Directed Differentiation of Embryonic Stem Cells Into Cardiomyocytes by Bacterial Injection of Defined Transcription Factors.
Directed Differentiation of Embryonic Stem Cells Into Cardiomyocytes by Bacterial Injection of Defined Transcription Factors.
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通过细菌注射确定的转录因子将胚胎干细胞定向分化为心肌细胞
DOI：
10.1038/srep15014
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发表时间：
2015-10-09
影响因子：
4.6
通讯作者：
Jin S
中科院分区：
文献类型：
--
作者：
Bai F;Ho Lim C;Jia J;Santostefano K;Simmons C;Kasahara H;Wu W;Terada N;Jin S
已充分证明，限定的转录因子的强制表达是用于细胞重编程或定向分化的有效方法。然而，由于潜在的插入诱变，转基因表达不适于治疗应用。在这里，我们开发了一种基于细菌III型分泌系统（T3 SS）的蛋白质递送工具，并展示了其通过控制递送与早期心脏发育相关的转录因子来指导多能干细胞分化的应用。通过融合到用于T3 SS依赖性注射的N-末端分泌序列，三种转录因子，即Gata 4、Mef 2c和Tbx 5（缩写为GMT），被易位到鼠胚胎干细胞（ESC）中，其中蛋白质有效地靶向细胞核，平均细胞内半衰期为5.5小时。外源性GMT蛋白注射激活了心脏程序，多轮GMT蛋白递送显著提高了ESC向心肌细胞分化的效率。T3 SS介导的GMT递送和激活素A治疗的组合显示出叠加效应，导致平均60%的ESCs分化为心肌细胞。ESC衍生的心肌细胞显示自发的节律性收缩运动以及正常的激素反应。这项工作为细菌传递多种转录因子以指导细胞命运而不损害基因组完整性奠定了基础。
Forced expression of defined transcriptional factors has been well documented as an effective method for cellular reprogramming or directed differentiation. However, transgene expression is not amenable for therapeutic application due to potential insertional mutagenesis. Here, we have developed a bacterial type III secretion system (T3SS)-based protein delivery tool and shown its application in directing pluripotent stem cell differentiation by a controlled delivery of transcription factors relevant to early heart development. By fusing to an N-terminal secretion sequence for T3SS-dependent injection, three transcriptional factors, namely Gata4, Mef2c and Tbx5 (abbreviated as GMT), were translocated into murine embryonic stem cells (ESCs), where the proteins are effectively targeted to the nucleus with an average intracellular half-life of 5.5 hours. Exogenous GMT protein injection activated the cardiac program and multiple rounds of GMT protein delivery significantly improved the efficiency of ESC differentiation into cardiomyocytes. Combination of T3SS-mediated GMT delivery and Activin A treatment showed an additive effect, resulting in on average 60% of the ESCs differentiated into cardiomyocytes. ESC derived cardiomyocytes displayed spontaneous rhythmic contractile movement as well as normal hormonal responses. This work serves as a foundation for the bacterial delivery of multiple transcription factors to direct cell fate without jeopardizing genomic integrity.