Asymmetric cellular responses in primary human myoblasts using sera of different origin and specification

For successful growth and maintenance of primary myogenic cells in vitro, culture medium and addition of sera are the most important factors. At present it is not established as to what extent sera of different origin and composition, supplemented in media or serum-free media conditions influence myoblast function and responses to different stimuli. By assessing markers of proliferation, differentiation/fusion, quiescence, apoptosis and protein synthesis the aim of the current study was to elucidate how primary human myoblasts and myotubes are modulated by different commonly used serum using FCS (foetal calf serum), (CS-FCS charcoal-stripped FCS, a manufacturing process to remove hormones and growth factors from sera), HS (horse serum) as well as in serum free conditions (DMEM). To characterise the biological impact of the different serum, myoblasts were stimulated with Insulin (100 nM) and Vitamin D (100 nM; 1α,25(OH)2D3, 1α,25-Dihydroxycholecalciferol, Calcitriol), two factors with characterised effects on promoting fusion and protein synthesis or quiescence, respectively in human myoblasts/myotubes. We demonstrate that sera of different origin/formulation differentially affect myoblast proliferation and myotube protein synthesis. Importantly, we showed that quantifying the extent to which Insulin effects myoblasts in vitro is highly dependent upon serum addition and which type is present in the media. Upregulation of mRNA markers for myogenic fusion, Myogenin, with Insulin stimulation, relative to DMEM, appeared dampened at varying degrees with serum addition and effects on p70S6K phosphorylation as a marker of protein synthesis could not be identified unless serum was removed from media. We propose that these asymmetric molecular and biochemical responses in human myoblasts reflect the variable composition of mitogenic and anabolic factors in each of the sera. The results have implications for both the reproducibility and interpretation of results from experimental models in myoblast cells/myotubes.体外原代肌源性细胞的成功培养与维持，培养基及血清添加方案是最为核心的影响因素。目前尚未明确，不同来源与组成的血清，无论是添加至培养基中还是应用于无血清培养体系，会在何种程度上影响成肌细胞的功能及其对不同刺激的应答反应。 本研究通过检测增殖、分化/融合、静息、凋亡及蛋白质合成相关标志物，旨在阐明不同常用血清——包括胎牛血清（FCS, foetal calf serum）、炭吸附胎牛血清（CS-FCS, charcoal-stripped FCS，即通过纯化工艺去除血清中激素与生长因子的制备方式）、马血清（HS, horse serum）——以及无血清培养体系（DMEM）对原代人成肌细胞及肌管的调控作用。 为表征不同血清的生物学效应，本研究采用胰岛素（100 nM）与维生素D（100 nM；1α,25-二羟维生素D3，即1α,25(OH)2D3、1α,25-Dihydroxycholecalciferol、骨化三醇Calcitriol）对成肌细胞进行刺激：这两种因子已被证实可分别促进人成肌细胞/肌管的融合与蛋白质合成，或诱导细胞进入静息状态。 本研究证实，不同来源/配方的血清对成肌细胞增殖及肌管蛋白质合成具有差异性影响。 尤为重要的是，本研究发现，体外实验中量化胰岛素对成肌细胞的作用效果，高度依赖于培养基中添加的血清种类与是否添加血清。相较于无血清培养体系（DMEM）对照组，胰岛素刺激下肌源性融合标志物肌生成素（Myogenin）的mRNA表达上调效应，会因血清添加而出现不同程度的减弱；而作为蛋白质合成标志物的p70S6K磷酸化水平变化，仅在无血清培养条件下才可被检测到。 本研究认为，人成肌细胞中出现的这些不对称分子与生化应答反应，反映了不同血清中促有丝分裂与合成代谢因子组成的差异性。 本研究结果对成肌细胞/肌管实验模型的结果可重复性与结果解读均具有重要指导意义。