Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest

Most cell culture studies have been performed at atmospheric oxygen tension of 21%, however the physiological oxygen tension is much lower and is a factor that may affect skeletal muscle myoblasts. In this study we have compared activation of G0 arrested myoblasts in 21% O2 and in 1% O2 in order to see how oxygen tension affects activation and proliferation of human myoblasts.Human myoblasts were isolated from skeletal muscle tissue and G0 arrested in vitro followed by reactivation at 21% O2 and 1% O2. The effect was assesses by Real-time RT-PCR, immunocytochemistry and western blot.We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and β-catenin indicated that notch signaling may be induced in 21% O2, while the canonical Wnt signaling may be induced in 1% O2 during activation and proliferation of myoblasts

Injectable scaffold materials differ in their cell instructive effects on primary human myoblasts

Scaffolds are materials used for delivery of cells for regeneration of tissues. They support three-dimensional organization and improve cell survival. For the repair of small skeletal muscles, injections of small volumes of cells are attractive, and injectable scaffolds for delivery of cells offer a minimally invasive technique. In this study, we examined in vitro the cell instructive effects of three types of injectable scaffolds, fibrin, alginate, and poly(lactic-co-glycolic acid)-based microparticles on primary human myoblasts. The myoblast morphology and progression in the myogenic program differed, depending on the type of scaffold material. In alginate gel, the cells obtained a round morphology, they ceased to proliferate, and entered quiescence. In the fibrin gels, differentiation was promoted, and myotubes were observed within a few days in culture, while poly(lactic-co-glycolic acid)-based microparticles supported prolonged proliferation. Myoblasts released from the alginate and fibrin gels were studied, and cells released from these scaffolds had retained the ability to proliferate and differentiate. Thus, the study shows that human myogenic cells combined with injectable scaffold materials are guided into different states depending on the choice of scaffold. This opens for in vivo experiments, including testing of the significance of the cell state on regeneration potential of primary human myoblasts

Fibrocytes in early and long-standing rheumatoid arthritis: a 6-month trial with repeated synovial biopsy, imaging and lung function test

Objectives To correlate the level of fibrocytes in peripheral blood, synovial tissue and in vitro culture in rheumatoid arthritis (RA) with changes in disease activity, imaging and pulmonary function.Methods Twenty patients with early RA (ERA) and 20 patients with long-standing RA (LRA) were enrolled in a 6-month prospective study. Sixteen patients undergoing wrist arthroscopy were healthy controls. Patients with RA underwent pulmonary function tests, ultrasound and synovial ultrasound-guided needle biopsy of the same wrist at baseline and 6 months. Wrist MRI was performed at baseline (all) and 6 months (ERA). Circulating fibrocytes were measured by flow cytometry, in vitro by the number of monocytes that were differentiated to fibrocytes and in synovial biopsies by counting in histological sections.Results Fibrocytes were primarily located around vessels and in the subintimal area in the synovium. Fibrocyte levels did not decline during the trial despite effective RA treatment. In the ERA group, increased synovitis assessed by ultrasound was moderate and strongly correlated with an increase in circulating and synovial fibrocyte levels, respectively. Increased synovitis assessed by MRI during the trial in the ERA group was moderately correlated with both increased numbers of circulating and cultured fibrocytes. Absolute diffusion capacity level was overall weakly negatively correlated with the level of circulating and synovial fibrocytes. The decline in diffusion capacity during the trial was moderately correlated with increased levels of synovial fibrocytes.Conclusion Our findings suggest that fibrocytes are involved in RA pathogenesis, both in the synovium and the reduction in lung function seen in a part of patients with RA.Trial registration number NCT02652299

Immunostainings for NCAM and DES were performed on myoblasts activated from G₀ arrest under hypoxic and normoxic conditions.

<p>We found large number of cells expressing both NCAM and DES, however, when quantitating the fraction of NCAM and DES positive cells, we found no consistent result pointed towards an up regulation in 1% O2, as seen in the <i>NCAM</i> and <i>DES</i> gene expressions. Scale bar: 50 μm.</p

The expression of <i>PAX3</i>, <i>PAX7</i>, MRFs and genes involved in myogenesis were studied in human myoblasts re-activation from G₀-arrest in hypoxic and normoxic culture conditions.

<p>The up regulation of <i>PAX7</i> was highly significant in 1% O₂ for all three days in hypoxic conditions, while <i>PAX3</i> was increased only on day one. <i>MYOD</i> and <i>MYOG</i> were up regulated in 1% O₂, whereas <i>MYF5</i> and <i>MYF6</i> had similar expression levels at 1% O₂ and 21% O₂. The myogenesis related genes <i>C-MET</i>, <i>MEF2A</i>, <i>MEF2C</i>, <i>NCAM</i>, <i>DES</i> and <i>CDH15</i> all had increased expression levels in 1% O₂. Thus, most of the genes involved in the myogenic programme were induced at 1% O₂. Filled symbols: 1% O₂; Empty symbols: 21% O₂. Significance level: * p<0.05, ** p<0.01.</p

Overview of myogenic cell culture studies conducted with different oxygen tensions.

<p>Overview of myogenic cell culture studies conducted with different oxygen tensions.</p

Myoblast purity and proliferation rate.

<p>The purity of the isolated myoblasts was tested by desmin stainings and differentiation assays (A). Almost all of the isolated cells were desmin positive confirming a high level of myoblast purity and the myoblasts were able to form large multinucleated myofiber when cultured in differentiation medium. Proliferation rate of myoblasts in 1% O₂ and 21% O₂, respective, was measured by proliferation assays (three days) and colony forming assays (14 days). The short-term proliferation rate demonstrated no difference in myoblast proliferation (B). The colony forming assays (crystal violet staining) (C) demonstrated no difference in the number of colonies formed by myoblasts in 1% and 21% O₂, however, the colonies formed in 1% O₂ were bigger and had a higher cell density, thus demonstrating an induced proliferation. Scale bar: 400 μm.</p