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.

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 Schneider's 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.

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.

Cell therapy of primary myopathies.

Mesoangioblasts are multipotent progenitors of mesodermal tissues. In vitro mesoangioblasts differentiate into many mesoderm cell types, such as smooth, cardiac and striated muscle, bone and endothelium. After transplantation mesoangioblasts colonize mostly mesoderm tissues and differentiate into many cell types of the mesoderm. When delivered through the arterial circulation, mesoangioblasts significantly restore skeletal muscle structure and function in a mouse model of muscular dystrophy. Their ability to extensively self-renew in vitro, while retaining multipotency, qualifies mesoangioblasts as a novel class of stem cells. Phenotype, properties and possible origin of mesoangioblasts are addressed in the first part of this paper. In the second part we will focus on the cell therapy approach for the treatment of Muscular Dystrophy and we will describe why mesangioblasts appear to be promising candidates for this strategy.

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 alpha-subunit of the lysosomal enzyme beta-hexosaminidase A (E.C. 3.2.1.52). Our results demonstrate that transduced Tay-Sachs bone marrow-derived stromal cells have beta-hexosaminidase A comparable to that of bone marrow-derived stromal cells from wild-type mice. Moreover, beta-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.

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 homogeneous 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.

Determination, diversification and multipotency of mammalian myogenic cells.

In amniotes, myogenic commitment appears to be dependent upon signaling from neural tube and dorsal ectoderm, that can be replaced by members of the Wnt family and by Sonic hedgehog. Once committed, myoblasts undergo different fates, in that they can differentiate immediately to form the myotome, or later to give rise to primary and secondary muscle fibers. With fiber maturation, satellite cells are first detected; these cells contribute to fiber growth and regeneration during post-natal life. We will describe recent data, mainly from our laboratory, that suggest a different origin for some of the cells that are incorporated into the muscle fibers during late development. We propose the possibility that these myogenic cells are derived from the vasculature, are multi-potent and become committed to myogenesis by local signaling, when ingressing a differentiating muscle tissue. The implications for fetal and perinatal development of the whole mesoderm will also be discussed.

Ultrastructural changes in human gingival fibroblasts in vitro after exposure to vapour phase smoke components.

Tobacco and some of its volatile and non-volatile components have been found to affect many types of cells including gingival fibroblasts. Because normal gingival fibroblast functioning is fundamental to the maintenance of the oral connective tissue as well as to wound healing, we examined the effect of two vapour phase smoke components (acrolein and acetaldehyde) on proliferation and ultrastructure of human gingival fibroblasts (HGFs) in culture. A human gingival fibroblast strain derived from healthy individuals was used in this study. The cells were incubated in the presence of different concentrations of acrolein and acetaldehyde and cell proliferation and fine morphology were evaluated. The results show that acrolein and acetaldehyde produced dose dependent inhibition of HGF viability and alteration of cytoplasmic organelles. The main ultrastructural finding for the HGF cytoplasm was the presence of vacuoles and lysosomal structures which became prominent with increasing concentration of acrolein and acetaldehyde. Our results suggest that the ultrastructural alterations we observed in HGFs may be due to the uptake and storage of acrolein and acetaldehyde by the cells.

Skeletal myogenic progenitors originating from embryonic dorsal aorta coexpress endothelial and myogenic markers and contribute to postnatal muscle growth and regeneration.

Skeletal muscle in vertebrates is derived from somites, epithelial structures of the paraxial mesoderm, yet many unrelated reports describe the occasional appearance of myogenic cells from tissues of nonsomite origin, suggesting either transdifferentiation or the persistence of a multipotent progenitor. Here, we show that clonable skeletal myogenic cells are present in the embryonic dorsal aorta of mouse embryos. This finding is based on a detailed clonal analysis of different tissue anlagen at various developmental stages. In vitro, these myogenic cells show the same morphology as satellite cells derived from adult skeletal muscle, and express a number of myogenic and endothelial markers. Surprisingly, the latter are also expressed by adult satellite cells. Furthermore, it is possible to clone myogenic cells from limbs of mutant c-Met-/- embryos, which lack appendicular muscles, but have a normal vascular system. Upon transplantation, aorta-derived myogenic cells participate in postnatal muscle growth and regeneration, and fuse with resident satellite cells.The potential of the vascular system to generate skeletal muscle cells may explain observations of nonsomite skeletal myogenesis and raises the possibility that a subset of satellite cells may derive from the vascular system.

High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies.

Ex vivo gene therapy of primary myopathies, based on autologous transplantation of genetically modified myogenic cells, is seriously limited by the number of primary myogenic cells that can be isolated, expanded, transduced, and reimplanted into the patient's muscles. We explored the possibility of using the MyoD gene to induce myogenic conversion of nonmuscle, primary cells in a quantitatively relevant fashion. Primary human and murine fibroblasts from skin, muscle, or bone marrow were infected by an E1-deleted adenoviral vector carrying a retroviral long terminal repeat-promoted MyoD cDNA. Expression of MyoD caused irreversible withdrawal from the cell cycle and myogenic differentiation in the majority (from 60 to 90%) of cultured fibroblasts, as defined by activation of muscle-specific genes, fusion into contractile myotubes, and appearance of ultrastructurally normal sarcomagenesis in culture. 24 h after adenoviral exposure, MyoD-converted cultures were injected into regenerating muscle of immunodeficient (severe combined immunodeficiency/beige) mice, where they gave rise to beta-galactosidase positive, centrally nucleated fibers expressing human myosin heavy chains. Fibers originating from converted fibroblasts were indistinguishable from those obtained by injection of control cultures of lacZ-transduced satellite cells. MyoD-converted murine fibroblasts participated to muscle regeneration also in immunocompetent, syngeneic mice. Although antibodies from these mice bound to adenoviral infected cells in vitro, no inflammatory infiltrate was present in the graft site throughout the 3-wk study period. These data support the feasibility of an alternative approach to gene therapy of primary myopathies, based on implantation of large numbers of genetically modified primary fibroblasts massively converted to myogenesis by adenoviral delivery of MyoD ex vivo.

Absence of MEF2 binding to the A/T-rich element in the muscle creatine kinase (MCK) enhancer correlates with lack of early expression of the MCK gene in embryonic mammalian muscle.

During skeletal muscle development, different types of muscle fibers are generated, which express different combinations of muscle-specific gene products. For example, the muscle creatine kinase gene (MCK) is highly expressed in fetal but not embryonic myotubes. We performed transient transfections of CAT reporter constructs, driven by the MCK promoter with variable lengths of 5'-flanking sequence, into primary cultures of embryonic and fetal muscle cells. Reporter activity was observed in fetal but not embryonic muscle cells. We assayed the ability of nuclear extracts prepared from embryonic and fetal muscle and C2C12 myotubes to bind specific regulatory elements in the MCK enhancer. The profile of DNA/protein complexes resulting from electrophoretic mobility shift assays was qualitatively the same with all extracts used when the oligonucleotide probes represented the MCK-E-box, MHox site, CArG-box, and AP2 site. In contrast, no binding activity to the MEF2 site was observed with embryonic nuclear extract. Interestingly, MEF2 mRNAs and proteins were detected in both fetal and embryonic muscle, with the exception of the MEF2D1b isoform, which is restricted to fetal muscle. Furthermore, we found that protein phosphatase inhibitors included in the preparation of embryonic nuclear extracts or added to the medium of transfected embryonic myotubes can restore MEF2 DNA binding activity, as well as reporter activity driven by the MCK promoter and partial transcriptional activation of the endogenous MCK gene. We propose that phosphorylation of MEF2 regulates its activity and represents an important aspect of the mechanism controlling stage-specific transcription during skeletal myogenesis.

Enhanced expression of myogenic regulatory genes in aging skeletal muscle.

MyoD, myogenin, myf-5, and MRF4, belonging to the family of basic helix-loop-helix (bHLH) myogenic regulatory factors (MRFs), control muscle cell differentiation, in concert with other transcription factors such as MEF-2, yet their role in age-related skeletal muscle alteration has not been addressed. We here report that MyoD and myogenin transcripts are expressed at high levels in the hind limb muscles of newborn mice and their level of expression continuously declines throughout postnatal life to become virtually undetectable in the adult mouse. However, these transcripts are again expressed at high levels in the muscles of older mice. MRF4 transcript, on the other hand, is present at a constant level throughout the life span of the animal. Conversely, the expressions of myf-5 and MEF-2C, components of the autoregulatory loop for the activation of bHLH gene expression, conspicuously increase in adult and senile muscle. In order to establish whether these transcripts are functioning in the aged muscle we investigated the expression of bHLH inhibitory factor Id mRNA showing that it does not present significant changes during aging. Immunofluorescence analysis with an anti-myogenin antibody revealed nuclear accumulation of the protein in the muscle fibers of old, but not of adult, mice. Muscle-specific genes transactivated by MyoD and myogenin such as AChR, MLC, and MCK are also up-regulated during aging, albeit at a lower level. Significant changes in the size and ratio of type I/type II fibers are detectable in senile muscle. These findings show that all members of the MRF family are expressed to a high extent and are likely active in senile muscle. It is conceivable that these changes might operate as a compensatory mechanism in maintaining the expression of differentiated muscle products in senile muscle at a steady-state level.

Differential response of embryonic and fetal myoblasts to TGF beta: a possible regulatory mechanism of skeletal muscle histogenesis.

Embryonic and fetal skeletal myoblasts were grown in culture in the presence of TGF beta. Under the conditions employed, TGF beta inhibited differentiation of fetal but not of embryonic myoblasts. To investigate the possible relevance of these data to skeletal muscle histogenesis in vivo, we studied the proliferation/differentiation state of mesodermal cells in the proximal region of the limb bud at the time of primary fiber formation. BrdU labeling and immunostaining for myosin heavy chains revealed that very few mesodermal cells enter the S phase of the cycle when differentiated primary fibers first appear. However, a few hours later, many cells in S phase surround newly formed muscle fibers, suggesting that the latter may be a source of mitogens for undifferentiated myoblasts. Co-culture experiments supported this hypothesis, showing that medium conditioned by fiber-containing explants can stimulate myoblast proliferation. Taken together these data suggested a possible mechanism for the regulation of muscle fiber formation. The model assumes that fibers form in the proximal region of the limb bud, where TGF beta is known to be present, and BrdU labeling experiments did not reveal cells in S phase. It is conceivable that non-dividing embryonic myoblasts (which do not respond to TGF beta) can undergo differentiation, while fetal myoblasts are inhibited by TGF beta. Once formed, primary fibers may stimulate a new wave of proliferation in fetal myoblasts, in order to expand the pool of cells needed to form secondary fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Pro-opiomelanocortin gene is expressed in post-implantation mouse embryos and enhances growth potential of myogenic cells.

We previously showed that the adrenocorticotropin hormone (ACTH) is a mitogen for myoblasts and is present in post-implantation mammalian embryos (Cossu et al. [1989] Dev. Biol. 131:331-336; De Angelis et al. [1992] Dev. Biol. 151:446-458). In this paper, we investigated the expression of the corresponding gene, pro-opiomelanocortin (POMC), by in situ hybridization and polymerase chain reaction. In situ analysis revealed low level expression in the basal layer of 10.5 d.p.c. neural tube and in several discrete areas around the dorsal aorta. By more sensitive Reverse-transcription polymerase chain reaction (RT-PCR) analysis, expression was detected also in developing limb buds and in cultured myogenic cells, but not in fibroblasts. To investigate the possible role of POMC gene expression in myogenesis, we induced its over-expression in proliferating myoblasts. Upon sub-optimal growth conditions, over-expressing cells were found to give rise to clones larger than control cells. The differentiation potential of POMC over-expressing myogenic cells was unchanged.

ACTH-like peptides in postimplantation mouse embryos: a possible role in myoblast proliferation and muscle histogenesis.

ACTH and related peptides are mitogens for certain mesodermal cell types such as adrenocortical cells, T-lymphocytes, and skeletal myoblasts. In order to postulate a possible physiological role for these peptides in skeletal muscle histogenesis, it is necessary to establish whether they are present in muscle forming anlagens of postimplantation mouse embryos. By radioimmunoassay and immunofluorescence with antibodies specific for ACTH, we have detected these peptides in many areas of mouse embryos including neural tube, limb buds, eye lens, and myotomal muscles. During fetal development, immunoreactivity decreased in muscle tissue and appeared in visceral ganglia. Furthermore, primary myotubes or C2C12 myotubes, but not muscle or 3T3 fibroblasts, release significant levels of ACTH immunoreactive peptides into the culture medium. Using a microassay for mitogen production, primary myotubes or C2C12 myotubes, but not other mesodermal cells (with the exception of dermal fibroblasts) were shown to release factors into the medium which support myoblast proliferation. Neutralizing antibodies against ACTH inhibit myoblast but not fibroblast proliferation in a dose-dependent fashion. Based on these results, we propose that myotube-derived mitogens (including ACTH-like peptides) promote the proliferation of surrounding myoblast during muscle histogenesis in vivo.

The effects of AZT and DDI on pre- and postimplantation mammalian embryos: an in vivo and in vitro study.

This study reports the effects of the nucleoside analogs dideoxyinosine (DDI) and 3'-azido-3'-deoxythymidine (AZT) on mammalian embryonic development. When administered to pregnant mice (at concentrations ranging from 10 to 300 mg/kg/day), through all or part of gestation, AZT and DDI did not result in any visible effect on mouse embryos nor did they cause any obvious malformation or defect at birth or during postnatal growth. Similarly, when embryonic or fetal mouse or human cells (from brain, limb buds, or different organ rudiments) were exposed to AZT or DDI in vitro, cytotoxicity was observed only in the mM range, with AZT showing slightly higher cytotoxicity and brain cells appearing slightly more sensitive to both nucleosides. However, even in cultures treated with very high concentrations of AZT or DDI, the reduction in the number of terminally differentiated skeletal myotubes, cardiocytes, neurons, and chondrocytes was similar to the reduction in the total number of cells, indicating that AZT and DDI did not selectively inhibit differentiation of any of the above-mentioned cell types. Finally, preimplantation mouse embryos (at the 2-cell or 4-cell stage), treated in vitro with micromolar concentrations of AZT, were arrested at the 4-cell stage. DDI or other nucleoside analogs tested did not have this effect.

MyoD, myogenin independent differentiation of primordial myoblasts in mouse somites.

The accumulation of two myogenic regulatory proteins, MyoD and myogenin, was investigated by double-immunocytochemistry and correlated with myosin heavy chain expression in different classes of myoblasts in culture and during early myogenesis in vivo. During in vitro differentiation of fetal myoblasts, MyoD-positive cells were detected first, followed by the appearance of cells positive for both MyoD and myogenin and finally by the appearance of differentiated myocytes and myotubes expressing myosin heavy chain (MHC). A similar pattern of expression was observed in cultures of embryonic and satellite cells. In contrast, most myogenic cells isolated from newly formed somites, expressed MHC in the absence of detectable levels of myogenin or MyoD. In vivo, the appearance of both myogenin and MyoD proteins was only detected at 10.5 d postcoitum (d.p.c.), when terminally differentiated muscle cells could already be identified in the myotome. Parasagittal sections of the caudal myotomes of 10.5-d-old embryos showed that expression of contractile proteins preceded the expression of myogenin or MyoD and, when coexpressed, MHC and myogenin did not co-localize within all the cells of the myotome. In the limb bud, however, many myogenin (or MyoD) positive/MHC negative cells could be observed in the proximal region at day 11. During further embryonic development the expression of these proteins remained constant in all the muscle anlagens examined, decreasing to a low level during the late fetal period. Western and Northern analysis confirmed that the myogenin protein could only be detected after 10.5 d.p.c. while the corresponding message was clearly present at 9.5 d.p.c., strongly suggesting a posttranscriptional regulation of myogenin during this stage of embryonic development. These data show that the first myogenic cells which appear in the mouse myotome, and can be cultured from it, accumulate muscle structural proteins in their cytoplasm without expressing detectable levels of myogenin protein (although the message is clearly accumulated). Neither MyoD message or protein are detectable in these cells, which may represent a distinct myogenic population whose role in development remains to be established.

Adrenocorticotropin is a specific mitogen for mammalian myogenic cells.

Peptides derived from proopiomelanocortin (POMC) have been found to stimulate the proliferation of murine myogenic cells. Among these peptides, adrenocorticotropin (ACTH) and alpha-, beta-, and gamma-melanocyte-stimulating hormones (MSH) were found to be active, whereas the opioid peptides were not. At clonal density, both ACTH and MSH caused a three- to fourfold increase in the average number of cells per clone in myogenic but not in fibroblast colonies. At high cell density, ACTH and MSH caused a three- to fourfold increase in proliferation of myogenic cells, reflected by an increased accumulation of skeletal myosin. On the other hand mouse embryo skin or muscle fibroblasts or vertebral chondroblasts did not increase proliferation in response to POMC-derived peptides. The half-maximal dose at which ACTH stimulated myoblast proliferation was around 5 nM, and the mitogenic effect was doubled by suboptimal doses of fibroblast growth factor. The possible physiological significance of the mitogenic effect of ACTH on myogenic cells is discussed.