Underlying Data for Overcoming the Silencing of Doxycycline-Inducible Promoters in hiPSC-derived Cardiomyocytes

Background: Human induced pluripotent stem cells (hiPSCs) hold great promise for studying human development, perform disease modeling, and develop regenerative medicine approaches. Effective control of transgene expression is crucial to achieve temporal and quantitative precision in all of these contexts. The use of doxycycline (dox)-inducible systems provides a promising approach. In particular, the OPTi-OX platform achieves optimal expression in hiPSCs by separately integrating the constitutively expressed third generation reverse tetracycline-controlled transactivator (Tet-ON 3G) and the dox-responsive transgene at two different genomic safe habors (GSH), the human ROSA26 and AAVS1 loci, respectively. However, transgene silencing during differentiation of OPTi-OX hiPSCs, for instance in hiPSC-derived cardiomyocytes (hiPSC-CMs), remains a major challenge.

Methods: To address this issue, we tested various strategies for enhancing dox-inducible transgene expression in OPTi-OX hiPSCs and hiPSC-CMs. Two promoters, TRE3VG and a modified variant T11, were evaluated for activity and stability. We also examined the addition of a Ubiquitous Chromatin Opening Element (UCOE) to prevent silencing. Furthermore, we explored relocating the inducible transgene cassette to an alternative GSH, the CLYBL locus. Finally, we tested inhibition of histone deacetylases (HDAC) using sodium butyrate (SB) to restore the activity of silenced promoters. We assessed inducible promoter activity via flow cytometry of an inducible reporter gene in both hiPSCs and hiPSC-CMs. Additionally, we quantified Tet-ON 3G expression with real-time quantitative PCR (RT-qPCR).

Results: TRE3VG demonstrated stronger activity than T11, but both were prone to silencing. UCOE did not increase promoter activity in hiPSCs, but modestly prevented silencing in hiPSC-CMs. Targeting the CLYBL GSH improved promoter activity compared to the AAVS1 locus in both hiPSCs and hiPSC-CMs. SB treatment successfully restored silenced promoter activity in hiPSC-CMs, but negatively impacted hiPSC viability.

Conclusions: This study highlights the complex nature of inducible promoter silencing during hiPSC differentiation and suggests that combined strategies, including GSH selection, optimal cassette, and chromatin modifications, are necessary to achieve stable transgene expression.