Single-construct polycistronic doxycycline-inducible vectors improve direct cardiac reprogramming and can be used to identify the critical timing of transgene expression

Tomohiko C. Umei, Hiroyuki Yamakawa, Naoto Muraoka, Taketaro Sadahiro, Mari Isomi, Sho Haginiwa, Hidenori Kojima, Shota Kurotsu, Fumiya Tamura, Rina Osakabe, Hidenori Tani, Kaori Nara, Hiroyuki Miyoshi, Keiichi Fukuda, Masaki Ieda

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

ssDirect 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.

Original languageEnglish
Article number1805
JournalInternational Journal of Molecular Sciences
Volume18
Issue number8
DOIs
Publication statusPublished - 2017 Aug 19

    Fingerprint

Keywords

  • Cardiomyocyte
  • Cell cycle
  • Doxycycline-inducible
  • Fibroblast
  • Reprogramming

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Computer Science Applications
  • Spectroscopy
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

Cite this

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. (2017). Single-construct polycistronic doxycycline-inducible vectors improve direct cardiac reprogramming and can be used to identify the critical timing of transgene expression. International Journal of Molecular Sciences, 18(8), [1805]. https://doi.org/10.3390/ijms18081805