Abstract 189: HuR, RNA Stabilizing Protein, Regulation of Pluripotency and Differentiation in iPSCs

Abstract

Background: The Hu family of RNA-binding proteins, HuR (also known as ELAVL1 or human embryonic lethal abnormal vision-like protein), binds to the 3’-untranslated region of mRNAs and regulates transcript stability and translation. Global deletion of HuR is embryonically lethal in mice and plays a critical role in progenitor cell survival and biology. Induced-pluripotent stem cells (iPSC) have distinct transcriptional machinery for the maintenance of pluripotency and achievement of differentiation. However, the exact role of HuR in pluripotency or differentiation of iPSC to cardiomyocytes (iCM) remains unclear. Methods: HuR knockdown in human dermal fibroblast-derived iPSCs was achieved by CRISPR/Cas9 or lentiviral shRNA transduction and subsequently differentiated into cardiomyocytes (iCM). Then, the expression of HuR, pluripotency and cardiomyocyte markers were evaluated on days 0, 1, 3, 6, 8 and 17 following the initiation of differentiation. Results: At basal level, HuR expression was higher in the iPSCs compared to dermal fibroblasts. Upon differentiation of iPSCs into iCM, HuR mRNA expression gradually reduced with significantly lower levels on day 17. As expected, pluripotency markers gradually reduced upon differentiation with significantly lower levels from day 6 onwards. We observed a corresponding increase in ISL1, MESP1 (mesoderm/cardiac progenitor markers) from day 3 through day 8 with a steep fall from day 8 to day 17. This was associated with Myosin light chain-2V and GATA4 expression increases from day 8 through day 17. Interestingly, knockdown of HuR resulted in clumps of colonies with differentiated cells and a corresponding increase in cardiac-troponin positive cells. However, as a general observation, HuR knockdown reduced beating intensity compared to wild type cells. Conclusions: Based on these data, we could speculate that HuR might be necessary for maintenance of pluripotency and loss of which renders cells to differentiate in culture. HuR knockdown yields higher number of c-troponin positive cells but its effect on functional maturity of iCM needs to be further evaluated.