Department of Biomedical Engineering, School of Medicine and School of Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Department of Medicine/Cardiovascular Diseases, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Int J Mol Sci. 2024 Aug 9;25(16):8714. 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.
先前的研究表明，当细胞周期调控蛋白 cyclin D2 (CCND2) 在人诱导多能干细胞 (hiPSC) 中过度表达时，这些 CCND2 过表达 hiPSC 分化而来的心肌细胞 (CM) 在移植到梗死心脏后可以增殖，这显著提高了细胞的心肌再生能力。然而，CM 的持续增殖可能导致肿瘤生长或心律失常并发症的发展；因此，本研究的目的是生成一条可严格控制 CCND2 过表达的 hiPSC 系。首先，我们用编码四环素诱导型 Tet-On 转录激活剂和与 VPR 激活结构域融合的 dCas9 的载体转染 hiPSC；然后，对相同的 hiPSC 进行工程改造，使其表达靶向 CCND2 启动子的 guide RNA。因此，用强力霉素 (dox) 处理激活了 dCas9-VPR 的表达，guide RNA 则将 dCas9-VPR 引导到 CCND2 启动子，从而激活了 CCND2 的表达。随后的实验证实，在这条新构建的 hiPSC 系（CCND2-hiPSCs）中，CCND2 的表达是依赖 dox 的：用 dox 处理 48 小时后，CCND2 蛋白大量表达，在 dox 处理停止约 96 小时后，CCND2 蛋白的表达水平下降到接近基线水平。
