Background & Aim: MicroRNAs (miRs) are small non-coding RNA molecules, which modulate gene expression. A given miR can cause large scale of switching of gene expression programmes, hence may have a role in directing cell fate decisions. Our aim was to identify differentially expressed miRs in human embryonic stem cell (hESC) derived cardiomyocytes.
Methods: We had previously generated a reporter cardiac cell line NKX2-5GFP/w in which green fluorescent protein (GFP) expression is under control of the cardiac-specific transcription factor NKX2.5 promoter. This facilitates the separation and comparison of hESC derived cardiomyocytes. NKX2-5GFP/w hESCs were differentiated for 7 days, then separated into GFP+ and GFP- fractions by flow cytometry. The differential micro-RNA expression profile of GFP+ and GFP- fraction was examined by micro-array (Affymetrix) and changes confirmed using RT-PCR Taqman microRNA assays (Applied Biosystems). Finally, functional analysis was performed using miR knock-down experiments in embryoid bodies.
Results: A number of miRs, previously associated with pluripotency exhibited lower levels in the GFP positive fraction including miRs -200c,-302a, -302c*,-302d, 371-5p,-372, and-373. Similarly, there were a number of miRs with higher expression in the GFP+ fraction, most notably known muscle markers miR-1 and -133a. Preliminary experiments demonstrated a decrease in GFP expression upon knocking down identified miR expression.
Conclusions: Cardiomyocytes derived from human embryonic stem cells have a distinctive miRNA profile. Further functional experiments are required to determine if the differentially expressed miRs are involved in the mechanism of cardiac differentiation.