Sequential introduction and dosage balance of defined transcription factors affect reprogramming efficiency from pancreatic duct cells into insulin-producing cells
Sequential introduction and dosage balance of defined transcription factors affect reprogramming efficiency from pancreatic duct cells into insulin-producing cells
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DOI：
10.1016/j.bbrc.2014.01.083
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发表时间：
2014-02-21
影响因子：
3.1
通讯作者：
Shimomura, Iichiro
中科院分区：
文献类型：
--
作者：
Miyashita, Kazuyuki;Miyatsuka, Takeshi;Shimomura, Iichiro
虽然已经显示转录因子组合的外源表达诱导非β细胞转化为胰岛素产生细胞，但重编程效率仍然很低。为了开发用于优化重编程方案的体外筛选系统，我们产生了报告细胞系mPac-MIP-RFP，其中可以用在胰岛素启动子控制下表达的红色荧光蛋白定量重编程效率。用表达Pdx 1、Neurog 3和Mafa的腺病毒依次感染mPac-MIP-RFP细胞的分析显示，在Neurog 3或Mafa之前表达Pdx 1增加了重编程效率。接下来，与同时感染携带每种转录因子的三种腺病毒相比，用表达Pdx 1、Neurog 3和Mafa的多顺反子腺病毒载体感染显著增加了胰岛素的表达水平，尽管Mafa与多顺反子载体一起的过度表达显著抑制了重编程为胰岛素产生细胞。因此，用mPac-MIP-RFP报告细胞系进行的体外筛选表明，用确定的转录因子进行基因递送的时间和剂量影响重编程效率。进一步的研究应优化重编程条件，为未来的糖尿病细胞治疗。(C)2014爱思唯尔公司All rights reserved.
While the exogenous expression of a combination of transcription factors have been shown to induce the conversion of non-beta cells into insulin-producing cells, the reprogramming efficiency remains still low. In order to develop an in vitro screening system for an optimized reprogramming protocol, we generated the reporter cell line mPac-MIP-RFP in which the reprogramming efficiency can be quantified with red fluorescent protein expressed under the control of the insulin promoter. Analysis with mPac-MIP-RFP cells sequentially infected with adenoviruses expressing Pdx1, Neurog3, and Mafa revealed that expression of Pdx1 prior to Neurog3 or Mafa augments the reprogramming efficiency. Next, infection with a polycistronic adenoviral vector expressing Pdx1, Neurog3 and Mafa significantly increased the expression level of insulin compared with the simultaneous infection of three adenoviruses carrying each transcription factor, although excessive expression of Mafa together with the polycistronic vector dramatically inhibited the reprogramming into insulin-producing cells. Thus, in vitro screening with the mPac-MIP-RFP reporter cell line demonstrated that the timing and dosage of gene delivery with defined transcription factors influence the reprogramming efficiency. Further investigation should optimize the reprogramming conditions for the future cell therapy of diabetes. (C) 2014 Elsevier Inc. All rights reserved.