Myoblasts and Embryonic Stem Cells Differentially Engraft in a Mouse Model of Genetic Dilated Cardiomyopathy

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.Cell therapies are progressively emerging as promising tools for the treatment of heart failure. In an attempt to achieve cardiac cell-based replacement therapy in the setting of postischemic cardiomyopathies (ICM), a variety of adult cell types have been tested up to preclinical stages in small and large animal models, including skeletal myoblasts (Mb), muscle-derived stem cells, adipose-derived stem cells, bone marrow mononuclear cells, hematopoietic stem cells, circulating endothelial progenitors, mesenchymal stem cells, smooth muscle cells, cardiac stem cells, and most of these approaches have demonstrated some degree of efficacy.^{1,2,3,4,5,6,7} Except for some specific populations of cardiac stem cells, most categories of adult stem cells show partial or complete inability to produce bona fide cardiomyocytes and to participate to true myocardial tissue formation, with respect to homogeneity of electrical conduction.⁸ Their functional benefits would be linked, essentially, to the mechanical strengthening of the scar tissue, and/or to the promotion of myocardial cell survival through paracrine synthesis of trophic factors and/or improved local angiogenesis.^{1,4,7,8,9,10,11} Indeed, phase II randomized clinical trials developed using adult stem cells have provided encouraging but still limited results.^12,13 However, the applicability and therapeutic relevance of cell therapies remain under-explored for nonischemic heart failure (dilated cardiomyopathy (DCM), myocarditis), probably due to the progressive nature of the disease and extension of fibrotic remodeling, which make the targeting of a specific area more difficult than when considering a delineated scar formed upon myocardial infarction. A few preclinical studies have been carried out using Mb,^14,15 smooth muscle cells or ventricular heart cells¹⁶ in cardiomyopathic hamsters, or mesenchymal stem cells,¹⁷ mixed mesenchymal stem cells and Mb,¹⁸ or bone marrow cells in rat models of DCM.¹⁹ Among those studies, Mb seem to have the best potential of integration in the dilated myocardium, and represent a “gold standard” for cell-based therapy, although these cells are not able to differentiate into cardiomyocyte lineage.In contrast, embryonic stem cells (ESC) are pluripotent and can be readily committed towards the cardiogenic lineage in vitro. There is also increasing evidence that cardiac-committed ESC can engraft into the scar tissue within the infarcted myocardium and differentiate into cardiomyocytes, thereby operating a regeneration of the myocardium, eliminating fibrotic scar tissue, and promoting sustained improvement of left ventricular function.^{7,8,11,20,21,22,23,24,25,26} This confers the potential ability to rebuild true cardiac tissue, to replenish areas that have been depopulated following ischemic accidents or the progression of fibrosis.⁸ In contrast, this positive benefit is limited by the formation of teratomas²⁷ or if too many cells are in uncommitted state at the time of injection.²⁴In the present study, we have compared the integration and functional efficacy of the CGR8 line of murine ESC with the D7LNB1 line of murine Mb, in the myocardium of Lmna^H222P/H222P mice. This genetic model of laminopathy reproduces a human missense mutation in the Lamin A/C gene causing Emery-Dreifuss muscular dystrophy. This model exhibits a rapidly progressive and lethal DCM,²⁸ showing pathophysiological evolution and conduction defects comparable to the human situation. Of note, these animals are immunocompetent.The CGR8 cell line of ESC was chosen because it can be grown feeder-free, and it is efficiently committed toward cardiogenic differentiation in vitro upon treatment with bone morphogenic protein 2 (BMP-2),^23,24,29,30 a treatment that indirectly lowers the risk of teratoma formation in vivo by decreasing the proportion of pluripotent cells.^24,27 The committed CGR8 cells, whether selected or not, have been previously shown to efficiently improve cardiac function following injection into the scar tissue in animal models of postischemic heart failure.^7,8,23,24,25 The time window for the addition of BMP-2 is of crucial importance,³⁰ therefore we pretreated CGR8 ESC for a short period of time, and we designed the experiments using limited amounts of cells to reach a compromise between myocardial differentiation and risk of teratoma formation (3 × 10⁵ per heart, at four different sites).The Mb have been assayed, in the present study, as a gold standard for validating the injection procedure, the efficacy of the immunosuppression regimen, the natural evolution of the implanted cells, the immunohistological procedures. Comparisons between Mb and ESC in murine models of postischemic heart failure have pinpointed important intrinsic differences in the efficacies and persistence of these two cell types, which now deserve a comparison in a DCM model.¹¹ The D7 Mb cell line was originally derived from the dy/dy mouse model of laminin-2 deficient congenital muscular dystrophy.³¹ It was engineered to express β-Galactosidase (β-Gal) constitutively and named D7LNB1. It showed no modification in its ability to form myotubes in vitro and in vivo. In addition, unlike C2 or G8 murine Mb, D7LNB1 Mb cell line did not produce carcinomas in vivo.³² CGR8 ESC and D7 Mb originate from close parental strains of 129 mice derived from the same animals in the 1970''s (129P/Ola background for ESC, 129/ReJ background for D7), and they share identical markers.³³ To avoid immune rejection triggered by murine ESC³⁴ or Mb³⁵ further expressing β-Gal in allogenic contexts, recipient mice were immunosuppressed using Tacrolimus.³⁶ We compared the engraftment of committed ESC and D7LNB1 Mb in terms of functional benefits and fate of engrafted cells in vivo. Our results suggest that cardiac-committed ESC failed to engraft into the dilated myocardium of the Lmna^H222P/H222P mice model, whereas Mb have a higher transplantation efficiency and greater functional improvement of cardiac function in this genetic model of dilated heart failure.     

Duodenal hematoma: the ring sign in MR imaging

Proper management of duodenal hematoma requires that an accurate diagnosis be made using noninvasive radiological methods. Conventional imaging may be nonspecific if there is no history of trauma or coagulopathy. Two cases of duodenal hematoma that were imaged by magnetic resonance (MR) and computed tomography (CT) are described. In both cases the hematoma had a well-defined concentric ring configuration on MR images, a finding which helped establish the diagnosis. MR imaging may provide tissue-specific characterization of duodenal hematomas.     

Biliary stricture dilatation: multicenter review of clinical management in 73 patients

Eighty-nine biliary strictures in 73 patients who had undergone percutaneous balloon dilatation were reviewed to determine long-term patency rates and clinical management problems. The majority of dilatations were performed in patients with anastomotic strictures (n = 44), iatrogenic strictures (n = 28), and strictures associated with sclerosing cholangitis (n = 17). Patency rates after 36 months or more were 67%, 76%, and 42%, respectively. Complications, mostly minor, occurred in less than 7% of patients. Of patients with significant biliary obstruction, 15% had little or no intrahepatic biliary duct dilatation demonstrated by cross-sectional imaging and/or direct cholangiography. No definite conclusions could be drawn about the utility of long-term internal/external stenting.     

Rapid MR imaging with suspended respiration: clinical application in the liver

The authors demonstrate that it is possible to obtain highly T1-weighted images of the abdomen using a suspended respiration partial saturation (SRPS) method in a breath-holding interval. T2*-weighted images, which reflect tissue T2 as well as variations in the static magnetic field, can also be rapidly obtained. The authors studied five healthy subjects and 19 patients with a variety of liver abnormalities, including benign and malignant hepatic neoplasms, fatty liver infiltration, ascites, and hematoma. On T1-weighted multisection acquisitions, the entire liver can be screened for mass lesions in a single 20-second breath-holding interval. Phase-contrast SRPS images are sensitive to fatty infiltration of the liver. SRPS images are more sensitive to variations in magnetic susceptibility than spin-echo images are, which has been proved to be of value in the detection of hemorrhage. With continued pulse sequence development and clinical study, this method has the potential to become the method of choice for evaluation of the upper abdomen.     

Quantification of normal dystrophin mRNA following myoblast transplantation in mdx mice

A mutagenesis RT-PCR method was used to detect normal dystrophin mRNA following the injection of normal myoblasts in mdx mice using two immunosuppressors. A specific sequence of the dystrophin mRNA (257 bp) was amplified by RT-PCR from the muscle total RNA. Maell digestion of the amplified products allowed us to distinguish the normal messenger of dystrophin from the dystrophic one and to establish the percentage of normal and of dystrophic (mdx) dystrophin mRNA. Normal dystrophin mRNA was detected using this technique in mdx muscles transplanted with histocompatible normal myoblasts. For this type of transplantation, no significant difference in the percentage of normal dystrophin mRNA was observed between immunosuppressed mice and those not immunosuppressed. No normal dystrophin mRNA was, however, observed in mdx mice following histoincompatible normal myoblast transplantation without immunosuppression. When such transplantations were done in mice immunosuppressed with cyclosporine or FK-506, normal dystrophin mRNA accounted for 31% and 36% of the total dystrophin mRNA, respectively. In fact, one animal immunosuppressed with FK-506 expressed as much as 57% of normal dystrophin mRNA. These results thus show that FK-506 makes it possible to restore dystrophin expression to a level comparable to that observed in DMD carriers that are usually asymptomatic. © 1995 John Wiley & Sons, Inc.     

Ferrite particles for bowel contrast in MR imaging: design issues and feasibility studies

Diverse materials with varying physical and magnetic properties have been evaluated as gastrointestinal contrast agents for magnetic resonance (MR) imaging. Uniform marking of the small bowel remains the greatest challenge. Ferrites are magnetically active iron oxide particles that are miscible with water and cause loss of signal on MR images. The decrease in MR signal intensity produced by ferrites occurs with a wide range of iron concentrations (0.1-10 mM) and with both T1- and T2-weighted pulse sequences. These effects of ferrites are explained by predominant T2 shortening with negligible T1 effects. The ferrite preparation used in this study was stable in vitro, with little iron solubilized by acid. Intragastric administration of ferrite (5 mg of iron per kg in 6 ml) routinely marked the small bowel of rats. The authors conclude that ferrites represent a promising new class of contrast agents for gastrointestinal MR imaging.     

Tetrahydrocannabinol vs. prochlorperazine. The effects of two antiemetics on patients undergoing radiotherapy

The authors tested the effectiveness of orally administered delta-9-tetrahydrocannabinol (THC) as compared to prochlorperazine for the alleviation of symptoms, such as vomiting and nausea, experienced by patients receiving radiotherapy. The test subjects rated the severity of their illness, as well as the extent of their subsequent moods, their level of concentration, their amount of physical activity, and their desire for social interaction. They chose the drug they preferred and recorded its side effects. The use of THC was slightly more beneficial than the use of prochlorperazine.