to the presence of polymorphic regions, referring to strains of
As almost 100% of the injected ES and EB cells were previously labeled with a red fluorescent dye, we analyzed the presence of these marked cells in histological sections of the injected mice. We identified their presence for longer periods (after 1 week) only in the muscle of the ES injected mice (Table 3 and Fig. 2). This may mean that the ES remained in the injected place.
In the animals Mdx 10, 11 and 12 (local injections of ES cells, after 1, 2 and 4 weeks), we noticed an increase of the muscles in the leg, and the hematoxilin-eosin (H&E) staining showed an intense degeneration and presence of several types of undifferentiated cells (Fig. 3). A histopathological examination of frozen sections identified the proliferation of various types of immature cells, suggesting the formation of a tumor, consistent with the embryonic nature of these cells. But we did not have clear evidences that it was teratoma.
To check if the new generated tissue included differentiation into muscle fibers, we probed the same region described above with the antibodies against fetal myosin. However, there was no immunoreactivity for fetal myosin and no evidence of formation of new fibers (Fig. 3).
3.4.1. ES cell quantification
To investigate the presence and try to quantity of injected ES and EB cells in the muscle we used PCR analysis with specific primers to polymorphic markers of each cell strain. This method detected the presence of stem cells only in the muscle of Mdx 11 (Fig. 4A), who also showed an enlargement of the leg, with significant mass growth.
The experiment revealed a 121 bp band corresponding to the molecular weight of
To estimate the relative amount of the injected cells and verify their possible multiplication in vivo, we performed a standard curve with known concentrations of DNA from the two different mice strains (Fig. 4B). The ES cells strain was only detected starting with the concentration of 30%. In Mdx 11, the number of ES cells in the mice injected muscle were in a similar concentration of the limit of detection of 30% (Fig. 4A).No 142 bp band was observed in other mice, suggesting lower concentration of the injected ES and EB cells in these muscles.
4. Efficient stem cell therapies for dystrophic muscle
Replacement therapies using stem cells are emerging as promising avenues for treatment of genetic diseases because the transplantation of healthy stem cells into affected individuals can potentially rescue defects and injuries.
The identification of mesenchymal stem cells (MSCs) as a sub-population of fibroblast-like adherent cells in the BM opened new perspectives for therapies for the different forma of muscular dystrophies, since these cells were able to differentiate into several other lineages, producing in vitro skeletal muscle, in addition to osteoblasts, chondroblasts, and adipocytes (Friedenstein et al., 1966; Prockop, 1997). Very recently, we showed that hMSC were not rejected when transferred to
Here we show that undifferentiated ES cells hold a better promise of success of retention than differentiated EB cells, since after intramuscular injections, ES cells, but not EB cells, were found in the muscle of animals after 1, 2 and 4 weeks post-injections. Additionally, systemic injection of both ES and EB cells were not directed nor retained by the dystrophic muscles.Two other reports on the use of ES cells in the treatment of DMD showed evidence for engraftment (Bhagavati & Xu, 2005; Darabi et al., 2008). In the first study, the transplantation of EB co-cultured with dissociated skeletal muscle into mdx mice was limited to qualitative detection of donor-derived cells in recipient muscle. However, in the second study, authors showed that paraxial mesoderm is not generated efficiently during ES differentiation induced by conventional protocols, which may further explain our lack of success in integrating ES cells into dystrophic muscles (Darabi et al., 2008).
A second important question relates to the extent to which stem cells are immunogenic, since the safe and successful clinical will be indirectly dependent on the strength of rejection reactions (Mimeault et al., 2007; Trounson 2006). The first studies on the immunologic properties of ES cells and their derivatives have suggested their ability to induce immunologic tolerance (Drukker et al., 2002; Bonde & Zavazava, 2006). Because of this, we investigated the myogenic potential of ES and EB cells when injected into muscle of immunosuppressed mdx mice. Our results showed that the non differentiated ES cells were retained, while pre-differentiated EB cells were all eliminated. Although this could happen because of the non-integration of the EB cells, this could also suggest that even with immunosupression, more differentiated cells may cause a stronger immunological response than undifferentiated ES cells, as also suggested by other group (Drukker et al., 2002). These data are in accordance with recent publication showing that hESCs were highly immunogenic, triggered both cellular and humoral-mediated pathways, and as a result, were rapidly rejected in xenogeneic hosts. These authors observed that combinations of immunosuppressive regimens are necessary for stem cell survival in vivo (Swijnenburg et al., 2008).
Several reports have already described that, when injected sub-cutaneousely into severe combined immunodeﬁcient mice, undifferentiated ES cells will produce teratoma or teratocarcinoma, with derivatives of the three germ layers (Bradley et al., 2002; Mimeault et al., 2007; Reubinoff et al., 2000; Andrews et al., 2005; Wu et al., 2007).Here we observed an increase of the leg of ES cells injected mice. Our histology findings suggest that the donor cells may have multiplied and have been embedded in the mice muscles, in a pattern similar to a teratoma.In one hand, the formation of tumor confirms the ability of these ES injected cells to multiplication in vivo. On the other hand, this new tissue had no muscle characteristics, since no dystrophin, nor developmental myosin positive fibers were identified. Ultimately, it is clear that the signaling environment of the severely dystrophic muscle was not able to induce the differentiation of stem cells into muscle cells.
The use of single injections of stem cells for future therapies in children would have the significant benefit of being less invasive, and less traumatic from a clinical and immunological stand point. But, it also leads to a limitation in the number of injected cells, which can be insufficient to promote an appropriate restoration of the degenerated muscle. However, the finding of the injected ES cells in the recipient muscle, even using only one application of the same quantity of 106 cells, indicates that this number was sufficient for their retention in the injected muscle. Using polymorphic markers for the ES cell line we identified a concentration of at least 30% of these cells in the injected muscle.This result indicates that muscle enlargement observed in injected mice was caused by at least this amount of cells. In the other locally injected mice with leg enlargement, probably a lower amount of these cells were sufficient to promote the tumor, but this amount was not sufficient to allow its detection by our methodology.
Our results suggest that the dystrophic muscle is an adverse ambient for the homing and maintenance of injected stem cells, since when the “still pluripotent” stem cells were injected they formed tumors, andwhen the muscle pre-differentiatedcells were injected they were cleared/migrated out of the tissue. Injected embryonic stem cells, therefore, must receive more or different stimulation from the dystrophic environment to differentiate or to fuse into muscle. Additional studies are necessary to increase the therapeutic potential of these cells in dystrophic murine models.
The authors would like to thank the following researchers, for scientific and technical support: Dra. Mayana Zatz, Dra. Helga C. Silva, Lydia Yamamoto, Dinorah Zilberztajn, Marta Canovas, Patrícia Kossugue, Adriano Senkevics, Priscila Clara Calyjur and André Santos. FVB transgenic mice carrying the enhanced green ﬂuorescent protein (eGFP) were generated and kindly donated by the Transgenic Unit of the Faculty of Medicine of the University of São Paulo. This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo - Centro de Pesquisa, Inovação e Difusão (FAPESP-CEPID), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Instituto Nacional de Ciencia e Tecnologia (INCT), FINEP, and Associação Brasileira de Distrofia Muscular (ABDIM).