M. G. Cusella De Angelis

Cardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: Implications for myocardium regeneration

The concept of tissue-restricted differentiation of postnatal stem cells has been challenged by recent evidence showing pluripotency for hematopoietic, mesenchymal, and neural stem cells. Furthermore, rare but well documented examples exist of already differentiated cells in developing mammals that change fate and trans-differentiate into another cell type. Here, we report that endothelial cells, either freshly isolated from embryonic vessels or established as homogenous cells in culture, differentiate into beating cardiomyocytes and express cardiac markers when cocultured with neonatal rat cardiomyocytes or when injected into postischemic adult mouse heart. Human umbilical vein endothelial cells also differentiate into cardiomyocytes under similar experimental conditions and transiently coexpress von Willebrand factor and sarcomeric myosin. In contrast, neural stem cells, which efficiently differentiate into skeletal muscle, differentiate into cardiomyocytes at a low rate. Fibroblast growth factor 2 and bone morphogenetic protein 4, which activate cardiac differentiation in embryonic cells, do not activate cardiogenesis in endothelial cells or stimulate trans-differentiation in coculture, suggesting that different signaling molecules are responsible for cardiac induction during embryogenesis and in successive periods of development. The fact that endothelial cells can generate cardiomyocytes sheds additional light on the plasticity of endothelial cells during development and opens perspectives for cell autologous replacement therapies.

The differentiation of human adipose-derived stem cells (hASCs) into osteoblasts is promoted by low amplitude, high frequency vibration treatment

Several studies have demonstrated that tissue culture conditions influence the differentiation of human adipose-derived stem cells (hASCs). Recently, studies performed on SAOS-2 and bone marrow stromal cells (BMSCs) have shown the effectiveness of high frequency vibration treatment on cell differentiation to osteoblasts. The aim of this study was to evaluate the effects of low amplitude, high frequency vibrations on the differentiation of hASCs toward bone tissue. In view of this goal, hASCs were cultured in proliferative or osteogenic media and stimulated daily at 30Hz for 45min for 28days. The state of calcification of the extracellular matrix was determined using the alizarin assay, while the expression of extracellular matrix and associated mRNA was determined by ELISA assays and quantitative RT-PCR (qRT-PCR). The results showed the osteogenic effect of high frequency vibration treatment in the early stages of hASC differentiation (after 14 and 21days). On the contrary, no additional significant differences were observed after 28days cell culture. Transmission Electron Microscopy (TEM) images performed on 21day samples showed evidence of structured collagen fibers in the treated samples. All together, these results demonstrate the effectiveness of high frequency vibration treatment on hASC differentiation toward osteoblasts.

The homeobox gene Arx is a novel positive regulator of embryonic myogenesis

Skeletal muscle fibers form in overlapping, but distinct phases that depend on the generation of temporally different lineages of myogenic cells. During primary myogenesis (E10.5–E12.5 in the mouse), embryonic myoblasts fuse homotypically to generate primary fibers, whereas during later development (E14.5–E17.5), fetal myoblasts differentiate into secondary fibers. How these myogenic waves are regulated remains largely unknown. Studies have been hampered by the lack of markers which would distinguish embryonic from fetal myoblast populations. We show here that the homeobox gene Arx is strongly expressed in differentiating embryonic muscle, downstream of myogenic basic helix–loop–helix (bHLH) genes. Its expression progressively decreases during development. When overexpressed in the C2C12 myogenic cell line, Arx enhances differentiation. Accordingly, it stimulates the transcriptional activity from the Myogenin promoter and from multimerized E-boxes when co-expressed with MyoD and Mef2C in CH310T1/2. Furthermore, Arx co-immunoprecipitates with Mef2C, suggesting that it participates in the transcriptional regulatory network acting in embryonic muscle. Finally, embryonic myoblasts isolated from Arx-deficient embryos show a delayed differentiation in vivo together with an enhanced clonogenic capacity in vitro. We propose here that Arx acts as a novel positive regulator of embryonic myogenesis by synergizing with Mef2C and MyoD and by establishing an activating loop with Myogenin.

Restoration of the GM2 Ganglioside Metabolism in Bone Marrow–Derived Stromal Cells from Tay-Sachs Disease Animal Model

The therapeutic potential of bone marrow–derived stromal cells for the therapy of Tay-Sachs disease is primarily related to the restoration of their own GM2 ganglioside storage. With this aim, we produced bone marrow–derived stromal cells from the adult Tay-Sachs animal model and transduced them with a retroviral vector encoding for the α-subunit of the lysosomal enzyme β-hexosaminidase A (E.C. 3.2.1.52). Our results demonstrate that transduced Tay-Sachs bone marrow–derived stromal cells have β-hexosaminidase A comparable to that of bone marrow-derived stromal cells from wild-type mice. Moreover, β-hexosaminidase A in transduced Tay-Sachs bone marrow-derived stromal cells was able to hydrolyze the GM2 ganglioside in a feeding experiment, thus demonstrating the correction of the altered phenotype.

Bone production by human maxillary sinus mucosa cells

The Schneider membrane is the mucosa that covers the inner part of the maxillary sinus cavities. The free surface is a ciliated pseudostratified epithelium, while the deeper portion is a highly vascularized connective tissue. The stromal fraction, bordering the bony wall of the sinus, after tooth loss can exhibit increased osteoclastic activity resulting in resorption of the bone in the posterior maxilla. Goal of our study was to isolate and characterize mesenchymal progenitors in the Schneiders membrane connective net and to evaluate their self ability to differentiate toward osteoblastic lineage, in absence of osteoinductive factors and osteoconductive biomaterials of support. This should indicate that maxillary sinus membrane represents an useful an approachable source of MSCs for bone tissue engineering and cell therapy and owns the intrinsic capacity to restore maxillary bone after tooth loss without the needing of biomaterials. J. Cell. Physiol. 227: 3278–3281, 2012.

High-Frequency Vibration Treatment of Human Bone Marrow Stromal Cells Increases Differentiation toward Bone Tissue

In order to verify whether differentiation of adult stem cells toward bone tissue is promoted by high-frequency vibration (HFV), bone marrow stromal cells (BMSCs) were mechanically stimulated with HFV (30 Hz) for 45 minutes a day for 21 or 40 days. Cells were seeded in osteogenic medium, which enhances differentiation towards bone tissue. The effects of the mechanical treatment on differentiation were measured by Alizarin Red test, (q) real-time PCR, and protein content of the extracellular matrix. In addition, we analyzed the proliferation rate and apoptosis of BMSC subjected to mechanical stimulation. A strong increase in all parameters characterizing differentiation was observed. Deposition of calcium was almost double in the treated samples; the expression of genes involved in later differentiation was significantly increased and protein content was higher for all osteogenic proteins. Lastly, proliferation results indicated that stimulated BMSCs have a decreased growth rate in comparison with controls, but both treated and untreated cells do not enter the apoptosis process. These findings could reduce the gap between research and clinical application for bone substitutes derived from patient cells by improving the differentiation protocol for autologous cells and a further implant of the bone graft into the patient.

High frequency vibration (HFV) induces muscle hypertrophy in newborn mice and enhances primary myoblasts fusion in satellite cells

The present study aimed at verifying “in vivo” and “in vitro” the effects of mechanical vibrations on muscle development and on differentiation of satellite cells, the “stem cells” of muscle tissue. We realized a bioreactor composed by an eccentric motor which produces a displacement of 11 mm at frequencies between 1 and 120 Hz on a plate connected to the motor. On the plate we fixed a cage used for animals and the dishes for satellite cells and linear acceleration provoked by the motor to samples was measured. We used 30 Hz as stimulating frequency and we treated newborn mice from their birth for the next four weeks one 1h/day and satellite cells 1 and 4 days 1h/day always at 30 Hz. Every week we collected from a control and a treated mouse tibialis anterior muscles and we performed Western Blot analysis and quantitative Real-Time PCR (qRT-PCR) to investigate proteins and genes involved in hypertrophy and atrophy pathways of skeletal muscle. On satellite cells we studied some genes involved in differentiation and fusion of primary myoblasts. Results demonstrate that mechanical vibration induces muscle hypertrophy within the first week of treatment and enhances terminal differentiation of myoblasts of treated satellite cells respect to control ones.

High frequency mechanical vibrations stimulate the bone matrix formation in hBMSCs (human Bone Marrow Stromal Cells)

The aim of this work is to test the effects of a specific mechanical stimulation (Low Amplitude, high Frequency Vibrations) on the bone matrix formation of hBMSCs. Previous studies demonstrated that chemical culture conditions could influence the differentiation of hBMSCs toward bone: by plating the cells in appropriate osteogenic culture medium hBMSCs differentiate into osteoblasts [1-3].

An “in vivo” Study of High Frequency Vibration on Muscle Development

The present study aimed at verifying “in vivo” the effects of mechanical vibrations on muscle development. We realized a bioreactor composed by an eccentric motor which produces a displacement of 11 mm at frequencies between 1 and 120 Hz on a plate connected to the motor. On the plate we fixed a cage used for animals and the linear acceleration provoked by the motor to the cage was measured. We used 30 Hz as stimulating frequency and we treated newborn mice from their birth for the next five weeks one hour/day. Every week we collected from a control and a treated mouse tibials and quadriceps anterior muscles and we performed morfometric analysis on muscle fibers. Moreover, with PCR and Real-Time PCR (RT-PCR) we analysed for the second and the third week the expression of genes involved in muscle differentiation. Preliminary results suggest that whole-vibration treatment increases the rate of proliferation of muscle fibers with respect to the control, while from the third week the total number of fibers of treated or non-treated mice tend to balance. The results of RT-PCR of some of the genes involved in terminal differentiation demonstrate that mechanical vibration seems to increase the rate of differentiation of the treated muscles with respect to the control ones.

A Comparison between the Proliferation Rate of SAOS-2 Human Osteoblasts and BMSCs (Bone Marrow Stromal Cells) Using Mathematical Models

The aim of our work is to find a time scale factor between the proliferation models of SAOS-2 human osteoblasts, a tumorigenic osteoblasts-like cell line, and BMSCs (bone marrow stromal cells). In order to perform this study we previously took the two cell lines to the same differentiation stage, by using the deposition of calcium on cellular matrix as indicator. Afterwards the same number of cells for both the SAOS-2 and BMSCs were plated, and we followed the growth in number of cells for four weeks, by stopping and counting them at 0, 7, 14, 21 and 28 days after the beginning of the experiment. The results were analyzed by Matlab 7.1 software and we tested a sigmoidal curve fitting and a linear model for the two cellular growths. The errors between the data and the curve fitting were calculated. In order to find a coefficient to represent the scale factor between the proliferation of SAOS-2 and BMSCs, the rate between the two amplification coefficients of the sigmoidal model and the rate between the two angular coefficients of the linear curves were calculated. The two scale factors are very similar (between 5.08 and 5.5) and we used this value to adapt the duration of the experiments previously completed on SAOS-2, to BMSCs.