Gabriella Cusella

Mesoangioblasts, Vessel-Associated Multipotent Stem Cells, Repair the Infarcted Heart by Multiple Cellular Mechanisms A Comparison With Bone Marrow Progenitors, Fibroblasts, and Endothelial Cells

Objective—To test the potential of mesoangioblasts (Mabs) in reducing postischemic injury in comparison with bone marrow progenitor cells (BMPCs), fibroblasts (Fbs), and embryonic stem cell–derived endothelial cells (ECs), and to identify putative cellular protective mechanisms. Methods and Results—Cells were injected percutaneously in the left ventricular (LV) chamber of C57BL/6 mice, 3 to 6 hours after coronary ligation, and detected in the hearts 2 days and 6 weeks later. Echocardiographic examinations were performed at 6 weeks. LV dilation was reduced and LV shortening fraction was improved with Mabs and BMPCs but not with ECs and Fbs. Donor cell colonization of the host myocardium was modest and predominantly in the smooth muscle layer of vessels. Capillary density was higher in the peripheral infarct area and apoptotic cardiomyocytes were fewer with Mabs and BMPCs. Mabs and BMPCs, but not Fbs or ECs, promoted survival of cultured cardiocytes under low-oxygen in culture. This activity was present in Mab-conditioned medium and could be replaced by a combination of basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF)-1, and hepatocyte growth factor (HGF), all of which are produced by these cells. Conditioned medium from Mabs, but not from Fbs, stimulated proliferation of smooth muscle cells in vitro. Conclusions—Mabs appear as effective as BMPCs in reducing postinfarction LV dysfunction, likely through production of antiapoptotic and angiogenic factors.

Metabolic correction in oligodendrocytes derived from metachromatic leukodystrophy mouse model by using encapsulated recombinant myoblasts

In an effort to develop an encapsulated cell-based system to deliver arylsulfatase A (ARSA) to the central nervous system of metachromatic leukodystrophy (MLD) patients, we engineered C2C12 mouse myoblasts with a retroviral vector containing a full-length human ARSA cDNA and evaluated the efficacy of the recombinant secreted enzyme to revert the MLD phenotype in oligodendrocytes (OL) of the As2-/- mouse model. After transduction, C2C12 cells showed a fifteen-fold increase in intracellular ARSA activity and five-fold increase in ARSA secretion. The secreted hARSA collected from transduced cells encapsulated in polyether-sulfone polymer, was taken up by enzyme-deficient OL derived from MLD mice and normally sorted to the lysosomal compartment, where transferred enzyme reached 80% of physiological levels, restoring the metabolism of sulfatide. To evaluate whether secreted enzyme could restore metabolic function in the brain, encapsulated cells and secreted ARSA were shown to be stable in CSF in vitro. Further, to test cell viability and enzyme release in vivo, encapsulated cells were implanted subcutaneously on the dorsal flank of DBA/2J mice. One month later, all retrieved implants released hARSA at rates similar to unencapsulated cells and contained well preserved myoblasts, demonstrating that encapsulation maintains differentiation of C2C12 cells, stable transgene expression and long-term cell viability in vivo. Thus, these results show the promising potential of developing an ARSA delivery system to the CNS based on the use of a polymer-encapsulated transduced xenogenic cell line for gene therapy of MLD.

Molecular signature of amniotic fluid derived stem cells in the fetal sheep model of myelomeningocele

Abnormal cord development results in spinal cord damage responsible for myelomeningocele (MMC). Amniotic fluid-derived stem cells (AFSCs) have emerged as a potential candidate for applications in regenerative medicine. However, their differentiation potential is largely unknown as well as the molecular signaling orchestrating the accurate spinal cord development. Fetal lambs underwent surgical creation of neural tube defect and its subsequent repair. AFSCs were isolated, cultured and characterized at the 12th (induction of MMC), 16th (repair of malformation), and 20th week of gestation (delivery). After performing open hysterectomy, AF collections on fetuses with sham procedures at the same time points as the MMC creation group have been used as controls. Cytological analyses with the colony forming unit assay, XTT and alkaline-phosphatase staining, qRT-PCR gene expression analyses (normalized with aged match controls) and NMR metabolomics profiling were performed. Here we show for the first time the metabolomics and molecular signature variation in AFSCs isolated in the sheep model of MMC, which may be used as diagnostic tools for the in utero identification of the neural tube damage. Intriguingly, PAX3 gene involved in the murine model for spina bifida is modulated in AFSCs reaching the peak of expression at 16 weeks of gestation, 4 weeks after the intervention. Our data strongly suggest that AFSCs reorganize their differentiation commitment in order to generate PAX3-expressing progenitors to counteract the MMC induced in the sheep model. The gene expression signature of AFSCs highlights the plasticity of these cells reflecting possible alterations of embryonic development.

Autologous Periosteum-Derived Micrografts and PLGA/HA Enhance the Bone Formation in Sinus Lift Augmentation

Sinus lift augmentation is a procedure required for the placement of a dental implant, whose success can be limited by the quantity or quality of available bone. To this purpose, the first aim of the current study was to evaluate the ability of autologous periosteum-derived micrografts and Poly(lactic-co-glycolic acid) (PLGA) supplemented with hydroxyl apatite (HA) to induce bone augmentation in the sinus lift procedure. Secondly, we compared the micrografts behavior with respect to biomaterial alone, including Bio-Oss® and PLGA/HA, commercially named Alos. Sinus lift procedure was performed on 24 patients who required dental implants and who, according to the study design and procedure performed, were divided into three groups: group A (Alos + periosteum-derived micrografts); group B (Alos alone); and group C (Bio-Oss® alone). Briefly, in group A, a small piece of periosteum was collected from each patient and mechanically disaggregated by Rigenera® protocol using the Rigeneracons medical device. This protocol allowed for the obtainment of autologous micrografts, which in turn were used to soak the Alos scaffold. At 6 months after the sinus lift procedure and before the installation of dental implants, histological and radiographic evaluations in all three groups were performed. In group A, where sinus lift augmentation was performed using periosteum-derived micrografts and Alos, the bone regeneration was much faster than in the control groups where it was performed with Alos or Bio-Oss® alone (groups B and C, respectively). In addition, the radiographic evaluation in the patients of group A showed a radio-opacity after 4 months, while after 6 months, the prosthetic rehabilitation was improved and was maintained after 2 years post-surgery. In summary, we report on the efficacy of periosteum-derived micrografts and Alos to augment sinus lift in patients requiring dental implants. This efficacy is supported by an increased percentage of vital mineralized tisssue in the group treated with both periosteum-derived micrografts and Alos, with respect to the control group of Alos or Bio-Oss® alone, as confirmed by histological analysis and radiographic evaluations at 6 months from treatment.

Mononucleated Cells to Regenerate Skeletal Muscle Syncytial Tissues

Skeletal muscle is one of the most plastic tissues of vertebrates since it may able upon exercises to double in size due to a physiological hypertrophy. Despite the fact that it is mainly a syncytial tissue, it contains a relevant number of mononucleated cells that can be involved in its homeostasis and repair. Although the mononuclear cell types with the highest myogenic potential are the satellite cells located underneath the basal lamina of muscle fibres, other interstitial cells have been shown to contribute to muscle regeneration. Adding complexity to this scenario is the fact that several authors revealed myogenic potential in pluripotent stem cells, which can be generated from patient somatic cells and eventually manipulated to correct the genetic defect. Notwithstanding the copiousness of myogenic cell types, their use in ex vivo cell therapies for muscular degenerative diseases is still questionable. However, new discovers on their biological properties have advanced our comprehension in handling myogenic stem cells significantly. In this review, we will focus on the myogenic potential of multi- and pluri-potent stem cells and their use in preclinical and clinical studies. New insights from direct reprogramming and epigenetic signalling to generate myogenic stem cells are also considered.

Reporter-based isolation of developmental myogenic progenitors

The formation and activity of mammalian tissues entail finely regulated processes, involving the concerted organization and interaction of multiple cell types. In recent years the prospective isolation of distinct progenitor and stem cell populations has become a powerful tool in the hands of developmental biologists and has rendered the investigation of their intrinsic properties possible. In this protocol, we describe how to purify progenitors with different lineage history and degree of differentiation from embryonic and fetal skeletal muscle by fluorescence-activated cell sorting (FACS). The approach takes advantage of a panel of murine strains expressing fluorescent reporter genes specifically in the myogenic progenitors. We provide a detailed description of the dissection procedures and of the enzymatic dissociation required to maximize the yield of mononucleated cells for subsequent FACS-based purification. The procedure takes ~6–7 h to complete and allows for the isolation and the subsequent molecular and phenotypic characterization of developmental myogenic progenitors.

Magic-F1 transgene cooperates with Pax 3 during early myogenesis to induce muscular hypertrophy

Met-Activating Genetically Improved Chimeric Factor-1 (Magic-F1) is a human recombinant protein derived from hepatocyte growth factor/scatter factor (HGF/ SF) and consists in two Met-binding domains repeated in tandem and separated by an artificial linker. It has a reduced affinity for Met and, in contrast to HGF, it elicits activation of the AKT but not the ERK signaling pathway. We recently showed that Magic-F1 induces muscle cell hypertrophy but not progenitor cell proliferation, both in vitro and in vivo where a transgenic mouse express the recombinant protein exclusively in skeletal muscle tissue [1]. Here, we examined the temporal and spatial expression pattern of Magic-F1 in comparison with Pax3 (paired box gene 3) transcription factor during embryogenesis [2]. Ranging from 9.5 to 17.5 dpc (days post coitum) mouse embryos were analyzed by in situ hybridization using whole mounts during early stages of development (9.5-10.5-11.5 dpc) and cryostat sections for later stages (11.5-13.5-15.5-17.5 dpc). We found that Magic-F1 is expressed in developing organs and tissues of mesenchymal origin, where Pax3 signal appears to be downregulated respect to the wt embryos. These data suggest that Magic-F1 could be responsible of muscular hypertrophy, cooperating with Pax3 signal pathway in skeletal muscle precursor cells.

Effect of global posture re-education on range of motion, pain and quality of life in adults individuals with chronic lower back pain: a randomized clinical trial

Global Postural Re-Education (RPG®) method, developed by the French physiotherapist Philippe E. Souchard, is based on progressing knowledge of human biomechanics and motor coordination, all applied to postural re-education and neuromusculoskeletal rehabilitation. It requires an active role to the patient can be applied to patients of any age, respecting each person’s capability and is indicated in for people presenting postural ailments and musculoskeletal disorder. In the same way, Mezieres Method end point is regain “body harmony”. Raggi® Method and Pancafit ® apply the principles previously theorized and combine them to diaphragmatic breathing. Our asses was to evaluate the effect of muscle chain stretching, as proposed by the global posture reeducation method, in the therapy of patients with chronic lower back pain. Eight patients (6 male and 2 female, aged 52.0 +/- 7.3 years old), performed global posture re-education using Pancafit according Raggi Method, once a week. The treatment program consisted of 1-hour individual sessions per week for eight weeks. Patients were evaluated before and after treatment and tested for Platform Lizard ( Stabilometry and postural assessment), pain intensity (by means of visual analog scale), range of motion (bending test), and health-related quality of life (by the SF-36 questionnaire). Significant pain relief and range of motion improvement were observed after treatment; quality of life also improved. Muscle chain stretching was effective in reducing pain and improving the range of motion and quality of life of patients with chronic lower back pain, suggesting that stretching exercises should be prescribed to chronic back pain patients.