Human embryonic stem cells : advancing biology and cardiogenesis towards functional applications l

Human embryonic stem cells (hESC) hold great potential as a model for human development, disease pathology, drug discovery and safety pharmacology. All these applications will depend on comprehensive knowledge of their biology and control of their signaling mechanisms and fate choices. To begin to address this, we developed a standardized feeder-free hESC culture protocol. This system is optimized and tested for 12 independently derived stem cell lines, and optimal for clonal growth and efficient gene transfer without loss of pluripotency (Chapter 2,3). Using these protocols we created stem cells ubiquitously expressing EGFP, showed efficient SOX2 knockdown and created a fluorescent reporter stem cell line for the stem cell regulator OCT4. Next we used mass spectroscopy to investigate the plasma membrane of hESC (Chapter 4). We were able to show that these cells express a uniform epithelial plasma membrane profile and that VIMENTIN, normally associated with mesenchymal cells is also expressed. This expression turned out to be related to stress and associated with hardness of the tissue culture plastic substrate rather than differentiation. We continued to investigate the plasma membrane of hESC and decided to focus on integrins, the cell surface receptors that bind extracellular matrix proteins. Functional analyses of their function showed human embryonic stem cells have the capacity to bind to a wide range of extracellular matrix proteins via specific integrin receptors. We were able to show that recombinant vitronectin robustly supports the maintenance of hESC in an undifferentiated state in completely defined culture medium. Having validated a completely defined culture protocol we began to investigate differentiation mechanisms under defined conditions (Chapter 5). We used Stable Isotope Labeling in Cell Culture (SILAC) and quantitative phopspho-proteomics to investigate how human embryonic stem cells exit the pluripotent state. BMP4 was used to trigger differentiation and protein samples were analyzed at 30 min, 60 min and 240 min. We showed that approximately 50% of the 3067 identified phosphosites were regulated within 1 hr of differentiation induction, revealing a complex interplay of phosphorylation networks spanning different signaling pathways. Among the phosphorylated proteins was the pluripotency-associated protein SOX2, which was SUMOylated as a result of phosphorylation. Using the data to predict kinase-substrate relationships we reconstructed the hESC kinome, with CDK1/2 emerging as a key kinase in controlling self-renewal and lineage specification. Next we used gene targeting to create a fluorescent cardiac reporter cell line. EGFP was targeted into one allele of the NKX2-5 gene. EGFP fluorescence driven by the endogenous Nkx2-5 promoter faithfully reported cardiovascular lineage commitment of differentiating hESC under defined culture conditions. Using fluorescence activated cell sorting we showed that the early NKX2-5 positive cell population contained multipotent progenitor cells capable of directed differentiation to cardiomyocytes, endothelial and vascular smooth muscle cells (Chapter 7). Finally, we used the cardiomcyocytes from hESC to develop a system for cardiac safety pharmacology (Chapter 8). Recent withdrawals of prescription drugs from clinical use because of unexpected side effects on the heart have highlighted the need for more reliable cardiac safety pharmacology assays. In particular, blocking of the human Ether-a-go go Related Gene ion channel is associated with life-threatening arrhythmias, such as Torsade de Pointes. We demonstrated that, as predicted, patient serum levels of drugs and known responses on QT interval overlapped with field potential duration values derived from hESC-CM,. On this basis, we propose field potential duration prolongation as a directly applicable safety criterion for pre-clinical evaluation of new drugs in development. In conclusion, the availability of human cardiomyocytes from stem cell sources is now expected to accelerate cardiac drug discovery and safety pharmacology by offering more clinically relevant human culture models than presently available (Chapter 9,10)