Lee Kunwoo, Yu Pengzhi, Lingampalli Nithya, Kim Hyun Jin, Tang Richard, Murthy Niren
Department of Bioengineering, University of California, Berkeley, CA, USA ; UC Berkeley and UCSF Joint Graduate Program in Bioengineering, Berkeley/San Francisco, CA, USA.
Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA.
Int J Nanomedicine. 2015 Mar 6;10:1841-54. doi: 10.2147/IJN.S75124. eCollection 2015.
The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation.
由于心脏组织无法再生,心肌梗死的治疗是医学上的一项重大挑战。通过递送转录因子mRNA将内源性心脏成纤维细胞直接重编程为功能性心肌细胞,具有使心脏组织再生和治疗心力衰竭的潜力。尽管将mRNA递送至心脏成纤维细胞具有治疗潜力,但在心脏成纤维细胞中进行mRNA转染一直具有挑战性。在此,我们通过聚精氨酸融合的心脏靶向肽和脂质体复合物(称为C-Lipo),在培养的小鼠心脏成纤维细胞中开发了一种高效的mRNA转染方法,并证明了心脏成纤维细胞向心肌细胞的部分直接重编程。C-Lipo因其高效转染且毒性低,能够实现mRNA诱导的直接心脏重编程,从而允许在两周的时间内对Gata4、Mef2c和Tbx5(GMT)mRNA进行多次转染。诱导产生的心肌样细胞具有α-MHC启动子驱动的绿色荧光蛋白表达,并且通过α-辅肌动蛋白免疫组织化学显示出横纹肌结构。在转染两周后,通过C-Lipo对培养的小鼠心脏成纤维细胞进行GMT mRNA转染,显著增加了心肌细胞标记基因Actc1、Actn2、Gja1、Hand2和Tnnt2的表达。此外,本研究提供了首个直接证据,证明GMT重编程因子的化学计量会影响心肌细胞标记基因的表达。我们的结果表明,mRNA递送是产生心肌细胞的一种潜在方法。