Gene expression of cardiac differentiation of induced pluripotent stem cells.
Ho, Wai Seen.
Date of Issue2012
School of Biological Sciences
National Heart Centre Singapore
As there is a lack of information on the cellular transcriptional regulation of cardiomyogenesis in hiPSCs, gene expression was investigated in viral-free hiPSC MSnviPSNF2 (C2) and MSnviPSNF3 (C3), with a novel cardiopoietic treatment employed for cardiac differentiation. Together with an overall increase in spontaneous beat clusters formed in hiPSC-CMs, panel of 18 genetic markers has been evaluated using quantitative RNA expression analysis (qPCR). Pluripotency markers were down-regulated upon differentiation induction (d0-d2), followed by an up-regulation of mesodermal and cardiomesodermal genes (d2-d4), as well as cardiac-associated transcriptional factors (d2-d21). Then, an up-regulation of other cardiac-specific structural and ion channel markers was observed (d8-d21). Due to intrinsic and extrinsic factors during cardiac differentiation, both cell lines displayed distinctive variations and differentiation propensity in genetic expressions of the markers, such as the differential down regulation of Oct 4 and higher expression levels of Mesp1, Tbx5, Tbx20 and Isl1 in C3 differentiating EBs. Characterisation of undifferentiated hiPSC also revealed that both cell lines shared similar genetic and pluripotency capabilities to human embryonic stem cells (hESCs), thus proving to be a possible alternative to circumvent ethical and immunological issues of hESCs. Overall, these results provided genetic information for further modifications and optimizations for cardiac differentiation protocols. With higher quality and yield of hiPSC-CM, it will be easier to comply with clinical requirements for downstream processes in future personalized cardiac cell therapy treatments.
Final Year Project (FYP)
Nanyang Technological University