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

A novel in vitro model for studying quiescence and activation of primary isolated human myoblasts

Skeletal muscle stem cells, satellite cells, are normally quiescent but become activated upon muscle injury. Recruitment of resident satellite cells may be a useful strategy for treatment of muscle disorders, but little is known about gene expression in quiescent human satellite cells or the mechanisms involved in their early activation. We have developed a method to induce quiescence in purified primary human myoblasts isolated from healthy individuals. Analysis of the resting state showed absence of BrdU incorporation and lack of KI67 expression, as well as the extended kinetics during synchronous reactivation into the cell cycle, confirming arrest in the G0 phase. Reactivation studies showed that the majority (>95%) of the G0 arrested cells were able to re-enter the cell cycle, confirming reversibility of arrest. Furthermore, a panel of important myogenic factors showed expression patterns similar to those reported for mouse satellite cells in G0, reactivated and differentiated cultures, supporting the applicability of the human model. In addition, gene expression profiling showed that a large number of genes (4598) were differentially expressed in cells activated from G0 compared to long term exponentially proliferating cultures normally used for in vitro studies. Human myoblasts cultured through many passages inevitably consist of a mixture of proliferating and non-proliferating cells, while cells activated from G0 are in a synchronously proliferating phase, and therefore may be a better model for in vivo proliferating satellite cells. Furthermore, the temporal propagation of proliferation in these synchronized cultures resembles the pattern seen in vivo during regeneration. We therefore present this culture model as a useful and novel condition for molecular analysis of quiescence and reactivation of human 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

Gene expression of cMET, FGFR1 and FGF2 during G₀ entrance, exit and differentiation.

<p><i>cMET</i> had a wave shaped expression during G₀ entrance and exit. <i>FGFR1</i> and its ligand <i>FGF2</i> were highly up regulated in the early phase of reactivation and down regulated in late phase and after differentiation. Immunocytochemical analyses of FGFR1 correlated with gene expression, with low levels of FGFR1 during G₀ arrest and up regulation immediate after reactivation followed by down regulation at GM48h (B). Only a few SGCA positive cells were observed at G₀ arrest (SM96h) and after replating (GM8h), however after differentiation both FGFR1 and SGCA were upregulated in myofibers. Scale bar: 100 µm.</p

Expression of KI67 and incorporation of BrdU during G₀ arrest and reactivation.

<p>(<b>A</b>) G₀ arrested cells were cytospinned on coverglasses, which caused aggregation of the cells. KI67 and BrdU, which was incorporated as 1 h pulse, were detected by immunocytochemistry. Selected time points (12, 24, 48 and 96 hours) during culture in SM are shown, and a down regulation in the expression of KI67 and incorporation of BrdU was observed. At SM96h no KI67 or BrdU were detected, thus the cells have entered the G₀ phase. (<b>B</b>) KI67 expression and BrdU incorporation is shown for selected time points after reactivation (GM12h, GM24h, GM32h and GM48h). KI67 expression was observed in a few cells at GM24h followed by a large up regulation during rest of the reactivation period and at GM48 most of cells were KI67 positive. During reactivation the cells were continuously exposed to BrdU, thus a cumulative BrdU incorporation is show. At GM12h only a few cells had incorporated BrdU but subsequently most of the cells became BrdU positive. Furthermore, the G₀ arrested cells were able to fully differentiate and form desmin positive myofibers (B, insert). Thus, the cells were able to enter the cell cycle after G₀ arrest. Scale bar: 50 µm.</p

The effect of low intensity shockwave treatment (Li-SWT) on human myoblasts and mouse skeletal muscle

Abstract Background Transplanting myogenic cells and scaffolds for tissue engineering in skeletal muscle have shown inconsistent results. One of the limiting factors is neovascularization at the recipient site. Low intensity shockwave therapy (Li-SWT) has been linked to increased tissue regeneration and vascularization, both integral to survival and integration of transplanted cells. This study was conducted to demonstrate the response of myoblasts and skeletal muscle to Li-SWT. Method Primary isolated human myoblasts and explants were treated with low intensity shockwaves and subsequently cell viability, proliferation and differentiation were tested. Cardiotoxin induced injury was created in tibialis anterior muscles of 28 mice, and two days later, the lesions were treated with 500 impulses of Li-SWT on one of the legs. The treatment was repeated every third day of the period and ended on day 14 after cardiotoxin injection.. The animals were followed up and documented up to 21 days after cardiotoxin injury. Results Li-SWT had no significant effect on cell death, proliferation, differentiation and migration, the explants however showed decreased adhesion. In the animal experiments, qPCR studies revealed a significantly increased expression of apoptotic, angiogenic and myogenic genes; expression of Bax, Bcl2, Casp3, eNOS, Pax7, Myf5 and Met was increased in the early phase of regeneration in the Li-SWT treated hind limbs. Furthermore, a late accumulative angiogenic effect was demonstrated in the Li-SWT treated limbs by a significantly increased expression of Angpt1, eNOS, iNOS, Vegfa, and Pecam1. Conclusion Treatment was associated with an early upregulation in expression of selected apoptotic, pro-inflammatory, angiogenic and satellite cell activating genes after muscle injury. It also showed a late incremental effect on expression of pro-angiogenic genes. However, we found no changes in the number of PAX7 positive cells or blood vessel density in Li-SWT treated and control muscle. Furthermore, Li-SWT in the selected doses did not decrease survival, proliferation or differentiation of myoblasts in vitro

Gene expression of early and late markers of myogenesis during G₀ entrance, exit and differentiation.

<p>(A) <i>MEF2A</i> and <i>MEF2C</i> were all expressed throughout G₀ arrest and re-activation, with peaks seen in SM and early GM samples followed by up regulation after differentiation. <i>NCAM</i>, <i>DESMIN</i> and <i>M-CAD</i> expressions were high in the early SM samples followed by down regulation and finally up regulation in the late GM samples and after differentiation. <i>MYH8</i> was up regulated during G₀ arrest but became down regulated in the reactivated samples after GM5h and largely up regulated after differentiation. (B,C) Protein expression of MYH8 was studied by immunocytochemistry and the fractions of MYH8 positive cells were determined during G₀ arrest. MYH8 seemed to be present in a small portion of the cells throughout G₀ entrance, however no correlation between gene and protein expression was observed. (D) Immunostainings of Fast Myosin during G₀ entrance showed a few positive cells, an expression similar to MYH8. Scale bar: 100 µm.</p

Expression levels of cell cycle related genes during G₀ entrance (SM), exit (GM) and differentiation (DM).

<p>Cell cultures A, B and C were cultured in suspension-, growth-, and differentiation medium, and qRT-PCR was performed at different time point during the study (A). <i>KI67</i> expression was highly down regulated during G₀ arrest between SM12h and SM24h and after reactivation we observed a major up regulation between GM16-GM48h followed by a large down regulation after differentiation. <i>CYCLIN D1</i> was expressed at low levels during G₀ arrest but after activation a large up regulation was detected already after 5 hours. In the late period of reactivation the expression was declining with further down regulation after differentiation. Expression levels of <i>P21</i>, <i>P27</i>, <i>P130</i> and <i>P53</i> were high during G₀ arrest, but after activation in GM the expression levels dropped followed by a small up regulation in the late GM samples. Furthermore, P21 and P53 were markedly up regulated after differentiation. The protein expression of P53 during G₀ arrest, reactivation and after differentiation is shown in (B). High levels of P53 were observed during G₀ arrest, followed by down regulation after activation. After differentiation the expression of P53 was again up regulated. Thus, the gene expression correlated with the protein expression. Scale bar: 100 µm.</p

Expression of <i>PAX</i> genes and MRFs during G₀ entrance (SM), exit (GM) and differentiation (DM). (A) <i>PAX3</i> and <i>PAX7</i> expression levels were high in G₀ arrested samples but became down regulated when cells were reactivated.

<p>Expression of MyoD1 seemed to increase during G₀ arrest followed by a drop in expression during early reactivation. In the later phase of reactivation the expression was up regulated and finally down regulation after differentiation. The markers <i>MYF5</i> and <i>MYF6</i> expressions were relatively stable during G₀ arrest for cultures A and C but after activation the expression of all three genes became up regulated followed by a large down regulation after differentiation in all three cultures. In cultures B and C, <i>MYOGENIN</i> expression was approx 2-fold up regulated during G₀ arrest, but after reactivation all three cultures had a drop in <i>MYOGENIN</i> expression followed by up regulation after differentiation. (B, C) The protein expression of MYOGENIN during G₀ entrance was studied by immunocytochemistry. After 6 h in SM only 2.5% (±1.1 SEM) of the cells were MYOGENIN positive and the fraction decreased further during culture in SM with a tendency for an increase up to 2.8% (±2.0 SEM). Thus, MYOGENIN protein expression did not seem to correlate completely with gene expression. Scale bar: 100 µm.</p

Comparison of gene expression in BG₀, G₀, AG₀ and Dif.

<p>Number of genes with ≥2-fold differential expression for the six comparisons are shown and the persentage of high and low expressed genes are calculated.</p