Flavio Ronzoni

Cellular mechanisms and local progenitor activation to regulate skeletal muscle mass

Skeletal muscle hypertrophy is a result of increased load, such as functional and stretch-overload. Activation of satellite cells and proliferation, differentiation and fusion are required for hypertrophy of overloaded skeletal muscles. On the contrary, a dramatic loss of skeletal muscle mass determines atrophy settings. The epigenetic changes involved in gene regulation at DNA and chromatin level are critical for the opposing phenomena, muscle growth and atrophy. Physiological properties of skeletal muscle tissue play a fundamental role in health and disease since it is the most abundant tissue in mammals. In fact, protein synthesis and degradation are finely modulated to maintain an appropriate muscle mass. When the molecular signaling is altered muscle wasting and weakness occurred, and this happened in most common inherited and acquired disorders such as muscular dystrophies, cachexia, and age-related wasting. To date, there is no accepted treatment to improve muscle size and strength, and these conditions pose a considerable anxiety to patients as well as to public health. Several molecules, including Magic-F1, myostatin inhibitor, IGF, glucocorticoids and microRNAs are currently investigated to interfere positively in the blueprint of skeletal muscle growth and regeneration.

miR669a and miR669q prevent skeletal muscle differentiation in postnatal cardiac progenitors

miR669a and miR669q inhibit postnatal cardiac progenitor differentiation by directly targeting the 3′UTR of MyoD.

Alpha sarcoglycan is required for FGF-dependent myogenic progenitor cell proliferation in vitro and in vivo

Mice deficient in α-sarcoglycan (Sgca-null mice) develop progressive muscular dystrophy and serve as a model for human limb girdle muscular dystrophy type 2D. Sgca-null mice suffer a more severe myopathy than that of mdx mice, the model for Duchenne muscular dystrophy. This is the opposite of what is observed in humans and the reason for this is unknown. In an attempt to understand the cellular basis of this severe muscular dystrophy, we isolated clonal populations of myogenic progenitor cells (MPCs), the resident postnatal muscle progenitors of dystrophic and wild-type mice. MPCs from Sgca-null mice generated much smaller clones than MPCs from wild-type or mdx dystrophic mice. Impaired proliferation of Sgca-null myogenic precursors was confirmed by single fiber analysis and this difference correlated with Sgca expression during MPC proliferation. In the absence of dystrophin and associated proteins, which are only expressed after differentiation, SGCA complexes with and stabilizes FGFR1. Deficiency of Sgca leads to an absence of FGFR1 expression at the membrane and impaired MPC proliferation in response to bFGF. The low proliferation rate of Sgca-null MPCs was rescued by transduction with Sgca-expressing lentiviral vectors. When transplanted into dystrophic muscle, Sgca-null MPCs exhibited reduced engraftment. The reduced proliferative ability of Sgca-null MPCs explains, at least in part, the severity of this muscular dystrophy and also why wild-type donor progenitor cells engraft efficiently and consequently ameliorate disease.

In vitro osteoblastic differentiation of human mesenchymal stem cells and human dental pulp stem cells on poly-L-lysine-treated titanium-6-aluminium-4-vanadium

Three-dimensional (3D) titanium-6-aluminium-4-vanadium (Ti6Al4V) is a widely used biomaterial for orthopedic prosthesis and dental implants; thanks to its very high-mechanical strength and resistance to corrosion. Human mesenchymal stem cells (hMSCs) and dental pulp stem cells (hDPSCs) are responsible for bone regeneration following colonization of prosthesis or dental implants. Both hMSCs and hDPSCs have lower ability to colonize this biomaterial in comparison with tissue culture-treated plastic. Both hMSCs and hDPSCs show lack of focal adhesion kinase (FAK) activation when grown on Ti6Al4V. This signal is restored in the presence of poly-L-lysine (poly-L-lys). Poly-L-lys has been used as part of organoapatite or together with zinc and calcium ions. Our results suggest that poly-L-lys alone induces FAK activation through β1-INTEGRIN, because the presence of β1-INTEGRIN blocking antibody avoided FAK autophosphorylation. Presence of poly-L-lys also increases expression of osteoblastic differentiation marker genes in hMSCs and hDPSCs grown on Ti6Al4V.

Perturbations of Heart Development and Function in Cardiomyocytes from Human Embryonic Stem Cells with Trisomy 21

Congenital heart defects (CHD) occur in approximately 50% of patients with Down syndrome (DS); the mechanisms for this occurrence however remain unknown. In order to understand how these defects evolve in early development in DS, we focused on the earliest stages of cardiogenesis to ascertain perturbations in development leading to CHD. Using a trisomy 21 (T21) sibling human embryonic stem cell (hESC) model of DS, we show that T21‐hESC display many significant differences in expression of genes and cell populations associated with mesodermal, and more notably, secondary heart field (SHF) development, in particular a reduced number of ISL1+ progenitor cells. Furthermore, we provide evidence for two candidate genes located on chromosome 21, ETS2 and ERG, whose overexpression during cardiac commitment likely account for the disruption of SHF development, as revealed by downregulation or overexpression experiments. Additionally, we uncover an abnormal electrophysiological phenotype in functional T21 cardiomyocytes, a result further supported by mRNA expression data acquired using RNA‐Seq. These data, in combination, revealed a cardiomyocyte‐specific phenotype in T21 cardiomyocytes, likely due to the overexpression of genes such as RYR2, NCX, and L‐type Ca2+ channel. These results contribute to the understanding of the mechanisms involved in the development of CHD. Stem Cells 2015;33:1434–1446

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.

In Vitro and In Vivo Studies of Alar-Nasal Cartilage Using Autologous Micro-Grafts: The Use of the Rigenera® Protocol in the Treatment of an Osteochondral Lesion of the Nose

Cartilage defects represent a serious problem due to the poor regenerative properties of this tissue. Regarding the nose, nasal valve collapse is associated with nasal blockage and persistent airway obstruction associated with a significant drop in the quality of life for patients. In addition to surgical techniques, several cell-based tissue-engineering strategies are studied to improve cartilage support in the nasal wall, that is, to ameliorate wall insufficiency. Nevertheless, there are no congruent data available on the benefit for patients during the follow-up time. In this manuscript, we propose an innovative approach in the treatment of cartilage defects in the nose (nasal valve collapse) based on autologous micro-grafts obtained by mechanical disaggregation of a small portion of cartilage tissue (Rigenera® protocol). In particular, we first analyzed in vitro murine and human cartilage micro-grafts; secondly, we analyzed the clinical results of a patient with pinched nose deformity treated with autologous micro-grafts of chondrocytes obtained by Rigenera® protocol. The use of autologous micro-graft produced promising results in surgery treatment of cartilage injuries and could be safely and easily administrated to patients with cartilage tissue defects.

Localization of Magic-F1 Transgene, Involved in Muscular Hypertrophy, during Early Myogenesis

We recently showed that Magic-F1 (Met-activating genetically improved chimeric factor 1), a human recombinant protein derived from hepatocyte growth factor/scatter factor (HGF/SF) induces muscle cell hypertrophy but not progenitor cell proliferation, both in vitro and in vivo. Here, we examined the temporal and spatial expression pattern of Magic-F1 in comparison with Pax3 (paired box gene 3) transcription factor during embryogenesis. 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.

Myogenic induction of adult and pluripotent stem cells using recombinant proteins

Met Activating Genetically Improved Chimeric Factor 1 (Magic-F1) is a human recombinant protein, derived from dimerization of the receptor-binding domain of hepatocyte growth factor. Previous experiments demonstrate that in transgenic mice, the skeletal muscle specific expression of Magic-F1 can induce a constitutive muscular hypertrophy, improving running performance and accelerating muscle regeneration after injury. In order to evaluate the therapeutic potential of Magic-F1, we tested its effect on multipotent and pluripotent stem cells. In murine mesoangioblasts (adult vessel-associated stem cells), the presence of Magic-F1 did not alter their osteogenic, adipogenic or smooth muscle differentiation ability. However, when analyzing their myogenic potential, mesoangioblasts expressing Magic-F1 differentiated spontaneously into myotubes. Finally, Magic-F1 inducible cassette was inserted into a murine embryonic stem cell line by homologous recombination. When embryonic stem cells were subjected to myogenic differentiation, the presence of Magic-F1 resulted in the upregulation of Pax3 and Pax7 that enhanced the myogenic commitment of transgenic pluripotent stem cells. Taken together our results candidate Magic-F1 as a potent myogenic stimulator, able to enhance muscular differentiation from both adult and pluripotent stem cells.

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.