Doxycycline-Mediated Control of Cyclin D2 Overexpression in Human-Induced Pluripotent Stem Cells

TL;DR

This study shows how doxycycline can control cyclin D2 levels in stem cells, which could help in safely regenerating heart tissue.

Contribution

The novel contribution is the development of a doxycycline-controlled system for cyclin D2 overexpression in hiPSCs.

Findings

Doxycycline treatment activates CCND2 expression in engineered hiPSCs.

CCND2 protein levels decrease after discontinuing doxycycline treatment.

The system allows tight control of CCND2 overexpression in hiPSC-derived cardiomyocytes.

Abstract

Previous studies have demonstrated that when the cyclin D2 (CCND2), a cell-cycle regulatory protein, is overexpressed in human-induced pluripotent stem cells (hiPSCs), cardiomyocytes (CMs) differentiated from these CCND2-overexpressing hiPSCs can proliferate after transplantation into infarcted hearts, which significantly improves the cells’ potency for myocardial regeneration. However, persistent CM proliferation could lead to tumor growth or the development of arrhythmogenic complications; thus, the goal of the current study was to generate a line of hiPSCs in which CCND2 overexpression could be tightly controlled. First, we transfected hiPSCs with vectors coding for a doxycycline-inducible Tet-On transactivator and S. pyogenes dCas9 fused to the VPR activation domain; then, the same hiPSCs were engineered to express guide RNAs targeting the CCND2 promotor. Thus, treatment with doxycycline (dox) activated dCas9-VPR expression, and the guide RNAs directed dCas9-VPR to the CCND2 promoter, which activated CCND2 expression. Subsequent experiments confirmed that CCND2 expression was dox-dependent in this newly engineered line of hiPSCs (doxCCND2-hiPSCs): CCND2 protein was abundantly expressed after 48 h of treatment with dox and declined to near baseline level ~96 h after dox treatment was discontinued.

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