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Stem Cell Rev and Rep (2010) 6:560–566DOI 10.1007/s12015-010-9187-5 Human Multipotent Mesenchymal Stromal Cellsfrom Distinct Sources Show Different In Vivo Potentialto Differentiate into Muscle Cells When Injectedin Dystrophic Mice N. M. Vieira & E. Zucconi & C. R. Bueno Jr. & M. Secco &M. F. Suzuki & P. Bartolini & M. Vainzof & M. Zatz # Springer Science+Business Media, LLC 2010 Abstract Limb-girdle muscular dystrophies are a hetero- differentiate in muscle cells in vivo or if this capability is geneous group of disorders characterized by progressive influenced by the niche from where they were obtained. In degeneration of skeletal muscle caused by the absence or order to address this question we injected human derived deficiency of muscle proteins. The murine model of umbilical cord tissue MSCs (hUCT MSCs) into the caudal Limb-Girdle Muscular Dystrophy 2B, the SJL mice, vein of SJL mice with the same protocol previously used carries a deletion in the dysferlin gene. Functionally, this for hASCs; we evaluated the ability of these cells to engraft mouse model shows discrete muscle weakness, starting at into recipient dystrophic muscle after systemic delivery, to the age of 4–6 weeks. The possibility to restore the express human muscle proteins in the dystrophic host and expression of the defective protein and improve muscular their effect in functional performance. These results are of performance by cell therapy is a promising approach for great interest for future therapeutic application.
the future treatment of progressive muscular dystrophies(PMD). We and others have recently shown that human Keywords Human multipotent mesenchymal stromal adipose multipotent mesenchymal stromal cells (hASCs) cells . Xenotransplantation . Muscular dystrophy . Therapy can differentiate into skeletal muscle when in contact withdystrophic muscle cells in vitro and in vivo. Umbilicalcord tissue and adipose tissue are known rich sources of multipotent mesenchymal stromal cells (MSCs), widelyused for cell-based therapy studies. The main objective of Multipotent mesenchymal stromal cells (MSCs) are potentially the present study is to evaluate if MSCs from these two useful for therapeutic approaches as well as models for different sources have the same potential to reach and developmental biology studies. MSC can be isolated fromdifferent tissues, such as adipose tissue, dental pulp, placenta,umbilical cord and fallopian tube []. However an N. M. Vieira and E. Zucconi contributed equally for this work.
important question is whether MSCs from different sourcesare comparable in their differentiation potential in vivo or N. M. Vieira : E. Zucconi : C. R. Bueno Jr. : M. Secco :M. Vainzof : M. Zatz (*) whether this potential is influenced by the niche from where Human Genome Research Center, Institute of Biosciences, Progressive muscular dystrophies (PMD) are a clinically Rua do Matão, n.106—Cidade Universitária, and genetically heterogeneous group of disorders caused by São Paulo, SP CEP: 05508-090, Brazile-mail: mayazatz@usp.br the deficiency or abnormal muscle proteins, resulting inprogressive degeneration and loss of skeletal muscle function.
As effective treatments for these diseases are still unavailable, Biotechnology Department, National Nuclear Energy they have been widely investigated as possible candidates for Among the different forms, the Limb Girdle Muscu- lar Dystrophies (LGMDs) constitute a sub-group char-acterized by the involvement of the pelvic and shouldergirdle musculature. A 171-bp in-frame deletion in themurine dysferlin cDNA was identified in a mousemodel, the SJL mice, with a corresponding reduction indysferlin levels to 15% of normal []. The SJL micedeletion is in-frame, and therefore does not cause a totalabsence of the protein.
The continuous and gradual muscle degeneration in PMDs leads to a depletion of satellite cells and,consequently, the capability to restore the skeletalmuscle is lost [, ]. Different sources of stem/progenitorcells that show extended proliferation in vitro and alsohave the ability to generate normal muscle fibers in vitroand in vivo have been described in several publications , –].
We recently showed that human adipose-derived stro- mal cells (hASCs) can differentiate into skeletal musclewhen in contact with dystrophic muscle cells in vitro and in vivo In addition we also observed that the humanumbilical cord tissue (hUCT) is a much richer source of Fig. 1 a Polymerase chain reaction analysis for human chromosome 7α-satellite sequences (H7) and mouse chromosome 8 centromeric MSC than umbilical cord blood and that they have repeat sequence (M8) of SJL mice. Muscles of the injected SJL mice, different expression profiles ]. However it is not known if samples shown are the following: 1–Distal foreleg muscle; 2– all MSCs show the same capacity in vivo. Do MSCs from Proximal foreleg muscle; 3–Distal hindleg muscle; 4–Proximal hind- adipose and umbilical cord tissue have the same potential to leg muscle; H–Human DNA; M–Mouse DNA. b Western blot analysisfor human-dystrophin of the muscles of two injected animals. Samples reach and differentiate into muscle cells in vivo? Or, this shown are the following: 1–Distal foreleg muscle; 2–Proximal foreleg capability is influenced by the niche from where they were muscle; 3–Distal hindleg muscle; 4–Proximal hindleg muscle; H– Human muscle protein; M–Mouse muscle protein. Myosin = myosin In order to address this question we have injected hUCT band detected in the Ponceau S pre-stained blot, for the evaluation ofloaded muscle proteins MSCs intravenously into the SJL mice, aiming to comparetheir potential to differentiate into skeletal muscle with ourprevious data with hASCs []. Differently from hASCs, Muscle Differentiation in the Host Muscle hUCT MSCs reached the muscle but did not differentiateinto muscle cells. These results suggest that according to the To explore the myogenic differentiation followed by the source from which MSCs were obtained they may show a engraftment of hUCT MSCs we analyzed the expression of greater potential to differentiate into determined cell lineages.
dysferlin and human-dystrophin in the host muscle.
This may have important implications depending on the The analysis of dysferlin is not sufficient to infer if the injected muscles are expressing human or mouse proteins]. Therefore, we assessed the presence of human-dystrophin, using a specific anti-human-dystrophin anti-body [Through western blot (WB) analysis, no human dystrophin was found in the muscles of the injected animals(Fig. hUCT MSCs Capacity to Reach and Engraft at the HostMuscle In order to assess the potential of hUCT MSCs to reach and We performed three standardized motor ability tests colonize the host muscle we injected undifferentiated, and compared the performance of each SJL mouse previously characterized, hUCT MSCs, into the caudal (injected and uninjected) before (2-months of age) and vein of SJL mice (n=7). PCR analysis detected human after (9-months of age) the injections period, in blind test DNA in the foreleg and hindleg muscles of all seven (Table We observed that for the tests that required trunk strength (inclined plane and wire hanging tests) the Table 1 Results of 3 motor ability tests in injected (n =7) and uninjected mice (n=7) before and after 6 months of injection At the inclined plane test the uninjected animals worsened their performance (p=0.008, t-Student test, n=7) while in the injected animals it did notdiffer (p=0.33, t-Student test, n=7) For the wire hanging test the uninjected animals worsened their performance (p=0.0012, t-Student test, n=7) while the injected animals showedno significant difference (p=0.07, t-Student test, n=7) At the ambulation test there was no difference in the performance of uninjected animals (p=0.11, t-Student test, n=7) and injected animals (p=0.16, t-Student test, n=7) after the injection period uninjected animals showed a significantly worse perfor- mice (30–90%) ]. Since the immunosupressive drug mance while in the injected animals there were no efficiently controlled the humoral and cellular immune statistically significant changes (Table The deambula- reactions, the authors concluded that the immune rejection tion test did not show a significant difference before and is not the cause of the low myoblast transplantation success after the injections period in both groups.
We have previously shown that systemic delivery of hASCs into the SJL mice, without immunosuppression, resulted in human muscle proteins expression in the hostmuscles and functional amelioration [ The successful use of stem cells for clinical applications in MSCs may be found in different tissues which are therapy for PMD requires the finding of a rich and easily obtainable source of cells, which must have the ability to important question to be addressed is whether stem-cells reach the entire body musculature, engraft and restore the obtained from different sources have the same potential to defective protein in the dystrophic muscle.
differentiate into different cell lineages or if there is already Sampaolesi et al (2006) ] reported that systemic a pre-commitment depending on the niche from which they injections of normal dog mesoangioblasts to the muscle of dystrophic dogs resulted in the restoration of dystrophin Since umbilical cord is a rich source of MSCs, we expression. However all transplanted dogs were maintained investigated their ability to originate muscle proteins and on steroids and received immunosuppressant drugs, which ameliorate functional parameters using the same animal makes difficult to evaluate functional results, since it is model and methodology proved to be successful in our known that immunosuppressive and anti-inflammatory drugs can ameliorate the phenotype in muscular dystrophy DNA analysis showed that the hUCT MSCs were able to reach the host muscle through systemic delivery. However Leriche-Guérin et al. (2002) ] investigated the effect we did not find human muscle proteins in the same muscle of myoblast transplantation into the SJL mice muscle with samples where the human DNA was present.
immunosupression. The percentage of dysferlin positive The functional ability in the previous and current study labeled fibers obtained in their study was lower than the was evaluated by standardized motor ability tests [ percentage of dystrophin-positive fibers usually observed ]. However, for the SJL model, the most affected muscles following the transplantation of normal myoblasts in mdx are the ones that are responsible for trunk strength [].
In opposition to our previous study with hASCs [] the In short, here we compared, for the first time, the ability injected animals with hUCT MSCs did not show clinical of MSCs obtained from human umbilical cord tissue and improvement, but, surprisingly, the performance of non- adipose tissue to engraft into recipient dystrophic muscle injected animals was significantly worse than in the “treated” after systemic delivery; express human muscle proteins in animals. The mice from the latter group maintained their the dystrophic host and their effect in functional perfor- performance at the end of the injection period, in particular mance using the same animal model and protocol. Our for the wire hanging test, which requires most trunk strength, results showed that although umbilical cord MSCs appar- suggesting an apparent stabilization of the dystrophic ently do not have the same potential to differentiate in process. That is, even without differentiating in muscle cells, human muscle proteins in vivo as hASCs they were able to the injected hUCT MSCs may have a positive effect when reach the muscle and showed an apparent therapeutic interacting with the host muscle. Indeed there are growing benefit in injected animals as compared to the control evidences in the literature describing the immunosuppressive group, probably due to their immunomodulatory effect.
properties of MSCS [Inflammatory infiltration is The present investigation suggests that although MSC from observed in the dystrophic muscle but little is known about different sources show apparently similar properties in vitro the mechanisms involved in mesenchymal immunomodula- they may be more or less efficient to differentiate into specific tion. It is possible that secreted known cytokines factors cell lineages in vivo according to the niche from where they (TNF-α, IFN-γ and IL-12) could act, by protecting the were obtained. Preclinical studies in different animal models, dystrophic muscle. Several authors showed that mesenchy- which are currently underway, will be essential to corroborate mal stem cells suppress proliferation of activated lympho- the present observations, which will have important implica- cytes in vitro in a dose-dependent, non-HLA-restricted tions aiming future cell therapy replacement.
manner [–]. Antibody-mediated depletion of CD4+and CD8+ T cells in mdx mice has been found to result ina reduction in muscle pathology []. MSCs are also being tested in clinical trials aiming to ameliorate graft-versus-hostdisease after haemopoietic-stem-cell transplantation in humans ]. Therefore, the immunomodulation effect ofMSCs in patients affected by progressive muscular dystro- This study was approved by the human research ethics phies could be a promising additional benefit to cell therapy.
committee (Comitê de ética em pesquisa—seres humanos— Although MSCs from different sources show similar ability CEP) and by the animal research ethics committee (Comissão to differentiate into muscle cells in vitro [, , ] preclinical de ética no uso de animais em experimentação—CEUA) of studies are of utmost importance to verify it this also happens Institute of Bioscience and University Hospital of University in vivo. The apparent greater potential of adipose tissue than of São Paulo. hUCT MSCs were collected from donated umbilical cord derived MSCs to differentiate into muscle umbilical cord units (UC), after all mothers signed the writhen cells here observed could be explained by a recently informed consent, in accordance with the ethical committee of described population of mesenchymal progenitors, distinct Institute of Bioscience and University Hospital of University from satellite cells, in the skeletal muscle These of São Paulo (CEP), permit number 040/2005. SJL mice were progenitors have many similarities with hASCs and accord- purchased from the Jackson Laboratory. Animal care and ing to the authors they may have the same origin. These cells experiments were performed in accordance with the animal do not generate myofibers but enhance the rate of differen- research ethics committee (CEUA) of the Biosciences tiation of primary myogenic progenitors, and have adipo- Institute, University of São Paulo, permit number 034/2005.
genic differentiation potential both in vitro and in vivo. Theinteraction between muscle cells and these mesenchymal progenitors has a considerable impact on muscle homeostasissince adipogenesis is strongly inhibited by the presence of UCs were filled with 0.1% collagenase (Sigma-Aldrich, satellite cell-derived myofibres ]. It remains unclear however which cell population participates in the regenera- incubated at 37°C for 20 min. Each UC was washed with tion process by fusing to the degenerated myotubes or proliferation medium (DMEM low glucose, 10% fetal forming new myofibers. The identification of this sub- bovine serum), and the detached cells were harvested after population will be extremely important for the establishment gentle massage of the UC. Cells were centrifuged at 300 g of clinical trial protocols. Interestingly, it has been recently for 10 min, resuspended in proliferation medium, and shown that there is an epigenetic memory in induced seeded in 25-cm2 flasks at a density of 5 × 107 cells per ml.
pluripotent stem-cells according to the tissue of origin After 24 h of incubation, non-adherent cells were removed which might occur also with adult MSCS derived cells.
were fixed with 4% paraformaldehyde for 30 min, washed, andstained with a working solution of 0.16% oil red O for 20 min.
To analyze cell-surface expression of specific markers,adherent cells were incubated with the following anti-human primary antibodies: CD29-PECy5, CD34-PerCP, CD31-phycoerythrin (PE), CD45-fluorescein isothiocyanate (FITC), A pellet culture system was used for chondrogenesis.
CD90-R-PE, CD73-PE, CD13-PE, CD44-PE, CD117-PE, Cells (2,5×105) were centrifuged in a 15-ml polypropylene human leukocyte antigen (HLA)-ABC-FITC, HLA-DR-R-PE tube at 500 g for 5 min, and the pellet was resuspended in (Becton, Dickinson and Company, Franklin Lakes, NJ, 10 ml of basal medium consisting of DMEM-LG supple- ). A total of 10,000 labeled cells were analyzed mented with 100 nM dexamethasone, 50 μM ascorbic acid-2 using a Guava EasyCyte flow cytometer running Guava phosphate (Sigma-Aldrich), 1 mM sodium pyruvate ExpressPlus software (Guava Technologies Hayward, CA, (Invitrogen-Gibco), and 1% ITS-Premix (Becton Dickinson).
Without disturbing the pellet, cells were resuspended in 0.5 mlof chondrogenic differentiation medium consisting of basal medium supplemented with 10 ng/ml transforming growthfactor-B1 (R&D Systems Inc., Minneapolis, The evaluation of MSCs properties included immunopheno- ). On day 1, tubes were flipped gently to typing by flow cytometric analysis, using a panel of surface acquire a single floating cell sphere. Medium was changed markers. hUCT MSCs were negative for CD31 (endothelial every 3-4 days, and cells were fixed on day 21 with 4% cell marker), CD34, CD45, CD117 (hematopoietic cell paraformaldehyde. Cryosections (10 um thick) were stained markers), and HLA-DR (human leukocyte differentiation with toluidine blue to demonstrate extracellular matrix antigen class II), whereas they were positive for CD29, CD44 (adhesion markers), CD90, CD73, CD13 (mesenchy-mal markers), and HLA-ABC (human leukocyte differentia- tion antigen class I) [] (data not shown).
The plasticity of hUCT MSCs was assessed by in vitro To promote osteogenic differentiation, subconfluent cells differentiation capacity, after three weeks of lineage were treated with proliferation medium supplemented with induction Myogenic, adipogenic, chondrogenic and 50 μM ascorbate-2 phosphate, 10 mM B-glycerophosphate osteogenic differentiation was demonstrated by the expres- (Sigma-Aldrich) and 0.1 μM dexamethasone, for 21 days.
sion of myogenic markers (myosin and desmin), lipid Osteogenesis was demonstrated by accumulation of mineral- vacuoles, mucopolysaccharide-rich extracellular matrix and ized calcium phosphate assessed by von Kossa stain. Briefly, calcium deposits, respectively. These results confirmed the cells were stained with 1% silver nitrate (Sigma-Aldrich) for mesenchymal nature of the isolated cells as well as their 45 min under ultraviolet light, followed by 3% sodium multipotent potential (data not shown).
thiosulfate (Sigma-Aldrich) for 5 min, and then counterstainedwith van Gieson stain.
To evaluate MSCs properties, hUCT MSCs (third passage,at 80%–90% confluence) were subjected to adipogenic, Fourteen two-months SJL mice were divided into two groups chondrogenic, myogenic, and osteogenic differentiation in of 7: transplanted animals (group A) and control group B vitro, according to established protocols []. Normal human (uninjected animals). Each animal from group A was injected dermal fibroblasts were used as a negative control in the in the tail vein with 1 ✕ 106 of hUCT MSC in 0.1 ml of Hank’s Buffered Salt Solution (HBSS). The animals wereinjected for 6 months, weekly in the first month and then monthly. All results were analyzed blindly. The code foreach of the mice groups was disclosed only after completion Subconfluent cells were cultured in proliferation medium of all the studies. Two months after the last cell transplan- supplemented with 1 μM dexamethasone (Sigma-Aldrich), tation, the animals were euthanatized using a CO2 chamber.
500 μM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 60 μMindomethacin (Sigma-Aldrich), and 5 μg/ml insulin (Sigma- Aldrich). Adipogenic differentiation was confirmed on day 21by intracellular accumulation of lipid-rich vacuoles stainable The DNA was obtained using DNeasy Blood & Tissue Kit with oil red O (Sigma-Aldrich). For the oil red O stain, cells (Qiagen). The presence of human DNA in the host samples were evaluated as described in Pelz et al (2005) ].
significance level of p=0.05 and the results were expressed Centromeric region of human chromosome 7 and mice by the percentage variation between their performance chromosome 8 was amplified by PCR (35 cycles, annealing before and after hUCT MSCs transfer period.
at 59°C). The PCR products were separated by electropho-resis on 2% agarose gels and stained with ethidium Marcos Valadares, Tatiana Jazedje, Amanda Assoni, Mayra Pelatti, bromide. Non-saturated digital images were obtained using Juliana Gomes, Gabriela Polster, Camila Almeida, Agnes Nishimura, an ImageQuant imaging system (GE HealthCare).
Natale Cavaçana, Miguel Mitne-Neto, Monize Lazar, Constancia Urbani,David Schlesinger, Daniela Bueno, Roberto Fanganiello, Antonia M P Cerqueira, Marta Canovas, Paula Onofre and Dr. Maria Rita Passos-Bueno for helpful suggestions. We thank Dr. Glenn Morris from theCenter for Inherited Neuromuscular Disease (CIND), RJAH Orthopaedic Muscle sample proteins were extracted through treatment with Hospital, Oswestry, Shropshire, UK for providing anti-human dystrophin a buffer containing 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 and 60 mM octylglucoside.
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