Transdifferentiation Of Human Dermal Fibroblasts Towards The Cardiac Cell Lineage

Transdifferentiation has been recently described as a novel method for converting human fibroblasts into induced cardiomyocyte-like cells. Such an approach can produce differentiated cells to study physiology or pathophysiology, examine drug interactions or toxicities, and engineer tissues. Here we describe the transdifferentiation of human dermal fibroblasts towards the cardiac cell lineage via the induced expression of transcription factors (TFs) GATA4, TBX5, MEF2C, MYOCD, NKX2-5, and delivery of microRNAs miR-1 and miR-133a. Cells undergoing transdifferentiation expressed ACTN2 and TNNT2 and partially organized their cytoskeleton in a cross-striated manner. The conversion process was associated with significant upregulation of a cohort of cardiac-specific genes, activation of pathways associated with muscle contraction and physiology, and downregulation of fibroblastic markers. We used a genetically encoded calcium indicator and readily detected active calcium transients although no spontaneous contractions were observed in transdifferentiated cells. Finally, we determined that inhibition of Janus kinase 1, inhibition of glycogen synthase kinase 3, or addition of NRG1 significantly enhanced the efficiency of transdifferentiation. Overall, we describe a method for achieving transdifferentiation of human dermal fibroblasts into induced cardiomyocyte-like cells via transcription factor overexpression, microRNA delivery, and molecular pathway manipulation. Human dermal fibroblasts (HDFs) were induced to transdifferentiate for 2 weeks. Group 1: Cardiac TF and microRNA, Group 2: Cardiac TF only, Group 3: Control (M2rtTA only). Conversion occurred using transdifferentiation medium supplemented with JAK1 inhibitor and sodium butyrate. Total RNA was isolated using the RNeasy Mini kit (Qiagen, 74104). Its quality was assessed using the Agilent 2100 Bioanalyzer G2939A (Agilent Technologies, Santa Clara, CA) and NanoDrop 8000 spectrophotometer (Thermo Scientific / NanoDrop, Wilmington, DE). Hybridization targets were prepared with MessageAmp™ Premier RNA Amplification Kit (Applied Biosystems / Ambion, Austin, TX) from total RNA, hybridized to GeneChip® Human Genome U133A 2.0 arrays in Affymetrix GeneChip® hybridization oven 645, washed in Affymetrix GeneChip® Fluidics Station 450 and scanned with Affymetrix GeneChip® Scanner 7G according to standard Affymetrix GeneChip® Hybridization, Wash, and Stain protocols (Affymetrix, Santa Clara, CA). This work was performed at the Duke University microarray core facility.

转分化（transdifferentiation）作为一项新兴技术，近来被证实可将人类成纤维细胞转化为诱导型心肌细胞样细胞。该方法可获取分化细胞，用于开展生理学或病理生理学研究、检测药物相互作用与毒性，以及构建组织工程样本。本研究通过诱导转录因子（transcription factors, TFs）GATA4、TBX5、MEF2C、MYOCD、NKX2-5的表达，并递送微RNA（microRNAs, miRNAs）miR-1与miR-133a，实现了人类真皮成纤维细胞向心肌细胞谱系的转分化。接受转分化处理的细胞可表达ACTN2与TNNT2，并能部分形成横纹状的细胞骨架结构。该转化过程伴随一系列心肌特异性基因的显著上调、肌肉收缩与生理相关通路的激活，以及成纤维细胞标志物的下调。本研究使用基因编码的钙指示剂，可轻松检测到活跃的钙瞬变，尽管转分化后的细胞未观察到自发收缩。最终，本研究证实：抑制贾纳斯激酶1（Janus kinase 1, JAK1）、糖原合酶激酶3（glycogen synthase kinase 3, GSK3），或添加NRG1，可显著提升转分化效率。综上，本研究报道了一种通过转录因子过表达、微RNA递送与分子通路调控，将人类真皮成纤维细胞转化为诱导型心肌细胞样细胞的方法。人类真皮成纤维细胞（human dermal fibroblasts, HDFs）被诱导转分化时长为2周。实验分为三组：组1：心肌转录因子与微RNA联合处理；组2：仅心肌转录因子处理；组3：对照组（仅转染M2rtTA）。转化过程使用添加了JAK1抑制剂与丁酸钠的转分化培养基完成。总RNA提取采用RNeasy Mini试剂盒（Qiagen，74104）。RNA质量通过Agilent 2100生物分析仪G2939A（安捷伦科技公司，加利福尼亚州圣克拉拉）与NanoDrop 8000分光光度计（赛默飞世尔科技/NanoDrop公司，特拉华州威尔明顿）进行检测。杂交靶点制备采用MessageAmp™ Premier RNA扩增试剂盒（应用生物系统公司/安比昂公司，德克萨斯州奥斯汀），以总RNA为模板。将其与GeneChip®人类基因组U133A 2.0芯片在Affymetrix GeneChip®杂交炉645中进行杂交，随后在Affymetrix GeneChip®流体工作站450中完成洗涤，并使用Affymetrix GeneChip®扫描仪7G进行扫描，实验流程严格遵循Affymetrix GeneChip®杂交、洗涤与染色标准操作方案（安捷伦科技公司，加利福尼亚州圣克拉拉）。本实验在杜克大学微阵列核心实验室完成。