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Open AccessArticle

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

1
Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
2
Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2017, 18(8), 1805; https://doi.org/10.3390/ijms18081805
Received: 27 July 2017 / Revised: 14 August 2017 / Accepted: 18 August 2017 / Published: 19 August 2017
(This article belongs to the Special Issue Stem Cell Research)
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. View Full-Text
Keywords: reprogramming; cardiomyocyte; doxycycline-inducible; cell cycle; fibroblast reprogramming; cardiomyocyte; doxycycline-inducible; cell cycle; fibroblast
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Umei, T.C.; Yamakawa, H.; Muraoka, N.; Sadahiro, T.; Isomi, M.; Haginiwa, S.; Kojima, H.; Kurotsu, S.; Tamura, F.; Osakabe, R.; Tani, H.; Nara, K.; Miyoshi, H.; Fukuda, K.; Ieda, M. Single-Construct Polycistronic Doxycycline-Inducible Vectors Improve Direct Cardiac Reprogramming and Can Be Used to Identify the Critical Timing of Transgene Expression. Int. J. Mol. Sci. 2017, 18, 1805.

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