Possible application of muscle specific conditional mouse-derived induced pluripotent stem cells for muscle research
Possible application of muscle specific conditional mouse-derived induced pluripotent stem cells for muscle research
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DOI：
10.1016/j.bbrep.2020.100744
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发表时间：
2020-03-01
影响因子：
2.7
通讯作者：
Hosoyama, Tohru
中科院分区：
文献类型：
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作者：
Hosoyama, Tohru
Cre-driver小鼠系允许在特定细胞中体内调节靶基因，是最近肌肉研究不可或缺的工具。本研究旨在探索肌肉特异性Cre-driver小鼠品系在肌肉研究中的新应用。为此，我从肌纤维特异性条件小鼠产生了具有他莫昔芬诱导的GFP表达的iPS细胞，然后我检查了他莫昔芬给药是否在iPS衍生的肌原性细胞中诱导了同源重组。从Myf 6（CE/wt）：：CAG-EGFP小鼠的尾部分离成纤维细胞，其通过注射他莫昔芬在Myf 6谱系中特异性表达GFP，然后通过用基于仙台病毒并含有OSKM基因的载体转染产生iPS细胞。肌肉特异性条件性小鼠iPS细胞（mCM-iPSC）在肌源性分化条件下成功分化为肌源性细胞，如Pax 7（+）肌肉祖细胞、MyoD（+）成肌细胞和MHC+肌管。使用该模型，我检查了4-羟基他莫昔芬（4 OH-TAM）施用是否在mCM-iPSC衍生的肌管中诱导同源重组。结果，多核肌管显示GFP表达，而在Pax 7（+）肌肉祖细胞和非肌源性细胞中均未检测到GFP信号。这些结果表明，同源重组可以通过他莫昔芬给药在mCM-iPSC衍生的肌管中诱导，并且该系统即使在重编程细胞中也正常运行。此外，我证明了GFP报告基因表达的成肌细胞除了多核肌管时，他莫昔芬脉冲施加在早期阶段的肌发生。总之，Myf 6（CE/wt）：：CAG-EGFP小鼠衍生的iPS细胞在小鼠肌发生期间至少部分地再现Myf 6表达。本研究证明了肌肉特异性条件性小鼠除了体内应用之外的新应用，并且mCM-iPSCs也可以用于肌肉特异性条件性敲除小鼠的体外研究。
The Cre-driver mouse line, which allows for in vivo regulation of target gene(s) in specific cells, is an indispensable tool for recent muscle research. In this study, I aimed to explore new applications of muscle specific Cre-driver mouse line in muscle research. For this purpose, I generated an iPS cells from a myofiber specific conditional mouse with tamoxifen inducible GFP expression, and then I checked whether homologous recombination was induced in the iPS-derived myogenic cells by tamoxifen administration. Fibroblasts were isolated from the tails of Myf6(CE/wt)::CAG-EGFP mice, which expressed GFP specifically in Myf6 lineages by tamoxifen injection, and then iPS cells was generated by transfection with a vector based on sendai-virus and containing OSKM genes. Muscle specific conditional mouse-derived iPS cells (mCM-iPSCs) were successfully differentiated to myogenic cells, such as Pax7(+) muscle progenitors, MyoD(+) myoblasts, and MHC+ myotubes, under myogenic differentiation conditions. Using this model, I examined whether homologous recombination was induced in mCM-iPSC-derived myotubes by 4-hydroxytamoxifen (4OH-TAM) administration. As a result, multinucleated myotubes showed GFP expression, while no GFP signals were detected in both Pax7(+) muscle progenitor and non-myogenic cells. These results indicated that homologous recombination could be induced in mCM-iPSC-derived myotubes by tamoxifen administration, and that this system operated normally even in reprogrammed cells. Also, I evidenced that GFP reporter was expressed in myoblasts in addition to multinucleated myotubes when tamoxifen-pulse was applied at an early phase of myogenesis. Taken together, Myf6(CE/wt)::CAG-EGFP mouse-derived iPS cells reproduced at least in part Myf6 expression during mouse myogenesis. This study demonstrated a novel application of muscle specific conditional mouse in addition to in vivo application, and mCM-iPSCs could also be used in in vitro investigations with muscle specific conditional knock-out mouse.