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

• Published in: International journal of molecular sciences Vol. 25; no. 16; p. 8714
• Main Authors: Qiao, Aijun, Wei, Yuhua, Liu, Yanwen, Kahn-Krell, Asher, Ye, Lei, Nguyen, Thanh, Zhang, Jianyi
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 09.08.2024; MDPI
• Subjects: Cell Differentiation - drug effects; Cloning; Communication; cyclin D2; Cyclin D2 - genetics; Cyclin D2 - metabolism; Doxycycline - pharmacology; doxycycline-inducible Tet-On transactivator; Gene Expression Regulation - drug effects; Genomes; human-induced pluripotent stem cells; Humans; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - drug effects; Induced Pluripotent Stem Cells - metabolism; myocardial regeneration; Myocytes, Cardiac - drug effects; Myocytes, Cardiac - metabolism; Plasmids; Promoter Regions, Genetic; Proteins; RNA, Guide, CRISPR-Cas Systems; Stem cells; United States > US; cyclin D2; human-induced pluripotent stem cells; doxycycline-inducible Tet-On transactivator; myocardial regeneration
• ISSN: 1661-6596; 1422-0067; 1422-0067
• DOI: 10.3390/ijms25168714

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 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 ( CCND2-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.