Single-Construct Polycistronic Doxycycline-Inducible Vectors Improve Direct Cardiac Reprogramming and Can Be Used to Identify the Critical Timing of Transgene Expression

被引：18

作者：

Umei, Tomohiko C. ^{[1
]}

Yamakawa, Hiroyuki ^{[1
]}

Muraoka, Naoto ^{[1
]}

Sadahiro, Taketaro ^{[1
]}

Isomi, Mari ^{[1
]}

Haginiwa, Sho ^{[1
]}

Kojima, Hidenori ^{[1
]}

Kurotsu, Shota ^{[1
]}

Tamura, Fumiya ^{[1
]}

Osakabe, Rina ^{[1
]}

Tani, Hidenori ^{[1
]}

Nara, Kaori ^{[1
]}

Miyoshi, Hiroyuki ^{[2
]}

Fukuda, Keiichi ^{[1
]}

Ieda, Masaki ^{[1
]}

机构：

[1] Keio Univ, Dept Cardiol, Sch Med, Shinjuku Ku, 35 Shinanomachi, Tokyo 1608582, Japan

[2] Keio Univ, Dept Physiol, Sch Med, Shinjuku Ku, 35 Shinanomachi, Tokyo 1608582, Japan

来源：

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES | 2017年 / 18卷 / 08期

关键词：

reprogramming; cardiomyocyte; doxycycline-inducible; cell cycle; fibroblast; PLURIPOTENT STEM-CELLS; TRANSCRIPTION FACTORS; FUNCTIONAL CARDIOMYOCYTES; FIBROBLASTS; GENERATION; GATA4; MEF2C; TBX5; MOUSE; HEART;

D O I：

10.3390/ijms18081805

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming.

引用

页数：14