Investigating miRNA Function and Cardiac Cell Lineage Specification in Human Cardiogenesis Using Pluripotent Stem Cells

2016-12-05T03:10:39Z (GMT) by Deevina Arasaratnam
The ability of human embryonic stem cells (hESCs) to recapitulate many aspects of cardiac development and differentiate into various cardiac lineages offers an unprecedented renewable source of cells for basic medical research, pharmaceutical development and therapy for cardiovascular disease. However, the excitement engendered by the emergence of stem cell technology is tempered by our limited understanding of the molecular control of the differentiation, growth, maturation and physiology of stem cell derived cardiac cell lineages. <br><br>    This thesis attempts to address some of these questions, using cardiac reporter cell lines such as NKX2-5<sup>eGFP/w </sup>hESCs to facilitate the monitoring of cardiac cell differentiation in vitro. This cell line carries an eGFP expression cassette under the control of a master cardiac specific transcription factor, NKX2-5, thus permitting the identification and purification of card iac-cell lineages. To elucidate the molecular processes governing cardiac-cell fate specification, this study was particularly interested in identifying a class of small non-coding RNA molecules, known as miRNAs involved in cardiac differentiation. MicroRNAs are post-transcriptional gene regulators and are known to cause large scale of switching of gene expression programmes. Therefore, they may have a role in directing cell fate decisions of hESCs during differentiation. Using miRNA microarray, RNA sequencing and quantitative PCR, we identified a comprehensive set of small noncoding RNAs, known as miRNAs (e.g. miRNA-1, - 133, -208, -499, 125b and 670) involved during cardiac differentiation. In addition, the utilization of a second hESC line that is deficient in NKX2-5 (NKX2- 5<sup>eGFP/eGFP</sup>) demonstrated that the transcriptional regulation of these miRNAs was independent of NKX2-5.<br> <br>    In addition to miRNAs, the multipotency of hESC-derived cardiac progenitors (CPCs) is also explored in this study using a third cell line, in which ectopic expression of an oncogenic transcription factor, c-MYC is driven from the GAPDH locus of NKX2-5<sup>eGFP/w</sup> hESC (GAPDH<sup>cMYC-ER/w</sup>). The induction of cMYC-ER allowed the expansion of GFP+ CPCs and their subsequent differentiation into epicardial-like lineages when treated with additional growth factors such as BMP4 and FGF2.<br><br>    Collectively, these studies provide a novel platform for further investigation of the functional roles of cardiac-specific miRNAs and the derivation of cardiac-cell lineages from hESC-derived CPCs, in hopes for future application of hESCs within a clinical setting.