[Show abstract][Hide abstract] ABSTRACT: The functional and architectural benefits of embryonic stem cells (ESC) and myoblasts (Mb) transplantations into infarcted myocardium have been investigated extensively. Whereas ESC repopulated fibrotic areas and contributed to myocardial regeneration, Mb exerted their effects through paracrine secretions and scar remodeling. This therapeutic perspective, however, has been less explored in the setting of nonischemic dilated cardiomyopathies (DCMs). Our aim was to compare the integration and functional efficacy of ESC committed to cardiac fate by bone morphogenic protein 2 (BMP-2) pretreatment and Mb used as gold standard following their transplantation into the myocardium of a mouse model of laminopathy exhibiting a progressive and lethal DCM. After 4 and 8 weeks of transplantation, stabilization was observed in Mb-transplanted mice (P = 0.008) but not in groups of ESC-transplanted or medium-injected animals, where the left ventricular fractional shortening (LVFS) decreased by 32 ± 8% and 41 ± 8% respectively. Engrafted differentiated cells were consistently detected in myocardia of mice receiving Mb, whereas few or no cells were detected in the hearts of mice receiving ESC, except in two cases where teratomas were formed. These data suggest that committed ESC fail to integrate in DCM where scar tissue is absent to provide the appropriate niche, whereas the functional benefits of Mb transplantation might extend to nonischemic cardiomyopathy.Molecular Therapy (2013); doi:10.1038/mt.2013.15.
[Show abstract][Hide abstract] ABSTRACT: Duchenne muscular dystrophy (DMD) is a genetic progressive muscle disease resulting from the lack of dystrophin and without effective treatment. Adult stem cell populations have given new impetus to cell-based therapy of neuromuscular diseases. One of them, muscle-derived stem cells, isolated based on delayed adhesion properties, contributes to injured muscle repair. However, these data were collected in dystrophic mice that exhibit a relatively mild tissue phenotype and clinical features of DMD patients. Here, we characterized canine delayed adherent stem cells and investigated the efficacy of their systemic delivery in the clinically relevant DMD animal model to assess potential therapeutic application in humans. Delayed adherent stem cells, named MuStem cells (muscle stem cells), were isolated from healthy dog muscle using a preplating technique. In vitro, MuStem cells displayed a large expansion capacity, an ability to proliferate in suspension, and a multilineage differentiation potential. Phenotypically, they corresponded to early myogenic progenitors and uncommitted cells. When injected in immunosuppressed dystrophic dogs, they contributed to myofiber regeneration, satellite cell replenishment, and dystrophin expression. Importantly, their systemic delivery resulted in long-term dystrophin expression, muscle damage course limitation with an increased regeneration activity and an interstitial expansion restriction, and persisting stabilization of the dog's clinical status. These results demonstrate that MuStem cells could provide an attractive therapeutic avenue for DMD patients.
American Journal Of Pathology 09/2011; 179(5):2501-18. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Islet-1 expression identifies populations of progenitor cells in embryonic, fetal, and newborn murine hearts that are able to give rise to all cardiac cell lineages ex vivo and in vivo. Using systematic immunohistochemistry, we investigated whether islet-1-positive cells are present in adult mouse heart from the perspective of their potential therapeutic utility. The presence, localization, and nature of islet-1-positive cells were assessed in mice of different strains, ages, and conditions. Islet-1-positive cells were present in mouse heart from postnatal day 1 to young adulthood. Depending on the strain, these cells were organized in either 1 or 2 types of clusters localized to restricted areas, at a distance of 6%-35% of the heart length from the base. The first type of cluster was present in all strains and consisted of neural crest-derived cells that formed cardiac ganglia. The number of cells remained stable (a few hundred) from neonatal up to adult ages, and variations were noted between strains regarding their long-term persistency. The second type of cluster was essentially present in 129SvJ or Balb/C strains and absent from the other strains tested (C57BL/6J, C3H, SJL). It consisted of cells expressing highly ordered sarcomeric actin, consistent with their having cardiomyocyte identity. These cells disappeared in animals older than 4 months. Neither the number nor the type of islet-1-positive cells varied with time in a mouse model of dilated cardiomyopathy. Our studies demonstrate that islet-1-positive cells are relatively few in number in adult murine heart, being localized in restricted and rather inaccessible areas, and can represent both neural crest and cardiomyocyte lineages.
Stem cells and development 10/2010; 20(6):1043-52. · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human skeletal muscle is an essential source of various cellular progenitors with potential therapeutic perspectives. We first used extracellular markers to identify in situ the main cell types located in a satellite position or in the endomysium of the skeletal muscle. Immunohistology revealed labeling of cells by markers of mesenchymal (CD13, CD29, CD44, CD47, CD49, CD62, CD73, CD90, CD105, CD146, and CD15 in this study), myogenic (CD56), angiogenic (CD31, CD34, CD106, CD146), hematopoietic (CD10, CD15, CD34) lineages. We then analysed cell phenotypes and fates in short- and long-term cultures of dissociated muscle biopsies in a proliferation medium favouring the expansion of myogenic cells. While CD56(+) cells grew rapidly, a population of CD15(+) cells emerged, partly from CD56(+) cells, and became individualized. Both populations expressed mesenchymal markers similar to that harboured by human bone marrow-derived mesenchymal stem cells. In differentiation media, both CD56(+) and CD15(+) cells shared osteogenic and chondrogenic abilities, while CD56(+) cells presented a myogenic capacity and CD15(+) cells presented an adipogenic capacity. An important proportion of cells expressed the CD34 antigen in situ and immediately after muscle dissociation. However, CD34 antigen did not persist in culture and this initial population gave rise to adipogenic cells. These results underline the diversity of human muscle cells, and the shared or restricted commitment abilities of the main lineages under defined conditions.
Experimental Cell Research 09/2010; 316(15):2513-26. · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Muscular dystrophies are often associated with significant cardiac disease that can be the prominent feature associated with gene mutations in sarcoglycan. Cardiac cell death is a main feature of cardiomyopathy in sarcoglycan deficiency and may arise as a cardiomyocyte intrinsic process that remains unclear. Deficiency of delta-sarcoglycan (delta-SG) induces disruption of the dystrophin-associated glycoprotein complex, a known cause of membrane instability that may explain cardiomyocytes cytosolic Ca2+ increase. In this study we assessed the hypothesis that cytosolic Ca2+ increase triggers cardiomyocyte death through mitochondrial Ca2+ overload and dysfunction in the delta-SG-deficient CHF147 hamster. We showed that virtually all isolated CHF147 ventricular myocytes exhibited elevated cytosolic and mitochondrial Ca2+ levels by the use of the Fura-2 and Rhod-2 fluorescent probes. Observation of living cells with Mito-Tracker red lead to the conclusion that approximately 15% of isolated CHF147 cardiomyocytes had disorganized mitochondria. Transmission electron microscope imaging showed mitochondrial swelling associated with crest and membrane disruption. Analysis of the mitochondrial permeability transition pore (MPTP) activity using calcein revealed that mitochondria of CHF147 ventricular cells were twofold leakier than wild types, whereas reactive oxygen species production was unchanged. Bax, Bcl-2, and LC3 expression analysis by Western blot indicated that the intrinsic apoptosis and the cell death associated to autophagy pathways were not significantly activated in CHF147 hearts. Our results lead to conclusion that cardiomyocytes death in delta-SG-deficient animals is an intrinsic phenomenon, likely related to Ca2+-induced necrosis. In this process Ca2+ overload-induced MPTP activation and mitochondrial disorganization may have an important role.
[Show abstract][Hide abstract] ABSTRACT: Aldehyde dehydrogenase 1A1 (ALDH) activity is one hallmark of human bone marrow (BM), umbilical cord blood (UCB), and peripheral blood (PB) primitive progenitors presenting high reconstitution capacities in vivo. In this study, we have identified ALDH(+) cells within human skeletal muscles, and have analyzed their phenotypical and functional characteristics. Immunohistofluorescence analysis of human muscle tissue sections revealed rare endomysial cells. Flow cytometry analysis using the fluorescent substrate of ALDH, Aldefluor, identified brightly stained (ALDH(br)) cells with low side scatter (SSC(lo)), in enzymatically dissociated muscle biopsies, thereafter abbreviated as SMALD(+) (for skeletal muscle ALDH(+)) cells. Phenotypical analysis discriminated two sub-populations according to CD34 expression: SMALD(+)/CD34(-) and SMALD(+)/CD34(+) cells. These sub-populations did not initially express endothelial (CD31), hematopoietic (CD45), and myogenic (CD56) markers. Upon sorting, however, whereas SMALD(+)/CD34(+) cells developed in vitro as a heterogeneous population of CD56(-) cells able to differentiate in adipoblasts, the SMALD(+)/CD34(-) fraction developed in vitro as a highly enriched population of CD56(+) myoblasts able to form myotubes. Moreover, only the SMALD(+)/CD34(-) population maintained a strong myogenic potential in vivo upon intramuscular transplantation. Our results suggest that ALDH activity is a novel marker for a population of new human skeletal muscle progenitors presenting a potential for cell biology and cell therapy.
[Show abstract][Hide abstract] ABSTRACT: 1H-NMR (nuclear magnetic resonance) imaging is regularly proposed to non-invasively monitor cell therapy protocols. Prior to transplantation, cells must be loaded with an NMR contrast agent (CA). Most studies performed so far make use of superparamagnetic iron oxide particles (SPIOs), mainly for favorable detection sensitivity. However, in the case of labeled cell death, SPIO recapture by inflammatory cells might introduce severe bias. We investigated whether NMR signal changes induced by preloading with SPIOs or the low molecular weight gadolinium (Gd)-DTPA accurately monitored the outcome of transplanted cells in a murine model of acute immunologic rejection. CA-loaded human myoblasts were grafted in the tibialis anterior of C57BL/6 mice. NMR imaging was repeated regularly until 3 months post-transplantation. Label outcome was evaluated by the size of the labeled area and its relative contrast to surrounding tissue. In parallel, immunohistochemistry assessed the presence of human cells. Data analysis revealed that CA-induced signal changes did not strictly reflect the graft status. Gd-DTPA label disappeared rapidly yet with a 2-week delay compared with immunohistochemical evaluation. More problematically, SPIO label was still visible after 3 months, grossly overestimating cell survival (<1 week). SPIOs should be used with extreme caution to evaluate the presence of grafted cells in vivo and could hardly be recommended for the long-term monitoring of cell transplantation protocols.
[Show abstract][Hide abstract] ABSTRACT: Several hamster strains are commonly used as models for cardiomyopathic phenotypes evolving toward heart failure. However, little is known about heart rate variability (HRV) in this species. Prolonged surface ECG recording, a prerequisite to HRV studies, can be obtained either by telemetry or by restraints. Here, we performed long time ECG recording using telemetry on young adult Syrian hamsters and we analyzed time series of interbeat intervals. Standard statistics showed that the mean of normal R-R intervals slightly increased with age, with standard deviation of normal R-R intervals remaining stable over time. However, time domain analysis using Poincaré plots revealed dynamic changes in the HRV. Analysis of frequency domains revealed that the ratio of spectral components (low frequency/high frequency) exhibited a maturation pattern. Thus refined analysis of HRV revealed a more complex pattern than common statistical analysis would translate. Unlike other rodents, hamsters display a great spontaneous variability of their heart rate. As the complexity canvas of HRV might be the consequence of extracardiac regulation factors, we assessed the sympathovagal balance in both time and frequency domain of heart rate. Pharmacological tests revealed that both sympathetic and vagal tones contribute to HRV in Syrian hamsters. Thus Syrian hamsters have a broad intrinsic HRV with large influences of the neurovegetative system. However, the influence of the previous beat seems to prevail over the autonomic oscillators. These animals present a high sensitivity to artificially altered cardiac regulation and might be great models for the diagnosis of early alterations in the HRV related to pathology. Therefore, Syrian hamsters represent a unique model for HRV studies.
[Show abstract][Hide abstract] ABSTRACT: Human skeletal muscle is an essential source of cellular progenitors with potential therapeutic perspectives deserving further identification and characterization. Aldehyde dehydrogenase type 1A1 (ALDH) belongs to a large family of enzymes involved in oxidation and detoxification, and its activity is one hallmark of human primitive progenitors presenting broad regeneration capacities in vivo. We investigated for the presence and capacities of cell populations expressing ALDH in human skeletal muscles. ALDH expression was assessed by immunohistochemistry, and its activity was revealed and quantified using the reliable fluorescent substrate Aldefluor®, which allowed cell sorting by flow cytometry. The phenotypic and differentiation capacities of different sub-populations of ALDH+ cells were assessed in vitro and in vivo upon intramuscular implantation. Immunohistofluorescence study identified rare endomysial cells positively labeled for ALDH1. Using flow cytometry, we described welldefined sub-populations of SSClo/ALDHbr cells isolated from bulk dissociated human skeletal muscle biopsies, that we called SMALD (Skeletal Muscle ALDH-positive cells). Two main sub-populations were discriminated according to CD34 expression, and termed SMALD/34- and SMALD/34+ cells. These sub-populations did not express endothelial (CD31), hematopoietic (CD45) and myogenic (CD56) markers, and could be partly discriminated by the variable expression of associated markers (CD44, CD90, CD105, CD140b). Therefore, these cells may be distinct from satellite cells, pericytes or mesangioblasts, myo-endothelial, or hematopoietic stem cells. Upon sorting, SMALD populations were expanded and their differentiation abilities were compared in vitro. While SMALD/34+ cells developed as an heterogeneous population of CD56- and CD15+ cells able to differentiate in adipoblasts, the SMALD/34- cells developed in vitro as a highly enriched population of CD56+ /CD15- myoblasts able to form myotubes. Interestingly, the initial proportion of SMALD/34- cells in a given biopsy was a predictor of its final yield in myogenic cells. In vivo, FACS-sorted SMALD/34- cells participated efficiently to muscle regeneration upon intramuscular transplantation in SCID mice, while SMALD/34+ cells did not. SMALD cells may thus become a new player in the field of muscle homeostasis or repair in therapeutic perspectives.
[Show abstract][Hide abstract] ABSTRACT: Myoblast transplantation is being considered as a potential strategy to improve muscle function in myopathies; hence, it is important to identify the transplanted cells and to have available efficient reagents to track these cells. We first validated a human to mouse xenotransplantation model warranting the complete and rapid rejection of the cells. We then used this model to assess the appropriateness of a nanoparticle reagent to track the transplanted cells. Human myoblasts were loaded with ferrite nanoparticles and injected into the tibialis muscle of immunocompetent mice. Upon collection and histological analysis of muscle sections at different time points, we observed the total disappearance of the human cells within 6 days while ferrite particles remained detectable and colocalized with mouse infiltrating and neighboring cells at the injection site. These results suggest that the use of exogenous markers such as ferrite nanoparticles may lead to false-positive results and misinterpretation of cell fate.
[Show abstract][Hide abstract] ABSTRACT: In vivo electrophysiology remains a suitable method to monitor cardiac activity; however, surface electrocardiogram (ECG) monitoring remains complicated in the case of small animals. Sedation has helped to maintain the animal still; however, it is known that anesthetic drugs impair the regulation of the cardiac electrical activity. To circumvent this problem, ECG monitoring using telemetry or restraints has been developed. This study reports a new methodology, based on a restraining system without further sedation, for recording ECGs on small animal models. We investigated its efficacy in Syrian hamsters and in several strains of mice, and we compared these data to those obtained with telemetry devices. We show that this new system can easily be used in animals of different sizes ranging from adult hamsters to newborn mice. When compared to telemetry, this restrained ECG monitoring method shows a very good yield, as 65% of total beats can be used for further analysis. When recorded in the same animals, RR intervals distributions are identical for both techniques. In conclusion, this restrained ECG monitoring technique is a well-suited tool for exploring various aspects of cardiac electrophysiology in a wide variety of small animals including very young mice.
Pflügers Archiv - European Journal of Physiology 05/2007; 454(1):165-71. · 4.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inherited cardiopathies are leading to life-threatening conditions such as heart failure. Moreover, treatments currently available fail in altering the cardiac phenotype. Thus, gene therapy appears as an attracting alternative to conventional treatments. However, gene delivery remains a major hurdle in achieving this goal. To obtain regional delivery of plasmid DNA, intrapericardial administration seems to be an interesting approach. In order to improve retention time at the site of injection, formulations based on a thermosensitive gel of Poloxamer 407 were assessed. Protection and condensation of plasmid DNA was initially performed through complexation with polyethyleneimine (PEI), a widely used polymer. Characterization of the size and zeta potential of the complexes suggested interactions between the polyplexes and the Poloxamer gel through significant increase of the size of the polyplexes and shielding of the surface charges. In vivo evaluation has highlighted the toxicity of PEI/DNA polyplexes toward the myocardium. However, feasibility of intrapericardial injection of Poloxamer based formulations as well as their very low toxicity has been established.
International Journal of Pharmaceutics 04/2007; 331(2):220-3. · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The hamster strain CHF147 presents a progressive dilated cardiomyopathy (DCM) due to a large deletion of the delta-sarcoglycan gene that leads to heart failure. This cardiomyopathy induces premature death. We have previously shown that a short-term treatment using IGF-1 preserves cardiac structure and improves function of the CHF147 hamster.
In the current study, we measured long-term effects of short-term treatment with recombinant human IGF-1 (rhIGF-1) in CHF147 hamsters. CHF147 hamsters (7-8 months old) were implanted under the skin with an osmotic pump filled either with saline or with recombinant human IGF-1 at a total dose of 25 microg. The osmotic pump allowed a continuous delivery of the protein for a mean duration of 19 days.
We observed a significant increase in overall survival, as well as preservation of cardiac function, in the rhIGF-1-treated group. At the time of death, hearts of treated animals did not present any macroscopical or histological differences compared to those of sham hamsters. These results show that rhIGF-1 treatment slows down the evolution of the DCM in the CHF147 hamster. Moreover, the low dose treatment did not increase IGF-1 serum levels.
This study is the first one reporting beneficial effects of IGF-1 treatment on survival of an animal model presenting DCM. Our results raise hopes for a new therapeutic approach of this pathology.
The Journal of Gene Medicine 09/2006; 8(8):1048-55. · 2.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: So far gene transfer to the muscle has mainly been based on viral vectors, given their efficiency to transfer DNA in vivo. However, virus-derived vectors have numbers of limitations, such as insert size, tissue specificity or immunogenicity, therefore restricting the possibilities of repeated administrations. After intramuscular injection, major hurdles remain tissue diffusion and intracellular entry.To improve these parameters with reference to naked DNA, our approach consisted in designing synthetic vectors. The first step intended to condense and protect plasmid DNA (pCMV-βGal, Invitrogen) through its association with various polymers differing by their charge, i.e. Polyethyleneimine (PEI, Sigma), Tetronic 304 (BASF) and PE6400 (BASF). For each polymer/DNA formulation, the morphological properties of the vectors were assessed by cryo- Transmission Electron Microscopy, their size by Dynamic Light Scattering and their zeta potential by Laser Doppler Velocimetry. Characterization revealed a great diversity of objects in terms of size, shape and zeta potential.In vivo toxicity and efficiency of the systems were also evaluated after intramuscular injections into tibialis anterior and quadriceps muscles of wild type Syrian hamsters. X-Gal revelation and Haematein/Eosin staining were then performed on serial sections of each muscle. These experiments highlighted the extremely high cytotoxicity of PEI/DNA complexes towards skeletal muscle. On the contrary, no significant lesions were detected after injection of PE6400/DNA or Tetronic/DNA formulations. Both systems did significantly improve transfection with reference to naked DNA.In order to promote cellular entry of the DNA, a second step in our study consisted in combining the previous polymeric vectors with randomly methylated beta-cyclodextrin (Rameb) since this compound has demonstrated its ability to destabilize the cell membrane through cholesterol complexation. In vivo toxicity of Rameb after intramuscular injection has been assessed as well as efficiency when associated to polymer/DNA formulations. Addition of Rameb to the polymeric vectors did not allow a significant increase of transfection compared to the previously established references. Interestingly we observed a decrease of the transfected muscle fibers when Rameb was associated to Tetronic 304, suggesting a partial inhibition of transfection.In summary, polymer/DNA vectors demonstrated a large variety of physical characteristics depending on the type of polymer. In vivo experiments underlined improved transfection efficiency of DNA formulations based on Tetronic 304 and PE6400 when compared to naked DNA. Moreover, addition of methyl-beta- cyclodextrin to DNA in order to improve cell entry exhibited a complex behavior. In conjunction with naked DNA, cyclodextrin increased muscle transfection. However, combined with the polymer/DNA vectors this beneficial effect of cyclodextrin was lost. Thus, further investigations need to be performed in order to obtain rationally designed vectors, which will increase transfection levels in the muscle tissue.