Libero Vitiello

In vivo tissue engineering of functional skeletal muscle by freshly isolated satellite cells embedded in a photopolymerizable hydrogel

The success of skeletal muscle reconstruction depends on finding the most effective, clinically suitable strategy to engineer myogenic cells and biocompatible scaffolds. Satellite cells (SCs), freshly isolated or transplanted within their niche, are presently considered the best source for muscle regeneration. Here, we designed and developed the delivery of either SCs or muscle progenitor cells (MPCs) via an in situ photo‐cross‐linkable hyaluronan‐based hydrogel, hyaluronic acid‐photoinitiator (HA‐PI) complex. Partially ablated tibialis anterior (TA) of C57BL/6J mice engrafted with freshly isolated satellite cells embedded in hydrogel showed a major improvement in muscle structure and number of new myofibers, compared to muscles receiving hydrogel + MPCs or hydrogel alone. Notably, SCs embedded in HA‐PI also promoted functional recovery, as assessed by contractile force measurements. Tissue reconstruction was associated with the formation of both neural and vascular networks and the reconstitution of a functional SC niche. This innovative approach could overcome previous limitations in skeletal muscle tissue engineering.—Rossi, C. A., Flaibani, M., Blaauw, B., Pozzobon, M., Figallo, E., Reggiani, C., Vitiello, L., Elvassore, N., De Coppi, P. In vivo tissue engineering of functional skeletal muscle by freshly isolated satellite cells embedded in a photopolymerizable hydrogel. FASEB J. 25, 2296‐2304 (2011). www.fasebj.org

Cardiac involvement in becker muscular dystrophy

OBJECTIVES The purpose of this study was to assess the incidence of myocardial involvement and the relation of cardiac disease to the molecular defect at the deoxyribonucleic acid (DNA) or protein level in Becker muscular dystrophy. BACKGROUND Dystrophin gene mutations produce clinical manifestations of disease in the heart and skeletal muscle of patients with Becker muscular dystrophy. METHODS Thirty-one patients underwent electrocardiographic and echocardiographic examination and 24-h Holter monitoring. The diagnosis was established by neurologic examination, dystrophin immunohistochemical assays or Western blot on muscle biopsy, or both, and DNA analysis. RESULTS Electrocardiographic and echocardiographic findings were abnormal in 68% and 62% of the patients, respectively. Right ventricular involvement was detected in 52%. Left ventricular impairment was observed either as an isolated phenomenon (10%) or in association with right ventricular dysfunction (29%). Right ventricular disease was manifested in the teenagers, and an impairment of the left ventricle was observed in older patients. Right ventricular end-diastolic volumes were significantly increased compared with those in a control group. The left ventricular ejection fraction was significantly lower in older patients than in control subjects or younger patients. Life-threatening ventricular arrhythmias were detected in four patients. No correlations were found between skeletal muscle disease, cardiac involvement and dystrophin abnormalities. In our patients, exon 49 deletion was invariably associated with cardiac involvement. Exon 48 deletion was associated with cardiac disease in all but two patients. CONCLUSIONS The cardiac manifestation of Becker muscular dystrophy is characterized by early right ventricular involvement associated or not with left ventricular impairment. Exon 49 deletion is associated with cardiac disease.

Loss-of-Function Mutation of the GPR40 Gene Associates with Abnormal Stimulated Insulin Secretion by Acting on Intracellular Calcium Mobilization

BACKGROUND Free fatty acids (FFAs) acutely stimulate but chronically impair glucose-stimulated insulin secretion from beta-cells. The G protein-coupled transmembrane receptor 40 (GPR40) mediates both acute and chronic effects of FFAs on insulin secretion and plays a role in glucose homeostasis. Limited information is available on the effect of GPR40 genetic abnormalities on insulin secretion and metabolic regulation in human subjects. STUDY DESIGN AND RESULTS For in vivo studies, we screened 734 subjects for the coding region of GPR40 and identified a new single-nucleotide mutation (Gly180Ser). The mean allele frequency was 0.75%, which progressively increased (P < 0.05) from nonobese subjects (0.42%) to moderately obese (body mass index = 30-39.9 kg/m2, 1.07%) and severely obese patients (body mass index > or = 40 kg/m2, 2.60%). The relationship between the GPR40 mutation, insulin secretion, and metabolic alterations was studied in 11 Gly/Ser mutation carriers. In these subjects, insulin secretion (insulinogenic index derived from oral glucose tolerance test) was significantly lower than in 692 Gly/Gly carriers (86.0 +/- 48.2 vs. 183.7 +/- 134.4, P < 0.005). Moreover, a case-control study indicated that plasma insulin and C-peptide responses to a lipid load were significantly (P < 0.05) lower in six Gly/Ser than in 12 Gly/Gly carriers. In vitro experiments in HeLa cells cotransfected with aequorin and the mutated Gly/Ser GPR40 indicated that intracellular Ca2+ concentration increase after oleic acid was significantly lower than in Gly/Gly GPR40-transfected cells. This fact was confirmed using fura-2 acetoxymethyl ester. CONCLUSIONS This newly identified GPR40 variant results in a loss of function that prevents the beta-cell ability to adequately sense lipids as an insulin secretory stimulus because of impaired intracellular Ca2+ concentration increase.

Electrophysiologic stimulation improves myogenic potential of muscle precursor cells grown in a 3D collagen scaffold

Abstract The production of engineered three-dimensional (3D) skeletal muscle grafts holds promise for treatment of several diseases. An important factor in the development of such approach involves the capability of preserving myogenicity and regenerative potential during ex vivo culturing. We have previously shown that electrical stimulation of myogenic cells grown in monolayer could improve the differentiation process. Here we investigated the effect of exogenous electrical field, specifically designed to mimic part of the neuronal activity, on muscle precursor cells (MPCs) cultured within 3D collagen scaffolds. Our data showed that electric stimulation did not affect cell viability and increased by 65.6% the release rate of NOx, an early molecular activator of satellite cells in vivo. NOx release rate was decreased by an inhibitor of NO synthase, both in stimulated and non-stimulated cultures, confirming the endocrine origin of the measured NOx. Importantly, electrical stimulation also increased the expression of two myogenic markers, MyoD and desmin. We also carried out some preliminary experiments aimed at determining the biocompatibility of our seeded collagen scaffolds, implanting them in the tibialis anterior muscles of syngeneic mice. Ten days after transplantation, we could observe the formation of new myofibers both inside the scaffold and at the scaffold/muscle interface. Altogether, our findings indicate that electrical stimulation could be a new strategy for the effective 3D expansion of muscle precursor cells in vitro without losing myogenic potential and that 3D collagen matrices could be a promising tool for delivering myogenic cells in recipient muscles.

Prevalence of dystrophin-positive fibers in 85 duchenne muscular dystrophy patients

The prevalence of dystrophin-positive fibers in Duchenne muscular dystrophy (DMD) muscle was estimated by direct counting on immunostained sections in a series of biopsy specimens from 85 patients, 42 of which were also screened for intragenic deletions by cDNA probes. Dystrophin-positive fibres are normotrophic and occur in muscle sections at a frequency between 0.01 and 6.81%. Frequencies over 1% were found only in patients older than 6 yr. The prevalence of dystrophin-positive fibers is about the same in patients with detectable and with undetectable deletions. The occurrence of positive fibers in small clusters supports the hypothesis of their clonal origin, suggesting that they may result from genetic reversion. No clinical differences were noticed in DMD patients of similar age with respect to the occurrence of dystrophin-positive fibres in their muscle biopsies.

Familial temporal lobe epilepsy with psychic auras associated with a novel LGI1 mutation

Background: Autosomal dominant lateral temporal epilepsy (ADLTE) is characterized by focal seizures with auditory features or aphasia. Mutations in the LGI1 gene have been reported in up to 50% of ADLTE pedigrees. We report a family with temporal lobe epilepsy characterized by psychic symptoms associated with a novel LGI1 mutation. Methods: All participants were personally interviewed and underwent neurologic examination and video-EEG recordings. LGI1 exons were sequenced by standard methods. Mutant cDNA was transfected into human embryonic kidney 293 cells; both cell lysates and media were analyzed by Western blot. In silico modeling of the Lgi1 protein EPTP domain was carried out using the structure of WD repeat protein and manually refined. Results: Three affected family members were ascertained, 2 of whom had temporal epilepsy with psychic symptoms (déjà vu, fear) but no auditory or aphasic phenomena, while the third had complex partial seizures without any aura. In all patients, we found a novel LGI1 mutation, Arg407Cys, which did not hamper protein secretion in vitro. Mapping of the mutation on a 3-dimensional protein model showed that this mutation does not induce large structural rearrangements but could destabilize interactions of Lgi1 with target proteins. Conclusions: The Arg407Cys is the first mutation with no effect on Lgi1 protein secretion. The uncommon, isolated psychic symptoms associated with it suggests that ADLTE encompasses a wider range of auras of temporal origin than hitherto reported.

Patterns of deletions of the dystrophin gene in different European populations

The distribution of deletion breakpoints in the dystrophin gene was studied in a series of subjects belonging to different European populations. The data, obtained from the literature or directly from the present study, refer to population samples from France, Finland, Germany, Italy, Netherland, Switzerland, and U.K. (England, Scotland, Wales). In total, 1516 breakpoints were assigned to different introns, 359 in the region encompassing the first 40 exons and 1157 (76%) in the distal part of the gene. Intron 7 appears to be equally involved as the starting or ending breakpoint, whereas intron 44 is involved mostly as a starting breakpoint. Breakpoint distribution by intron seems to differ in different populations, reaching statistical significance in the case of introns 44, 49, and 53. This finding suggests that some intronic sequences might contain preferential breakpoints that might vary in different populations, possibly as a consequence of genetic drift.

Efficient delivery of human single fiber-derived muscle precursor cells via biocompatible scaffold

The success of cell therapy for skeletal muscle disorders depends upon two main factors: the cell source and the method of delivery. In this work we have explored the therapeutic potential of human muscle precursor cells (hMPCs), obtained from single human muscle fibers, implanted in vivo via micropatterned scaffolds. hMPCs were initially expanded and characterized in vitro by immunostaining and flow cytometric analysis. For in vivo studies, hMPCs were seeded onto micropatterned poly-lactic-glycolic acid 3D-scaffolds fabricated using soft-lithography and thermal membrane lamination. Seeded scaffolds were then implanted in predamaged tibialis anterior muscles of CD1 nude mice; hMPCs were also directly injected in contralateral limbs as controls. Similarly to what we previously described with mouse precursors cells, we found that hMPCs were able to participate in muscle regeneration and scaffold-implanted muscles contained a greater number of human nuclei, as revealed by immunostaining and Western blot analyses. These results indicate that hMPCs derived from single fibers could be a good and reliable cell source for the design of therapeutic protocols and that implantation of cellularized scaffolds is superior to direct injection for the delivery of myogenic cells into regenerating skeletal muscle.

A Novel Loss-of-Function LGI1 Mutation Linked to Autosomal Dominant Lateral Temporal Epilepsy

BACKGROUND Mutations responsible for autosomal dominant lateral temporal epilepsy have been found in the leucine-rich, glioma-inactivated 1 (LGI1) gene. OBJECTIVES To describe the clinical and genetic findings in a family with autosomal dominant lateral temporal epilepsy and to determine the functional effects of a novel LGI1 mutation in culture cells. DESIGN Clinical, genetic, and functional investigations. SETTING University hospital and laboratory. PATIENTS An Italian family with autosomal dominant lateral temporal epilepsy. MAIN OUTCOME MEASURE Mutation analysis. RESULTS A novel LGI1 mutation, c.365T>A (Ile122Lys), segregating with the disease was identified. The mutant Lgi1 protein was not secreted by culture cells. CONCLUSION Our data provide further evidence that mutations in LGI1 hamper secretion of the Lgi1 protein, thereby precluding its normal function.

Three‐dimensional porous scaffold allows long‐term wild‐type cell delivery in dystrophic muscle

Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin; affected muscles are characterized by continuous bouts of muscle degeneration, eventually leading to the exhaustion of the endogenous satellite cell pool. At present, only palliative treatments are available, although several gene and cell therapy‐based approaches are being studied. In this study we proposed to overcome the limitations hampering intramuscular cell injection by using a biomaterial‐based strategy. In particular, we used a three‐dimensional (3D) collagen porous scaffold to deliver myogenic precursor cells (MPCs) in vivo in the mdx murine model of DMD. MPCs, derived from single fibres of wild‐type donors, were expanded in vitro, seeded onto collagen scaffolds and implanted into the tibialis anterior muscles of normal and mdx mice. As a control, cells were delivered via direct intramuscular cell injection in the contralateral muscles. Scaffold‐delivered MPCs displayed lower apoptosis and higher proliferation than injected cells; in terms of dystrophin restoration, collagen scaffolds yielded better results than direct injections. Importantly, time‐course experiments indicated that the scaffolds acted as a cell reservoir, although cell migration was mostly contained within 400 µm from the scaffold–host tissue interface. Copyright