Single cell qPCR reveals that additional HAND2 and microRNA-1 facilitate the early reprogramming progress of seven-factor-induced human myocytes.

TitleSingle cell qPCR reveals that additional HAND2 and microRNA-1 facilitate the early reprogramming progress of seven-factor-induced human myocytes.
Publication TypeJournal Article
Year of Publication2017
AuthorsBektik, E, Dennis A, Prasanna P, Madabhushi A, Fu J-D
JournalPloS one
Date Published2017
KeywordsBasic Helix-Loop-Helix Transcription Factors, Cell Differentiation, Cell Line, Cellular Reprogramming, Cellular Reprogramming Techniques, Fibroblasts, Gene Expression Regulation, Human Embryonic Stem Cells, Humans, MicroRNAs, Myocytes, Cardiac, Real-Time Polymerase Chain Reaction, Transcriptome

The direct reprogramming of cardiac fibroblasts into induced cardiomyocyte (CM)-like cells (iCMs) holds great promise in restoring heart function. We previously found that human fibroblasts could be reprogrammed toward CM-like cells by 7 reprogramming factors; however, iCM reprogramming in human fibroblasts is both more difficult and more time-intensive than that in mouse cells. In this study, we investigated if additional reprogramming factors could quantitatively and/or qualitatively improve 7-factor-mediated human iCM reprogramming by single-cell quantitative PCR. We first validated 46 pairs of TaqMan® primers/probes that had sufficient efficiency and sensitivity to detect the significant difference of gene expression between individual H9 human embryonic stem cell (ESC)-differentiated CMs (H9CMs) and human fibroblasts. The expression profile of these 46 genes revealed an improved reprogramming in 12-week iCMs compared to 4-week iCMs reprogrammed by 7 factors, indicating a prolonged stochastic phase during human iCM reprogramming. Although none of additional one reprogramming factor yielded a greater number of iCMs, our single-cell qPCR revealed that additional HAND2 or microRNA-1 could facilitate the silencing of fibroblast genes and yield a better degree of reprogramming in more reprogrammed iCMs. Noticeably, the more HAND2 expressed, the higher-level were cardiac genes activated in 7Fs+HAND2-reprogrammed iCMs. In conclusion, HAND2 and microRNA-1 could help 7 factors to facilitate the early progress of iCM-reprogramming from human fibroblasts. Our study provides valuable information to further optimize a method of direct iCM-reprogramming in human cells.

PDF Link

Alternate JournalPLoS ONE

 *IEEE COPYRIGHT NOTICE: 1997 IEEE. * Personal use of this material is permitted. However, permission to reprint/ republish this material for advertising or promotional purposes or for creating new collective works for resale or redistribution to servers or lists, or to reuse any copyrighted component of this work in other works must be obtained from the IEEE.

*COPYRIGHT NOTICE:* These materials are presented to ensure timely dissemination of scholarly and technical work. Copyright and all rights therein are retained by authors or by other copyright holders. All persons copying this information are expected to adhere to the terms and constraints invoked by each author's copyright. In most cases, these works may not be reposted without the explicit permission of the copyright holder.