1.1 肌源性分化是一个由特定转录因子和信号分子调节的过程，已被证明可诱导肌源性表型。转录因子标记肌生成的各个阶段，并作为肌生成分析的基准。 1.2 本指南适用于哺乳动物细胞，但不适用于非哺乳动物细胞，因为非哺乳动物细胞的肌源性标记可能不同于此处描述的标记。 1.3 本指南提出了在进行肌源性分化分析时要测量的适当标记。本指南描述了多能干细胞向成肌细胞和肌管分化的阶段。本指南提供了有关确定肌源性分化的适当方法的信息。本指南不提供有关驱动肌源性表型分化的培养基、补充剂或基质的信息。 1.4 本指南的目的是为骨骼肌生成领域的工作提供帮助。使用本指南，研究人员应该能够了解哪些骨骼肌标记物最适合于实验。本指南将通过为导致心肌细胞分化的每个阶段确定适当的标记物，提高多能干细胞肌源性分化研究的一致性。值得注意的是，成肌细胞分化 体外 可能无法预测可能获得的结果 体内 . 1.5 本标准并非旨在解决与其使用相关的所有安全问题（如有）。本标准的用户有责任在使用前制定适当的安全、健康和环境实践，并确定监管限制的适用性。 1.6 本国际标准是根据世界贸易组织技术性贸易壁垒（TBT）委员会发布的《关于制定国际标准、指南和建议的原则的决定》中确立的国际公认标准化原则制定的。 ====意义和用途====== 5.1 本指南描述了与成肌细胞分化有关的标记物，这些标记物可用于筛选干细胞，以帮助定义成肌能力。干细胞包括多能干细胞和多能干细胞，能够分化成几种不同的间充质细胞，包括骨骼肌成肌细胞。 5.2 为了评估来源于和非来源于肌肉的细胞中的肌生成，测量标记物以准确定义调节分化、融合和肌管形成的转录和结构蛋白的变化。 讨论这些标记对于理解为什么推荐它们很重要。 5.3 肌源性分化: 5.3.1 肌源性分化是一个受配对盒（Pax）转录因子和肌源性调节因子（MRF）家族控制的高度调节过程。在成人的早期分化过程中，肌源性祖细胞如活化的卫星细胞或成肌细胞表达 Pax3 和 Pax7型 . Pax3 和 Pax7型 转录因子将细胞转向肌源性命运，并抑制心肌细胞分化 ( 2. ) ，为以后的MRF启动单元格。为了形成肌肉，需要MRF家族最终分化成肌细胞并形成肌纤维。这些调节蛋白属于碱性螺旋-环-螺旋转录因子超家族，由肌源性分化因子1组成( Myod1 )，肌源性因子5( Myf5 )，肌生成素( Myog公司 )，和肌生成因子6( Myf6 ). 在肌源性分化的初始阶段， Myod1 和 Myf5 是第一个要表达的MRF，并触发 Myog公司 和 Myf6 ( 3. ) . 细胞内增加 麦格 和 Myf6 诱导成肌细胞终末分化为肌细胞，形成融合的肌管。 5.4 形成肌管: 5.4.1 虽然肌源性标记描述分化，但融合到多核肌管是肌肉生物学中的一个重要因素。成肌细胞分化为具有多个融合标记的融合表型。值得注意的一个标志物是m-钙粘蛋白。据报道，M-cadherin参与成肌细胞融合并调节肌管发育 ( 4. ) . 因此，除了肌源性分化标记外，融合标记的评估将有利于形成肌肉的细胞表型。 为了支持这一点，研究表明，尽管存在肌源性分化基因的表达，但不表达m-cadherin的细胞无法融合并形成肌肉。这些结果表明，除了肌源性分化标记物外，融合标记物作为细胞是否能够融合的重要指标也应予以考虑 ( 5. ) . 本指南将列举已发表的测量和量化成肌细胞融合标记物的方法。

1.1 Myogenic differentiation is a process regulated by specific transcription factors and signaling molecules that have been shown to induce a myogenic phenotype. Transcription factors mark the stages of myogenesis and act as benchmarks for use in myogenic assays. 1.2 This guide applies to mammalian cells but does not apply to non-mammalian cells as the myogenic markers for non-mammalian cells can be different than those described here. 1.3 This guide proposes appropriate markers to measure when conducting myogenic differentiation assays. This guide describes the stages for multipotent stem cell differentiation toward myoblasts and myotubes. This guide provides information about the appropriate methods to determine myogenic differentiation. This guide does not provide information about media, supplements, or substrates that drive differentiation toward a myogenic phenotype. 1.4 The purpose of this guide is to act as an aid for work performed in the area of skeletal myogenesis. Using this guide, researchers should be able to understand which skeletal muscle markers are best suited for experiments. This guide will improve consistency for studies of myogenic differentiation of multipotent stem cells by identifying appropriate markers for each stage leading to myocyte differentiation. It should be noted that myoblast differentiation in vitro may not be predictive of results that may be obtained in vivo . 1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. 1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee. ====== Significance And Use ====== 5.1 This guide describes markers involved in myoblast differentiation that can be used to screen stem cells to help define myogenic capacity. Stem cells include pluripotent and multipotent stem cells capable of differentiating into several different mesenchymal cells, including skeletal muscle myoblasts. 5.2 To assess myogenesis in cells derived and not derived from muscle, markers are measured to accurately define the changes in transcription and structural proteins that regulate differentiation, fusion, and myotube formation. Discussion of these markers is important to understand why they are recommended. 5.3 Myogenic Differentiation: 5.3.1 Myogenic differentiation is a highly regulated process controlled by paired box (Pax) transcription factors and the myogenic regulatory factor (MRF) family. During early differentiation in adults, myogenic progenitors such as activated satellite cells or myoblasts express Pax3 and Pax7 . Pax3 and Pax7 transcription factors switch the cells toward a myogenic fate, and repress myocyte differentiation ( 2 ) , priming the cell for later MRFs. To form muscle, the family of MRFs is required to terminally differentiate myoblasts and form myofibers. These regulatory proteins belong to a superfamily of basic helix-loop-helix transcription factors that consists of myogenic differentiation factor 1 ( Myod1 ), myogenic factor 5 ( Myf5 ), myogenin ( Myog ), and myogenic factor 6 ( Myf6 ). In the initial stages of myogenic differentiation, Myod1 and Myf5 are the first MRFs to be expressed, and trigger increased production of Myog and Myf6 ( 3 ) . Increased intracellular Myog and Myf6 induces terminal differentiation of myoblasts into myocytes, leading to fused myotubes. 5.4 Forming Myotubes: 5.4.1 While myogenic markers describe differentiation, fusion into multinucleated myotubes is an important factor in muscle biology. Myoblasts differentiate into a fusogenic phenotype characterized by multiple fusion markers. One marker of note is m-cadherin. M-cadherin is reported to be involved in myoblast fusion and to regulate myotube development ( 4 ) . Therefore, assessment of fusion markers in addition to myogenic differentiation markers would favor a cell phenotype capable of forming muscle. In support of this, studies have shown that despite expression of myogenic differentiation genes, cells not expressing m-cadherin were unable to fuse and form muscle. These results suggest that in addition to myogenic differentiation markers, fusion markers should be considered given their importance as indicators of whether a cell is able to fuse ( 5 ) . This guide will enumerate published methods to measure and quantify myoblast fusion markers.