Summary
Human pluripotent stem cells have traditionally been derived from surplus embryos and termed human embryonic stem cells (hESCs). However, it has recently been shown that human dermal fibroblasts can be reprogrammed to a pluripotent state using viruses encoding (three or) four transcription factors, Klf-4, Sox2, Oct-4, and c-Myc [Takahashi et al. 2007;Yu et al. 2007]. The full extent to which such reprogrammed induced pluripotency (hiPS) cells and their differentiated derivatives are comparable to hESC now urgently needs assessment [Shi et al 2007]. In particular the epigenetic and phenotypic stability of hiPS derivatives needs to be compared with that of hESC and derivatives. Only then can the usefulness of hiPS cells for both basic and therapeutic applications (e.g. differentiation pathway analysis/disease modelling or transplantation) be fully assessed.  In addition, for applications requiring large cell numbers expansion of committed progenitors from pluripotent cells rather than undifferentiated cells themselves would be more convenient. Although able to expand indefinitely in culture hESC and hiPS cells have the disadvantage of less controlled differentiation. A degree of restriction may be preferable to true pluripotency for some applications. To address these issues, we will compare the proteomes of hESC and hiPS cells and their cardiomyocyte derivatives using high-throughput quantitative Mass Spectrometry (MS). We have shown previously that SILAC, which allows accurate quantification of protein and peptide ratios through metabolic incorporation of ‘light’ or ‘heavy’ forms of amino acids into the proteins, is feasible in cultured hESC without loss of pluripotency [van Hoof 2007]. We have also developed techniques for targeting hESC and can select cardiac precursor cells (CPCs) from hESC by FACS. We will target and select hiPS CPCs similarly and compare their epigenetic status and protein networks directly. CPCs can be induced to differentiate to cardiomyocytes by activation of Smad signaling via BMPs and repression of wnt signaling. We will compare the phosphoproteome of induced with control CPCs as previously for undifferentiated hESC to identify cell-type specific signaling networks mediating terminal differentiation and by mutagenesis confirm function of the pathways identified.