Myoblast deactivation within engineered human skeletal muscle creates a transcriptionally heterogeneous population of quiescent satellite-like cells.
Myoblast deactivation within engineered human skeletal muscle creates a transcriptionally heterogeneous population of quiescent satellite-like cells.
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DOI：
10.1016/j.biomaterials.2022.121508
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发表时间：
2022-05
期刊：
影响因子：
14
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中科院分区：
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--
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卫星细胞（SCs）是骨骼肌中表达pax7的成体干细胞，对肌肉修复至关重要。然而，由于分离诱导的SC激活、天然静止状态的丧失和向成肌细胞的分化，SC功能的体外研究具有挑战性。在本研究中，我们优化了在工程人类骨骼肌组织（“肌团”）的三维培养环境中体外扩增的人成肌细胞失活的方法。免疫染色和基因表达分析显示，肌束内的一部分成肌细胞采用静止表型（3D-SCs），其特征是Pax7表达增加、细胞周期退出和Notch信号激活。与天然SCs类似，3D-SC的休眠受Notch和Wnt信号的调控，而包括bFGF在内的生长因子可诱导3D-SC失去休眠和重新激活。蜂毒（melittin）引起的肌束损伤可导致肌纤维断裂、功能下降和3D-SC增殖。通过单细胞rna测序（scRNA-seq），我们发现了两个3D-SC亚群（静止和激活）的存在，利用2D成肌细胞和3D-SC之间的轨迹推断确定了失活相关的基因特征，并表征了重新激活的3D-SC对蜂蜂素诱导损伤的转录组变化。这些结果证明了体外工程的3D人类骨骼肌环境能够支持静止和异质SC群体的形成，概括了天然SC表型的几个方面，并为未来人类肌肉再生和疾病相关SC功能障碍的研究提供了平台。
Satellite cells (SCs), the adult Pax7-expressing stem cells of skeletal muscle, are essential for muscle repair. However, in vitro investigations of SC function are challenging due to isolation-induced SC activation, loss of native quiescent state, and differentiation to myoblasts. In the present study, we optimized methods to deactivate in vitro expanded human myoblasts within a 3D culture environment of engineered human skeletal muscle tissues (“myobundles”). Immunostaining and gene expression analyses revealed that a fraction of myoblasts within myobundles adopted a quiescent phenotype (3D-SCs) characterized by increased Pax7 expression, cell cycle exit, and activation of Notch signaling. Similar to native SCs, 3D-SC quiescence is regulated by Notch and Wnt signaling while loss of quiescence and reactivation of 3D-SCs can be induced by growth factors including bFGF. Myobundle injury with a bee toxin, melittin, induces robust myofiber fragmentation, functional decline, and 3D-SC proliferation. By applying single cell RNA-sequencing (scRNA-seq), we discover the existence of two 3D-SC subpopulations (quiescent and activated), identify deactivation-associated gene signature using trajectory inference between 2D myoblasts and 3D-SCs, and characterize the transcriptomic changes within reactivated 3D-SCs in response to melittin-induced injury. These results demonstrate the ability of an in vitro engineered 3D human skeletal muscle environment to support the formation of a quiescent and heterogeneous SC population recapitulating several aspects of the native SC phenotype, and provide a platform for future studies of human muscle regeneration and disease-associated SC dysfunction.
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DOI：
10.1083/jcb.201310035
发表时间：
2014-04-14
期刊：
The Journal of cell biology
影响因子：
--
作者：
Bentzinger CF;von Maltzahn J;Dumont NA;Stark DA;Wang YX;Nhan K;Frenette J;Cornelison DD;Rudnicki MA
通讯作者：
Rudnicki MA
影响因子：
4.8
作者：
Bian, Weining;Bursac, Nenad
通讯作者：
Bursac, Nenad
影响因子：
7.7
作者：
Barruet, Emilie;Garcia, Steven M.;Pomerantz, Jason H.
通讯作者：
Pomerantz, Jason H.
DOI：
10.1073/pnas.78.9.5623
发表时间：
1981-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
BLAU, HM;WEBSTER, C
通讯作者：
WEBSTER, C
影响因子：
23.9
作者：
Bentzinger, C. Florian;Wang, Yu Xin;von Maltzahn, Julia;Soleimani, Vahab D.;Yin, Hang;Rudnicki, Michael A.;Rudnicki, A.
通讯作者：
Rudnicki, A.