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Abstract

Myoblast transplantation represents a promising therapeutic strategy in the treatment of several genetic muscular disorders including Duchenne muscular dystrophy. Nevertheless, such an approach is impaired by the rapid death, limited migration, and rejection of transplanted myoblasts by the host. Low molecular weight dextran sulfate (DXS), a sulfated polysaccharide, has been reported to act as a cytoprotectant for various cell types. Therefore, we investigated whether DXS could act as a “myoblast protectant” either in vitro or in vivo after transplantation in immunodeficient mice. In vitro, DXS bound human myoblasts in a dose dependent manner and significantly inhibited staurosporine-mediated apoptosis and necrosis. DXS pretreatment also protected human myoblasts from natural killer cell-mediated cytotoxicity. When human myoblasts engineered to express the renilla luciferase transgene were transplanted in immunodeficient mice, bioluminescence imaging analysis revealed that the proportion of surviving myoblasts 1 and 3 days after transplantation was two times higher when cells were preincubated with DXS compared to control (77.9 ± 10.1% vs. 39.4 ± 4.9%, p = 0.0009 and 38.1 ± 8.5% vs. 15.1 ± 3.4%, p = 0.01, respectively). Taken together, we provide evidence that DXS acts as a myoblast protectant in vitro and is able in vivo to prevent the early death of transplanted myoblasts.

Keywords

Myoblast transplantation Cell survival Dextran sulfate (DXS)Bioluminescence Mice

Introduction

Myoblasts are skeletal muscle progenitor cells involved in the natural processes of skeletal muscle growth and regeneration. As such and because myoblasts face the complex challenges of clinical application of cell therapy (37), myoblast transplantation appears to be a promising approach for the treatment of extensive skeletal muscle destruction that occurs in severe myopathies such as Duchenne muscular dystrophy (DMD). After the initial demonstration that myoblast transplantation could restore dystrophin expression in a murine model of DMD (26), the subsequent clinical trials of myoblast transfer in Duchenne patients have been disappointing due to the rapid death, limited migration, and rejection of transplanted myoblasts (24,30,34). Various strategies were investigated to enhance the success of myoblast transplantation including the upregulation of antiapoptotic and/or anti-inflammatory genes in injected myoblasts (6,21,28), and the use of laminin-111 or of fibrin as coadjuvants in myoblast transplantation (9,11). Moreover, as suggested by Skuk et al., who identified a clear relationship between the formation of ischemic necrosis cell pockets and the number of myoblast transplanted per single site (33), new injection methodologies, that is, increasing the number of injections per volume of muscle, were developed and enhance the success of myoblast transplantation in DMD patients (32). However, the overall efficiencies of these protocols remain unsatisfactory.

Low molecular weight dextran sulfate (DXS), a sulfated polysaccharide of 5 kDa, inhibits complement activation as well as the coagulation cascade (39). DXS prevents natural killer (NK) cell-mediated cytotoxicity in vitro and favors, in combination with cyclosporine A, the long-term survival of cardiac xenografts (19,20). Moreover, DXS treatment significantly reduces endothelial cell apoptosis in a rat aortic clamping model (2). Taken together, these beneficial effects of DXS in modulating immunity suggest that this substance may be a possible candidate to improve the efficiency of myoblast transplantation.

In the present study, DXS was preincubated with human myoblasts for 6 h before in vitro experiments or transplantation in immunodeficient mice. In vitro, DXS protected human myoblasts from staurosporine or NK cell-mediated cell death. Using optical bioluminescence imaging (BLI), we observed that DXS enhanced the survival of the transplanted cells by a factor of two, 1 and 3 days postinjection. Our findings suggest that DXS may act as an effective myoblast protectant in vitro and in vivo.

Materials and Methods

Ethics Statement

Procedures undertaken with human samples and mice were in compliance with the institutional guidelines, as well as the national and international guidelines. We obtained ethical approval for the study (human blood sample, human skeletal muscle biopsies) from The University Hospital of Geneva Research Committee for the use of humans as experimental subjects. All human samples were collected anonymously with written consent from the parents of the four children involved in the study. The Canton of Geneva authority on animal care approved the research as part of animal protocol 1043/3376/2.

Materials

Low molecular weight DXS (MW 5000) was purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Fluorescein-labeled DXS (DXS-Fluo) was produced as described earlier (20). DXS and DXS-Fluo were used at the indicated concentrations and were incubated with adherent myoblasts 6 h before in vitro or in vivo experiments. In all experiments, cells were washed two times with PBS (Life Technologies, Zug, Switzerland) after DXS pretreatment. DXS pretreatment did not induce any irreversible injury to treated cells as assessed by a trypan blue (Sigma-Aldrich) exclusion test.

Cell Culture

Human muscle samples, cell dissociation, and clonal culture from satellite cells were prepared as previously described (1,4). Briefly, human muscle samples were obtained during corrective orthopedic surgery of four patients (15 months to 4 years old) without any known neuromuscular disease. After muscle dissociation, single cells were manually collected with a micropipette under a microscope and individually distributed in 96-well culture plates (BD Biosciences, Franklin Lakes, NJ, USA). Clones were expanded in growth medium (GM) consisting of Ham's F10 (Life Technologies) supplemented with 15% fetal calf serum (FCS; Life Technologies), bovine serum albumin (Sigma-Aldrich; 0.5 mg/ml), fetuin (Sigma-Aldrich; 0.5 mg/ml), epidermal growth factor (Life Technologies; 10 ng/ml), dexamethasone (Sigma- Aldrich; 0.39 μg/ml), insulin (Sigma-Aldrich; 0.04 mg/ml), creatine (Sigma-Aldrich; 1 mM), pyruvate (Sigma-Aldrich; 100 μg/ml), uridine (Sigma-Aldrich; 50 μg/ml), and gentamycin (Life Technologies; 5 μg/ml) up to 10⁶ cells (20 divisions) and stored at −80°C until use. All human myoblasts used in the study had achieved less than 30 divisions before in vitro and in vivo experiments and were 100% positive for the myogenic markers cluster of differentiation 56 (CD56; BD Biosciences) and desmin (D33, Dako, Schweiz AG, Baar, Switzerland) as assessed by flow cytometry (data not shown). NK cells were isolated from buffy coats of healthy blood donors after Ficoll-Paque (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) gradient and negative selection using NK cell isolation kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). NK (CD56+CD3^-) cell purity was >90% as evaluated by fluorescence-activated cell sorting (FACS) on a FACSCalibur analyzer from BD Biosciences. Purified human NK cells were activated after culture in proliferating medium containing 25 ng/ml interleukin (IL)-15 (PeproTech EC Ltd., London, UK) for 6 days.

NK Cell Cytotoxic Assay

Cytotoxic activity was assessed according to Johann et al. (17) with slight modifications. Human myoblasts were labeled with carbocyanine dye vybrant 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzene-sulfonate salt (DiD; Molecular Probes, Invitrogen, Eugene, OR, USA) and incubated for 6 h in GM containing DXS at concentrations of 0-25 mg/ml. After washing in PBS, activated NK cells were added on adherent myoblasts for 4 h at effector/target (E/T) ratios of 2:1 and 4:1. Cells were washed, stained with phycoerythrin (PE)-labeled anti-CD45 mAb (BD Biosciences), and analyzed in the presence of 5 μg/ml of 7-amino-actinomycin D (7-AAD, Sigma-Aldrich). Killed targets were defined as 7-AAD+/DiD+/CD45^- cells. Analyses were run on a FACSCalibur analyzer from BD Biosciences, in PBS containing 2% FCS, 0.02% sodium azide (Sigma-Aldrich).

Flow Cytometry Analysis

Surface Molecules

Human myoblasts, pretreated or not with DXS for 6 h, were washed and then incubated for 48 h with recombinant human interferon-γ (IFN-γ; PeproTech EC Ltd.). Cells were then analyzed by flow cytometry with PE-labeled mAb against CD45, human leukocyte antigen (HLA)-ABC, HLA-DR, intracellular adhesion molecule 1 (ICAM-1), B7 homolog 1 (B7-H1; also known as programmed cell death ligand 1) (all BD Biosciences), or isotype control IgG1 (BD Biosciences).

Renilla Luciferase Expression

Cells were permeabilized by incubation on ice for 20 min with Fix/Perm buffer (BD Biosciences). Cells were washed twice in Perm/Wash buffer 1× (BD Biosciences) and incubated with a mouse anti-renilla luciferase antibody for 60 min on ice (1/50; Millipore, Zug, Switzerland). After washing, cells were incubated for 30 min on ice with a goat anti-mouse IgG fluorescein isothiocyanate (FITC; 1/500, Sigma).

Detecting Apoptosis In Vitro

Human myoblasts were treated with or without DXS (range 0–5 mg/ml) in vitro for 6 h. They were then washed and incubated with 1 mM staurosporine (Sigma) for 24 h. After trypsinization, the cells were washed twice in cold PBS and suspended (1×10⁶/ml) in Annexin V binding buffer (BD Biosciences). Cells (1×10⁵) were incubated with 5 μl of Annexin V-FITC (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in the presence of 5 μg/ml 7-AAD for 15 min at room temperature (RT) in the dark. Apoptotic cells were defined as Annexin V-positive cells and 7-AAD-negative cells. Necrotic cells were defined as 7-AAD-positive cells.

Cell Proliferation

Control myoblasts and Rluc-transduced myoblasts were suspended at 5×10⁷ cells/ml in PBS/0.1% bovine serum albumin (Sigma-Aldrich), and 5-6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes) was added to a final concentration of 10 μM and incubated for 10 min at 37°C. Labeled suspensions were then washed three times in cold GM and cultured under proliferation conditions up to 7 days. Cell proliferation (CFSE level) was assessed at days 0, 1, 4, and 7 by flow cytometry.

All flow cytometry analyses were run on a FACScan or FACSCalibur analyzer from BD Biosciences.

Renilla Luciferase Lentivirus Production and Reporter Assay

A three-plasmid expression system was used to generate second-generation lentiviral vectors by transient transfection as already described (21). The three plasmids were (i) the lentiviral vector plasmid in which the renilla luciferase cDNA (pRL-TK vector, Promega) was cloned in a pLox-EW plasmid; (ii) the lentiviral packaging plasmid pCMV-R8.74 (plasmid 22036, Addgene, Cambridge, MA, USA); and (iii) the envelope plasmid pMD2G (plasmid 12259, Addgene). The vectors were produced by transfection of plasmid DNA into 293T cells (ATCC, Manassas, VA, USA) using a calcium phosphate method. Human myoblasts were transduced with the lentivirus Rluc at multiplicities of infection (MOIs) of 0.2, 1, and 5. Cells were maintained under proliferation conditions for 3 days and were then processed with the Dual Luciferase reporter assay kit (Promega, Madison, WI, USA).

Fusion Indexes

Confluent layer of control myoblasts and Rluc-transduced myoblasts were transferred to differentiation medium for 48 h. Their myogenic properties were evaluated by assessing their fusion index as previously described (16) using an antibody against troponin-T (Sigma-Aldrich).

Intramuscular Transplantation

Female nonobese diabetic severe combined immunodeficient (NOD.CB17-Prkdc^scid/J) mice, 8–10 weeks old (n = 10) (The Jackson Laboratory, Bar Harbor, ME, USA), were bred and purchased from Université de Genève, Plan les Ouates, Switzerland. These mice have no functional T-cells and B-cells, lack complement activity, and have a low residual NK cell activity. Isoflurane (Abbott, Baar, Switzerland) was used as the anesthetic in all procedures, supplemented with oxygen through a semiclosed circuit inhalation system. After the exposition of the tibialis anterior muscle of the mice by chirurgical intervention, either untreated or DXS pretreated (10 mg/ml) human myoblasts (1×10⁶ cells per injection in 20 ml of PBS) were transplanted using a 50-ml syringe with a 27-gauge needle (Hamilton Company, Bonaduz, Switzerland). After surgery, mice received one intraperitoneal injection of Temgesic (0.05 mg/kg, Essex Chemie, Luzern, Switzerland).

Bioluminescence Analysis

For bioluminescence imaging (BLI), a Xenogen-IVIS 200 device was used (Caliper Life Sciences AG, Oftringen, Switzerland). For in vitro studies, coelenterazine (Biotium, Hayward, CA, USA), diluted in cold PBS⁺⁺ (containing Ca²⁺ and Mg²⁺) at a concentration of 1 mg/ml, was added to the cells, and BLI was acquired immediately (1-s acquisition time). For in vivo studies, coelenterazine (1 mg/kg body weight in 100 ml of PBS⁺⁺) was injected intravenously, and images were acquired continuously for 12 min (3-min acquisition time) and stored for subsequent analysis. We analyzed images at 6 min after coelenterazine injection (maximal signal intensity). A region of interest (ROI) was manually selected over the signal intensity. The area of the ROI was kept constant, and signal was recorded as maximum (photons/s). Bioluminescent signals lower than 2×10⁴ photons/s were defined as background. For technical reasons and despite the fact that cell death may occur in the first hours postinjection, the 3-h point postinjection was selected and defined as the reference 100% survival (day 0). BLI values measured at days 1, 3, 7, and 14 were related to the bioluminescence data obtained at day 0.

Immunofluorescence

To examine the presence of human nuclei in the muscle of immunodeficient mice at days 1 and 7 posttransplantation (n = 2 per group, control and DXS treated), tibialis anterior muscle samples were embedded in Tissue Tek (Sakura Finetek, Alphen aan den Rijn, the Netherlands) and stored at −80°C until use. For analysis, 5-μm cryosections were fixed in PBS-4% formal-dehyde (Sigma-Aldrich) for 15 min, washed in PBS, and blocked in PBS-5% goat serum (Life Technologies), 0.3% Triton X-100 (Sigma-Aldrich) for 1 h at RT. To follow the distribution of human cells, sections were stained with a mouse anti-human lamin A/C antibody (Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C and a goat anti-mouse PE (Molecular Probes) for 1 h at RT. Sections were then stained with an antibody against dystrophin (Abcam, Cambridge, UK) and a goat anti-rabbit FITC (Molecular Probes). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich), and sections were mounted using polyvinyl alcohol mounting medium with DABCO (Sigma-Aldrich).

Statistical Analysis

Correlation data were done using Pearson's correlation test (r). Statistical analyses were performed using unpaired Student's t test with Bonferroni correction (α=0.05) when necessary. Results were considered statistically significant when p < 0.05.

Results

DXS Binds to Human Myoblasts and Protects Them From Staurosporine-Induced Cell Death In Vitro

Semiconfluent monolayers of human myoblasts were incubated at 37°C for 6 h with various concentrations of DXS-Fluo (0-5 mg/ml). After washing, cells were trypsinized, and the amount of residual DXS-Fluo was analyzed by flow cytometry. Figure 1A shows that DXS-Fluo binds in a dose dependent manner to human myoblasts with a maximal mean fluorescence intensity ratio (MFIR) of 30 at a concentration of 5 mg/ml. Pretreatment of the cells with DXS prior to staurosporine incubation significantly inhibited the percentage of apoptotic cells (AnnexinV⁺/7-AAD^-) at DXS concentrations of 1 and 5 mg/ml (p < 0.001 as compared to untreated cells; left panel) (Fig. 1B). DXS pretreatment was also efficient in inhibiting the percentage of necrotic cells (7-AAD⁺) as compared to untreated cells at a DXS concentration of 5 mg/ml (p < 0.01 as compared to untreated cells; right panel) (Fig. 1B). A significant correlation was found between the proportion of apoptotic cells or the proportion of necrotic cells and the level of DXS-Fluo associated with human myoblasts (r = −0.92 and r = −0.97, respectively; p < 0.05) (Fig. 1C).

Figure 1.

Binding of DXS to human myoblasts correlates with inhibition of staurosporine-induced cell death in vitro. Dextran sulfate (DXS) bound dose dependently to human myoblasts (A) and inhibited staurosporine induced apoptosis and necrosis in vitro (B) as assessed by flow cytometry for Annexin V/7-amino-actinomycin D (7-AAD). Results were expressed either as mean fluorescence intensity ratios (MFIR) for binding of fluorescein-labeled DXS (DXS-Fluo) (A) or as percentage of apoptotic cells (Annexin V⁺/7-AAD^–) or necrotic cells (7-AAD⁺) (B). Bars show the means ± SD from three independent experiments. Statistical significance of DXS pretreated myoblasts versus untreated myoblasts prior to staurosporine incubation is indicated by ∗p < 0.05 using unpaired Student's t test with Bonferroni correction. (C) A significant correlation was found between the percentage of apoptotic cells and the MFIR for binding of DXS-Fluo to human myoblasts (r = −0.92, p < 0.05) and also between the percentage of necrotic cells and the MFIR for binding of DXS-Fluo to human myoblasts (r = −0.97, p < 0.05) using Pearson's correlation coefficient and unpaired Student's t test.

DXS Inhibits IFN-γ-Induced Phenotypic Maturation of Human Myoblasts

Incubation of human myoblasts with IFN-γ for 48 h significantly increased the expression of major histocompatibility complex (MHC) class I (HLA-ABC) and class II (HLA-DR) and of the adhesion molecule ICAM-1, but did not modify B7-H1 expression as compared to untreated myoblasts (p < 0.02) (Fig. 2). Preincubation of the cells with DXS for 6 h, prior to IFN-γ stimulation, significantly inhibited the upregulation of HLA-DR and ICAM-1 expression when DXS concentrations were >5 and >0.2 mg/ml, respectively (p < 0.05) (Fig. 2). DXS pretreatment did not affect IFN-γ-induced MHC class I upregulation.

Figure 2.

DXS prevents IFN-γ-induced phenotypic maturation of human myoblasts in a dose dependent manner. Human myoblasts, pretreated or not with DXS for 6 h, were washed and then analyzed by flow cytometry for various surface molecules [human leukocyte antigen (HLA)-ABC, HLA-DR, intracellular adhesion molecule-1 (ICAM-1), B7 homologue 1 (B7-H1)] after 48 h of interferon-γ (IFN-γ) stimulation. To compare the expression levels of the indicated surface molecules, the median fluorescence intensity ratios (MFIR) were calculated by dividing the median fluorescence of IFN-γ- or of DXS + IFN-γ-treated myoblasts by the median fluorescence of untreated control myoblasts. Values shown are the means ± SEM of four independent experiments. #p < 0.02, IFN-γ-treated myoblasts versus untreated myoblasts. ∗p < 0.05, DXS + IFN-γ-treated myoblasts versus IFN-γ-treated myoblasts, using unpaired Student's t test.

DXS Inhibits NK Cell-Mediated Cytotoxicity

The cytotoxicity of highly purified allogenic NK cells upon in vitro amplified myoblasts was assessed by flow cytometry. NK cells cultured in the absence of stimulatory cytokines did not lyse target cells (data not shown). However, after a 6-day activation with IL-15, human NK cells lysed, at an E/T ratio of 2:1, human myoblasts with 17% efficiency. Myoblast killing by allogenic NK cells was inhibited in a dose dependent manner when myoblasts were pretreated for 6 h prior to cytotoxic assay with DXS concentrations >1mg/ml (Fig. 3). A similar inhibition of NK killing by DXS was observed at an E/T ratio of 4:1 (data not shown).

Figure 3.

DXS protects human myoblasts from NK cell mediated killing. Adherent DiD⁺ myoblasts were preincubated with DXS (0–25 mg/ml) for 6 h, washed, and incubated in the presence of IL-15-activated human natural killer (NK) cells. After 4 h of incubation, myoblasts were trypsinized and analyzed by flow cytometry to determine the percentage of cell death, defined as 7-AAD⁺/DiD⁺/CD45^– cells. One representative experiment is presented in (A). In (B), graph shows the percentage relative to the maximal cell death observed; means ± SD from three independent experiments. ∗p < 0.05, DXS-pretreated myoblasts versus untreated myoblasts, using unpaired Student's t test.

Rluc Transgene Expression Has No Detrimental Effect on Myoblast Proliferation and Differentiation In Vitro

To allow the detection by bioluminescence imaging (BLI), human myoblasts were transduced with a lentivirus encoding the renilla luciferase (Rluc) gene under a HSV-TK promoter. We determined by flow cytometry that 80% of human myoblasts expressed the Rluc transgene at an MOI of 1 (Fig. 4A) and maintained their capacity to proliferate as assessed by CFSE staining (Fig. 4B). The enzymatic activity and the specificity of the renilla luciferase were demonstrated in a dual luciferase reporter assay (Fig. 4C), and we observed a dose dependent activity of the Rluc with increasing dose of the virus. We also tested if Rluc-transduced myoblasts kept their myogenic capacity to differentiate in vitro as assessed by fusion indexes (Fig. 4D). Rluc-transduced myoblasts exhibited fusion indexes similar to control cells at MOIs of 0.2 and of 1 but not at an MOI of 5.

Figure 4.

Human myoblasts express the renilla luciferase (Rluc) transgene without loss of myogenicity in vitro. (A) At a multiplicity of infection (MOI) of 1, 80% of the cells express the Rluc transgene as assessed by flow cytometry. (B) A similar pattern of cell proliferation was observed by 5-6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining after 1, 4, and 7 days under proliferation conditions between control (Ctrl) and Rluc-transduced cells. (C) Rluc activity and specificity was confirmed in a dual luciferase assay. Rluc activity increased dose dependently with increasing dose of virus (MOIs of 0.2, 1, and 5). (D) Human myoblasts were induced to differentiate for 2 days. Myotubes were stained with an antibody against troponin-T (red), and nuclei were stained with DAPI (blue). Rluc myoblasts had similar differentiation pattern as compared to control cell up to an MOI of 1. Scale bar: 50 μm. Data shown are representative of three independent experiments with similar results for each.

Linearity of Bioluminescence Imaging In Vitro and In Vivo

Increasing number of Rluc myoblasts (MOI = 1) were seeded in a 96-well plate. After addition of the Rluc substrate coelenterazine, we observed that bioluminescence values significantly correlated with cell number in vitro (r = 0.99, p = 0.007) (Fig. 5A). Moreover, we can follow the proliferation of Rluc-transduced myoblasts in vitro, leading to an increase in bioluminescence values with culture time (Fig. 5B), an observation that confirmed that construct integration was stable in human myoblasts. Rluc myoblast numbers and bioluminescence also significantly correlated in vivo (r = 0.96, p = 0.02) as assessed 3 h after transplantation (Fig. 5C). Altogether, these experiments allowed establishing that the minimum number of myoblast detectable in our in vivo model was 50,000 cells (background level).

Figure 5.

Linearity of bioluminescence imaging (BLI) in vitro and in vivo. (A) Relationship of signal intensities with the number of human myoblasts in vitro. Renilla luciferase-transduced (Rluc) myoblasts were seeded in a 96-well plate in quadruplicate, from 2,500 to 40,000 cells with a ×2 incremental increase. Graph of bioluminescence data represented as average ± SD (n = 4) with value of Pearson's correlation coefficient (r = 0.99, p = 0.007), and a representative bioluminescent image with the number of cells seeded indicated at the top. (B) The relationship of signal intensities with proliferation of human myoblasts in vitro. Control or Rluc myoblasts were seeded at day 0 in a six-well plate, and bioluminescence was analyzed at days 1, 3, and 7 after plating (left). Graph of bioluminescence data is presented on the right. (C) Increasing numbers of Rluc human myoblasts were injected into the tibialis anterior muscles of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice and imaged 3 h after injection. Shown is a graph of bioluminescence data with value of Pearson's correlation coefficient (r = 0.96, p = 0.02), and a bioluminescent image of representative injected mice, with the number of cells injected indicated at the top. In (B, C), data are representative of three independent experiments with similar results.

Improved Survival of Engrafted Human Myoblasts Pretreated with DXS

Injections of either control myoblasts or myoblasts preincubated with 10 mg/ml DXS for 6 h before transplantation were performed respectively in the exposed but intact left and right tibialis anterior muscle of NOD/SCID mice. Myoblasts were preincubated with 10 mg/ml DXS because we have observed, in vitro, a maximal inhibition of staurosporine- or NK cell-mediated myoblast death at this concentration (data not shown). BLI was measured at 3 h (day 0) and at 1, 3, 7, and 14 days post-cell transplantation. The number of Rluc surviving myoblasts was two times higher at 1 and 3 days postinjection in the DXS treated group as compared to the untreated group (77.9 ± 10.1% vs. 39.4 ± 4.9%, p = 0.0009 and 38.1 ± 8.5% vs. 15.1 ± 3.4%, p = 0.01, respectively, mean ± SEM; n = 7) (Fig. 6). No significant difference was observed at 7 days (8.0 ± 2.1% vs. 3.6 ± 0.7% of surviving cells for the DXS-treated group and the untreated group, respectively; p = 0.06) and at 14 days postinjection with values that were indistinguishable from background levels previously established at 50,000 cells. In parallel to the bioluminescent study, we checked by immunofluorescence the presence of donor human myoblasts at 1 and 7 days postinjection in the tibialis anterior muscles of immunodeficient mice. One day following injection, human myoblasts formed clusters in interstitial spaces at the site of injection, with a higher number of identifiable human nuclei in the DXS-treated group as compared to the control group (Fig. 7A). At day 7, only a few aligned lamin A/C-positive nuclei remained in the muscle tissue in both groups, indicating that only a few human myoblasts have differentiated in the host muscle tissue. Whether these cells are within muscle fibers or trapped in the endomysium is difficult to determine (Fig. 7B).

Figure 6.

Improved survival of engrafted human myoblasts pretreated with DXS. Representative pictures of cell survival are presented in (A) after transplantation of 1×10⁶ Rluc human myoblasts (control or DXS pretreated) into one location of the tibialis anterior of NOD/SCID mice. In vivo bioluminescent images were acquired 3 h (day 0), 1 day, 3 days, 7 days, and 14 days after transplantation. A region of interest (ROI) was manually selected over the signal intensity (photon/s). (B) Data obtained at day 0 were defined as the reference 100% survival for each group (control and DXS pretreated). Percentage of myoblasts survival at 1, 3, 7, and 14 days after transplantation was related to the maximal cell survival measured 3 h after injection (day 0) in each group. Data are represented as mean ± SEM (n = 7; ∗p < 0.05; ∗∗p < 0.001, using unpaired Student's t test).

Figure 7.

Immunofluorescence detection of donor human myoblasts in tibialis anterior muscle sections of immunodeficient mice. Immunofluorescence detection of donor human myoblasts in tibialis anterior muscle sections of immunodeficient mice transplanted with either control human myoblasts or human myoblasts pretreated with DXS at day 1 (A) and day 7 (B) posttransplantation. Nuclei double-labeled for human specific anti-lamin A/C antibody (red staining) and DAPI were identified to be of donor origin. Anti-dystrophin antibody (green staining) was used to visualize the dystrophin network in the muscle tissue. Scale bar: 50 μm. The white rectangle is displayed at higher magnification in the lower part of the figure. Representative pictures from one out of two mice are shown.

Discussion

The transplantation of culture-derived myoblasts in humans is confronted with the massive death of the grafted cells in the recipient, which limits the benefit of such therapeutic approach so far (24,31). Apoptosis, anoikis, and necrosis have been described as important mechanisms of cell death occurring early postinjection (5,6,21,33). Inflammatory cells that infiltrate the graft posttransplantation, such as NK cells, neutrophils, and macrophages, participate also to this mechanism (12,13,30, 31). Considering the potential benefits of low molecular weight DXS in preventing the activation of innate immunity in transplantation (3,19,20,35), we investigated its effect in an animal model comprising the transplantation of human myoblasts in unconditioned NOD.CB17-Prkdc^scid/J mice.

Our in vitro data showed that amplified human myoblasts were sensitive to staurosporine-induced apoptosis and necrosis, and that activated human NK cells killed them with the same efficiency (i.e., 17%) than other MHC class I-positive primary human cells such as primary mesenchymal stem cells or hematopoietic blasts derived from CD34⁺ cord blood cells (27). DXS pretreatment protected human myoblasts from staurosporine-induced apoptosis and necrosis and from allogenic NK cell killing in a dose dependent manner. Although the effects of DXS on innate immunity have been reported, the precise mechanisms of its action remain elusive (35). One of these mechanisms may consist in the inhibition of apoptosis via intracellular modulation of the mitogen-activated protein kinase (MAPK) signaling pathways (2), an effect related to the known capacity of DXS to be internalized into the cell (22,23). DXS may also modulate the death receptor pathway that activates apoptosis and/or programmed necrosis (18). Moreover, we show in this study that DXS was able, in vitro, to counteract the IFN-γ-induced upregulation of the MHC-II complex and ICAM-1, while it did not affect the upregulation of the MHC-I complex or of the inhibitory receptor B7-H1. Therefore, the shield provided by the DXS preincubation toward NK cell killing seems to not involve MHC-I recognition by NK cells. Among other possibilities, DXS may mask or prevent the upregulation of NK cell-activating ligands on target cells (20,25).

Noninvasive bioluminescence imaging, which requires beforehand the transduction of the renilla luciferase gene in the target cells, was used to quantify human myoblast survival after transplantation in immunodeficient mice. Human myoblasts were extremely permissive to the viral transduction, which allowed obtaining homogenous populations of myoblasts that produced a linear bioluminescence signal. Moreover, the random insertion of the lentiviral vector did not alter the biological function of the myoblasts, which proliferated and proceeded to myogenic fusions at the same rate than control cells. Preincubation of myoblasts with DXS prior to transplantation produced a clear-cut improvement of myoblast survival observed at days 1 and 3 after injection compared to untreated cells. Immunofluorescence analyses assessing the expression of human lamin A/C in the grafted tissue corroborated the quantitative BLI analysis on day 1. However, 7 days postinjection, only few aligned human nuclei were observed in host muscles, indicating that de novo muscular fiber generation was rather ineffective. This may be in part due to the fact that muscles were not injured before transplant; therefore, no major regenerative process was triggered in the presence of xenogenic myoblasts. Alternatively, the restricted number of aligned nuclei may also be linked to the premature elimination of myoblasts by the host before the process of myogenic fusion had time to be completed.

The causes of the enhanced in vivo survival observed in myoblasts preincubated with DXS before transfer may be multiple. However, the antiapoptotic and antinecrotic effects of DXS observed in vitro is certainly involved. Whether the inhibition of the NK cell activity exerted by DXS in vitro is involved in the prolonged survival in vivo is difficult to assess because the mouse provider refers only to “a markedly decreased NK cell activity in these animals” (Jackson Lab, internal report MGI Ref ID J.22026; http://www.informatics.jax.org/reference/J:22026) without referring to any quantitative data. Moreover, the xenogenic environment used here may further complicate the assessment of the intensity of the residual NK cell activity. Collectively, we cannot exclude that murine NK cells participate to myoblast rejection in our protocols and that DXS participated to the enhancement of graft survival by inhibiting such an activity (15,36).

Several studies have suggested that human myoblasts may act as facultative antigen-presenting cells during local immune reactions in muscle and, in this way, may favor the maintenance of inflammatory conditions at the site of transplantation and/or inflammation, a fact that could induce their rapid immune recognition and elimination (10,14,38). Consistent with this hypothesis is our observation that IFN-γ induced an upregulation of ICAM-1 and of both MHC-I and MHC-II expression on myoblasts. These data suggest that myoblasts could present antigens to both CD8⁺ and CD4⁺ T-cells once located in an inflammatory environment. This cannot be evaluated in our study due to the absence of functional B-cells and T-cells in the recipients. Nevertheless, one can speculate that DXS pretreatment, by inhibiting the upregulation of MHC-II and ICAM-1 molecules, may be of importance to reduce the capacity of myoblasts to interact with T-cells in immunocompetent recipients (7,38). Moreover, DXS, by inhibiting ICAM-1 upregulation, may reduce leukocyte function-associated antigen 1 (LFA-1)/ICAM-1 interaction and thus may reduce interaction between human myoblasts and inflammatory cells (12,13).

Although the present study was not mechanistically oriented, our results indicate that DXS might represent a useful therapeutic reagent to improve the success of myoblast/myogenic precursor cell transplantation. DXS has been injected in human with little side effects (8,29). More experiments are needed to evaluate if the pretreatment of human myoblasts with DXS, combined with systemic DXS administration, may be envisaged to enhance myoblast survival in the host. Moreover, it may be interesting in future investigations to test the efficacy of DXS in a dystrophic environment, which presents by itself a significant activation of the inflammatory process.

Footnotes

Acknowledgments

This work has been supported by the research funds of the Geneva Orthopaedic Service. The authors thank Robert Rieben, University of Bern, Switzerland, for providing fluorescein-labeled dextran sulfate. We also thank Stephane Konig, University of Geneva, for his help in the renilla luciferase lentiviral vector construction and Laurent Bernheim,

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Low Molecular Weight Dextran Sulfate Binds to Human Myoblasts and Improves their Survival after Transplantation in Mice

Abstract

Keywords

Introduction

Materials and Methods

Ethics Statement

Materials

Cell Culture

NK Cell Cytotoxic Assay

Flow Cytometry Analysis

Surface Molecules

Renilla Luciferase Expression

Detecting Apoptosis In Vitro

Cell Proliferation

Renilla Luciferase Lentivirus Production and Reporter Assay

Fusion Indexes

Intramuscular Transplantation

Bioluminescence Analysis

Immunofluorescence

Statistical Analysis

Results

DXS Binds to Human Myoblasts and Protects Them From Staurosporine-Induced Cell Death In Vitro

DXS Inhibits IFN-γ-Induced Phenotypic Maturation of Human Myoblasts

DXS Inhibits NK Cell-Mediated Cytotoxicity

Rluc Transgene Expression Has No Detrimental Effect on Myoblast Proliferation and Differentiation In Vitro

Linearity of Bioluminescence Imaging In Vitro and In Vivo

Improved Survival of Engrafted Human Myoblasts Pretreated with DXS

Discussion

Footnotes

Acknowledgments

References