Transdifferentiation Of Human Dermal Fibroblasts Towards The Cardiac Cell Lineage. Homo sapiens

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. Overall design: 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.