Transcription Factor Rational Design Improves Directed Differentiation of Human Mesenchymal Stem Cells Into Skeletal Myocytes
Transcription Factor Rational Design Improves Directed Differentiation of Human Mesenchymal Stem Cells Into Skeletal Myocytes
复制标题
DOI：
10.1038/mt.2010.308
复制
发表时间：
2011-07-01
影响因子：
12.4
通讯作者：
de Vries, Antoine A. F.
中科院分区：
文献类型：
--
作者：
Goncalves, Manuel A. F. V.;Janssen, Josephine M.;de Vries, Antoine A. F.
人们对将细胞从一个谱系转分化为另一个谱系的细胞以及通过部署天然存在的转录因子（TF）将成熟细胞去分化回到干细胞/祖细胞状态非常感兴趣。然而，通常，以可预测和有效的方式引导细胞分化途径仍然具有挑战性。在这里，我们研究了组合来自不同谱系特异性TF的结构域以改善定向细胞分化的原理。作为概念验证，我们设计了全人TF MyoDCD，其具有NH（2）-末端转录激活结构域（NH3）和邻近的MyoD COOH-末端融合至myocardin（MyoCD）的NH3的DNA结合基序。我们发现，通过报告基因和标记蛋白测定，以及通过细胞融合读出系统，针对MyoCD的转录因子的基因通常响应于骨骼肌特异性TF MyoD的实施更强大的肌源性重编程的非肌肉细胞比所实现的父母，原型主TF，MyoD。评估了用与合成横纹肌特异性启动子连接的密码子优化的微肌营养不良蛋白基因和/或MyoD或MyoDCD转导的人间充质干细胞（hMSC）通过异型细胞融合补充杜氏肌营养不良症（DMD）肌细胞中的遗传缺陷。用MyoDCD和微肌营养不良蛋白共转导hMSC导致含有最高肌营养不良蛋白水平的嵌合肌管。收稿日期：2010年9月29日;接受日期：2010年12月20日;在线发表日期：2011年1月25日。doi：10.1038/mt.2010.308
There is great interest in transdifferentiating cells from one lineage into those of another and in dedifferentiating mature cells back into a stem/progenitor cell state by deploying naturally occurring transcription factors (TFs). Often, however, steering cellular differentiation pathways in a predictable and efficient manner remains challenging. Here, we investigated the principle of combining domains from different lineage-specific TFs to improve directed cellular differentiation. As proof-of-concept, we engineered the whole-human TF MyoDCD, which has the NH(2)-terminal transcription activation domain (TAD) and adjacent DNA-binding motif of MyoD COOH-terminally fused to the TAD of myocardin (MyoCD). We found via reporter gene and marker protein assays as well as by a cell fusion readout system that, targeting the TAD of MyoCD to genes normally responsive to the skeletal muscle-specific TF MyoD enforces more robust myogenic reprogramming of nonmuscle cells than that achieved by the parental, prototypic master TF, MyoD. Human mesenchymal stem cells (hMSCs) transduced with a codon-optimized microdystrophin gene linked to a synthetic striated muscle-specific promoter and/or with MyoD or MyoDCD were evaluated for complementing the genetic defect in Duchenne muscular dystrophy (DMD) myocytes through heterotypic cell fusion. Cotransduction of hMSCs with MyoDCD and microdystrophin led to chimeric myotubes containing the highest dystrophin levels. Received 29 September 2010; accepted 20 December 2010; published online 25 January 2011. doi: 10.1038/mt.2010.308