Differentiation and sarcomere formation in skeletal myocytes directly prepared from human induced pluripotent stem cells using a sphere-based culture
Differentiation and sarcomere formation in skeletal myocytes directly prepared from human induced pluripotent stem cells using a sphere-based culture
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DOI：
10.1016/j.diff.2017.07.004
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发表时间：
2017-07-01
期刊：
影响因子：
2.9
通讯作者：
Suzuki, Masatoshi
中科院分区：
文献类型：
--
作者：
Jiwlawat, Saowanee;Lynch, Eileen;Suzuki, Masatoshi
人诱导多能干细胞（iPSC）是用于增殖肌源性祖细胞的有前途的资源。我们的小组最近报道了一个独特的协议衍生肌祖细胞直接（无遗传修饰）从人类多能细胞使用自由浮动球形培养。在这里，我们扩大我们以前的努力，并试图确定如何分化持续时间，培养表面涂层，营养补充剂中的培养基影响祖细胞分化和形成骨骼肌肌管含有肌节结构。长分化期（超过6周）促进iPSC衍生的肌源性祖细胞的分化和随后的肌管形成。这些iPSC衍生的肌管含有代表性的肌节结构，由有组织的肌球蛋白和肌动蛋白丝组成，并且可以自发收缩。我们还发现，使用三维（3D）人工肌肉结构的生物工程方法可以促进细长肌管的形成。最后，我们确定了培养表面涂层基质和不同补充剂如何影响终末分化。虽然Matrigel和层粘连蛋白涂层对肌肉分化显示出相当的效果，但与马血清相比，分化培养基中的B27无血清补充剂显著增强了肌生成。我们的研究结果支持建立一个收缩性肌节肌原纤维的体外模型的可能性，用于疾病建模和药物筛选，以研究神经肌肉疾病。
Human induced-pluripotent stem cells (iPSCs) are a promising resource for propagation of myogenic progenitors. Our group recently reported a unique protocol for the derivation of myogenic progenitors directly (without genetic modification) from human pluripotent cells using free-floating spherical culture. Here we expand our previous efforts and attempt to determine how differentiation duration, culture surface coatings, and nutrient supplements in the medium influence progenitor differentiation and formation of skeletal myotubes containing sarcomeric structures. A long differentiation period (over 6 weeks) promoted the differentiation of iPSC-derived myogenic progenitors and subsequent myotube formation. These iPSC-derived myotubes contained representative sarcomeric structures, consisting of organized myosin and actin filaments, and could spontaneously contract. We also found that a bioengineering approach using three-dimensional (3D) artificial muscle constructs could facilitate the formation of elongated myotubes. Lastly, we determined how culture surface coating matrices and different supplements would influence terminal differentiation. While both Matrigel and laminin coatings showed comparable effects on muscle differentiation, B27 serum-free supplement in the differentiation medium significantly enhanced myogenesis compared to horse serum. Our findings support the possibility to create an in vitro model of contractile sarcomeric myofibrils for disease modeling and drug screening to study neuromuscular diseases.